Paracoccidioides brasiliensis

Transcription

ALISSON LEONARDO MATSUO
Caracterização de proteinases, identificação de proteínas
associadas à parede celular diferencialmente expressas
e estudo preliminar de vesículas extracelulares de
Paracoccidioides brasiliensis.
Tese apresentada à Universidade
Federal de São Paulo – Escola Paulista
de Medicina, para obtenção do Título
de Doutor em Ciências
São Paulo
2007
ALISSON LEONARDO MATSUO
Caracterização de proteinases, identificação de proteínas
associadas à parede celular diferencialmente expressas
e estudo preliminar de vesículas extracelulares de
Paracoccidioides brasiliensis.
Orientadora: Profa. Dra. Rosana Puccia
Co-orientadora: Profa. Dra. Adriana K. Carmona
Tese apresentada à Universidade
Federal de São Paulo – Escola Paulista
de Medicina, para obtenção do Título
de Doutor em Ciências
São Paulo
2007
Matsuo, Alisson Leonardo
Caracterização de proteinases, identificação de proteínas associadas à parede
celular diferencialmente expressas e estudo preliminar de vesículas extracelulares
de Paracoccidioides brasiliensis / Alisson Leonardo Matsuo – São Paulo, 2007
xii, 165 fls
Tese (Doutorado) Universidade Federal de São Paulo. Escola Paulista de
Medicina. Programa de Pós-Graduação em Microbiologia e Imunologia.
Título em inglês: Caracterization of proteinases, identification of differentially
expressed associated cell wall proteins and preliminary studies of extracellular vesicles
from Paracoccidioides brasiliensis
1. Paracoccidioides brasiliensis; 2. proteinases; 3. serino-tiol proteinase; 4. Lon
proteinase; 5. parede celular; 6. vesículas/exossomos
Trabalho realizado na Disciplina de Biologia Celular do
Departamento de Microbiologia, Imunologia e parasitologia da
Universidade Federal de São Paulo – Escola Paulista de Medicina
com auxílio financeiro concedido pela
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
"Na maioria das vezes, um pepino é somente um pepino"
Freud
iv
Dedico este trabalho às pessoas mais importantes da minha vida...
...à minha querida esposa Leda e ao nosso filhinho Rafael, pela presença constante,
momentos alegres, paciência e apoio nas horas difíceis. Quando estou “incubado” no
laboratório sempre lembro de vocês, principalmente dos sorrisos e das travessuras do
Rafa. Amo vocês.
...aos meus pais Francisco e Lucia pela dedicação, apoio, educação, ensinamentos,
compreensão e carinho. Se hoje cheguei até aqui é porque vocês me deram este
privilégio. Admiro-os muito! Obrigado por tudo.
...à toda minha família em especial ao meu sobrinho Gustavo, aos meus irmãos Cleber e
Daniel, meus cunhados Claudia, Elaine, Henrique e Ieda e aos meus sogros Kiiti e
Alice, por comporem o tripé de nossa família e ajudar em momentos de necessidade.
Tenho certeza que são pessoas com quem sempre poderei contar.
v
Agradecimentos
Meu primeiro agradecimento é dedicado à minha orientadora Profa. Dra. Rosana
Puccia, também conhecida como Chefa, ou simplesmente Rô. Pela confiança, amizade,
paciência, honestidade e principalmente pelo meu desenvolvimento científico e correção
dos meus erros português. Acho que hoje já consigo escrever “um pouco” melhor.
À Profa. Dra. Adriana K. Carmona, minha co-orientadora, pela sua contribuição
na minha formação científica, confiança, alto astral e simpatia.
Ao Prof. Dr. Igor Correia de Almeida, pela oportunidade de intercâmbio na
UTEP, pelas sugestões científicas e bom humor.
Ao Prof. Dr. Ivarne Tersariol, pelos ensinamentos sobre enzimologia,
contribuição científica e idéias malucas.
A todos os professores dos departamentos de Microbiologia, Imunologia e
Parasitologia, Biofísica e Bioquímica da UNIFESP, pelos ensinamentos, equipamentos e
sugestões que ajudaram na minha formação. Em especial aos professores Travassos,
Elaine, Zoilo, Sérgio, Cida, Olga, Clara e Juliano, os quais admiro e são excelentes
pesquisadores.
Aos meu amigos de laboratório Luizão, Kátia, Luciane, Wagner, Antonio,
Milene, Graziela, Ernesto, Lívia, Milca, Thiago, Tânia, Flavia e Aguinaldo. Estes são
meus irmãos científicos, pessoas com quem convivi e compartilhei a maior parte do meu
tempo. Por isso agradeço pelos bons momentos, ajuda na hora do desespero, piadas com
ou sem a menor graça, seminários e é claro por me aturarem.
vi
Aos secretários Marcelo, Marcinha e Mércia, que estão sempre nos ajudando
com as burocracias, são muito eficientes e sempre sabem de tudo, até a matéria da capa
de jornal do dia seguinte.
Aos amigos de diversos laboratórios Rodrigo, Andrey, Thaysa, Xandão,
Hebeler, Geisa, Fabi, Luiz, Fizão, Bel, Ellen, Bianca, Mário, Ricardo, Simone,
Júnior, Cris, Roti, Patrícia, Rafael, Júlia, Teresa, Alberto, Fernando e Izaura por
tornar a nossa vida dentro de um laboratório muito mais alegre, ás vezes conversar sobre
outros assuntos além de resultados de experimentos faz muito bem. A compania de vocês
nestes anos com certeza foi muito importante.
A todos os funcionários da disciplina de Biologia Celular, em especial à Maria,
Américo, Claudeci, Claudio, Antonio e Sebastiana pela dedicação, esforço e
competência. Sem vocês o “andar não anda”. Obrigado por tudo.
Aos amigos da minha turma de graduação Bio 32 pelos 4 anos de convivência e
troca de experiência. Espero que se tornem grandes pesquisadores para um dia eu contar
para meu filho que eu tive a oportunidade de estudar com vocês.
Aos amigos do colegial, do clã e da faculdade Candiru, Danylo, Okano, César,
Yassumassa, Allan, Ogro, Fabrício, Bariri, Dragão, Kpone, Xarope, Ivan, Carlitos,
Luciano, Zé, Emperor, Henrique, Rodrigo, Marcelo, Shyvaia, Reclamador, Truz,
vii
Marcinho, Vagnão e Guto pelos momentos engraçados, viagens, bebedeiras, momentos
inusitados e alegria. Esses são meus amigos e com certeza contribuíram para a formação
da minha pessoa, meu caráter e até mesmo influenciaram na minha personalidade. A
segunda família.
Às bandas Pedreiros do Apocalipse e Rockcídio das quais sou integrante.
Através da música esqueço de todos os meus problemas, amarguras, tristezas e me sinto
muito mais confiante para enfrentar tudo. Também gostaria de agradecer ao Palmeiras, o
melhor time do mundo, pelas alegrias e títulos conquistados que me acompanham desde o
nascimento. Certamente ele não anda “bem das pernas” nos últimos anos, mas tenho
certeza que voltará a ganhar dezenas de títulos mundo afora.
À FAPESP e CNPq pelo financiamento deste trabalho.
viii
LISTA DE ABREVIATURAS
BSA: albumina sérica bovina
ELISA: “enzyme-linked immunosorbent assay”
Con-A: concanavalina A
DO: densidade ótica
PMSF: fluoreto de fenil-metil-sulfonil
F12: meio mínimo de cultura
IFN-γ: intérferon gama
IL: interleucina
NO: óxido nítrico
PBS: tampão salino tamponado com fosfato
PCM: paracoccidioidomicose
ATP: 5’-trifosfato de adenosina
Npys: “S-3-nitro-2-pyridinesulfenyl”
Abz: ácido orto-aminobenzóico
EDDnp: 2,4-dinitrofeniletilenodiamina
GalMan: galactomanana
HSP: “heat shock protein”
pHMB: para hidroximercuriobenzoato
EDTA: ácido etilenodiamino tetra-acético
E-64: “4-[(2S,3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido(4-guanidino)butane”
IPTG: isopropil-tiol-galactopiranosídeo
ix
LacZ: β-galactosidase
min: minuto
nt: nucleotídeo
mRNA: RNA mensageiro
PCR: reação de polimerização em cadeia
SDS: dodecil sulfato de sódio
SDS-PAGE: eletroforese em gel de poliacrilamida
sec: segundos
TBE: tampão tris-borato-EDTA
TEMED: N,N,N,N´-tetrametilenodiamina
UV: ultravioleta
PbST: serino-tiol proteinase extracelular de P. brasiliensis
x
ÍNDICE
LISTA DE ABREVIATURAS
ix
INTRODUÇÃO
1
1.1 Aspectos gerais sobre o Paracoccidioides brasiliensis
1
1.2 Paracoccidioidomicose humana
4
1.3 Parede celular, transição e virulência
10
1.4 Proteinases de P. brasiliensis
17
OBJETIVOS
29
CAPÍTULO 1: Proteinases de Paracoccidioides brasiliensis: serino-tiol
30
proteinase extracelular e Pb Lon mitocondrial.
1.1 Artigo: Modulation of the exocellular serine-thiol
32
proteinase activity of Paracoccidioides brasiliensis by
neutral polysaccharides
1.2 Artigo: C-Npys (S-3-nitro-2-pyridinesulfenyl) and
34
peptide derivatives can inhibit a serine-thiol proteinase activity
from Paracoccidioides brasiliensis
1.3 Artigo: The PbMDJ1 gene belongs to a conserved
MDJ1/LON locus in thermodimorphic pathogenic fungi
and encodes a heat shock protein that localizes to both the
mitochondria and cell wall of Paracoccidioides brasiliensis
xi
37
CONCLUSÕES: Capítulo 1
39
CAPÍTULO 2: Moléculas secretadas de Paracoccidioides
42
brasiliensis: proteínas associadas à parede celular diferencialmente
expressas e resultados preliminares sobre vesículas extracelulares
INTRODUÇÃO
43
2.1 Manuscrito: Proteomic analysis of differentially
49
expressed cell wall-associated proteins from the human
pathogen Paracoccidioides brasiliensis
2.2 Moléculas secretadas de Paracoccidioides brasiliensis:
80
resultados preliminares sobre vesículas extracelulares
Materiais e métodos
81
Resultados e Discussão
88
CONCLUSÕES: capítulo 2
102
REFERÊNCIAS BIBLIOGRÁFICAS
104
ANEXO 1
117
ANEXO 2
121
xii
Introdução
1.1 Aspectos gerais sobre o Paracoccidioides brasiliensis
Paracoccidioides brasiliensis é o fungo dimórfico térmico causador da
paracoccidioidomicose (PCM) humana. A PCM é uma micose sistêmica granulomatosa
que pode ser letal se não tratada adequadamente. O fungo foi originalmente descrito por
Adolpho Lutz (Figura 1) em 1908, quando isolou o fungo de lesões orais e de linfonodo
cervical no Instituto Biológico de São Paulo (Brasil). Observando os exames histológicos
de um de seus pacientes, declarou que a ausência de esférulas com esporos no seu interior
diferenciava-os de especimens característicos de coccidioidomicose, descrita previamente
na Argentina por Posadas em 1892 (Posadas A., 1892). Mesmo alguns anos após a
descoberta, o P. brasiliensis era comumente confundido com o fungo Coccidioides
immitis, justificando seu nome, já que o prefixo para significa “similiar a”. Análises de
sequências do gene do RNA ribossomal revelaram que os fungos causadores de micoses
granulomatosas Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis
e P. brasiliensis estão filogeneticamente relacionados e pertencem à mesma família
(Bialek et al., 2000; Peterson et al., 1998). Atualmente o P. brasiliensis é classificado
como Eukaryota do reino Fungi, filo Ascomycota, subfilo Pezizomicotina, classe
Eurotiomycetes, ordem Onygenales, família Onygenaceae, gênero Paracoccidioides e
espécie brasiliensis.
1
Figura 1. Retrato de Adolfo Lutz
Em condições ambientais (25oC), o fungo cresce na fase miceliana, na qual se
apresenta como hifas delgadas de 1 a 3 µm, possuindo como formas reprodutivas os
aleuroconídeos,
artroconídeos,
artroaleuroconídeos
e
clamidosporos
intercalares
(Restrepo-Moreno, 1994). Os conídeos são unicelulares, porém quando incubados a 37oC
ou alojados nos pulmões (McEwen et al., 1987) transformam-se em leveduras
multinucleadas (Figura 2). As leveduras do fungo são esféricas ou ovais de 4 – 30 µm de
diâmetro, com paredes refringentes, múltiplos brotamentos (Lacaz, 1994), múltiplos
vacúolos, corpos lipídicos e um grande número de mitocôndrias (Kashino et al., 1987). A
forma sexuada ainda não é conhecida e recentemente o P. brasiliensis foi classificado
como haplóide ou aneuplóide (Almeida et al., 2006). Estimativas mostram que o genoma
haploide varia entre 26 a 35 Mb, distribuído em 4 a 5 bandas cromossômicas de 2 a 10
Mb (Feitosa et al., 2000; Almeida et al., 2006).
2
Figura 2. Dimorfismo do P. brasiliensis
Evidências experimentais indicam que o tatu é um reservatório natural do P.
brasiliensis (revisão em Restrepo et al., 2001). O fungo foi isolado de duas espécies
diferentes (Figura 3), o Dasypus novemcintus (Bagagli et al., 1998) e o Cabassou
centralis (Corredor et al., 2005). Em áreas hiper-endêmicas da PCM, o fungo foi isolado
em cerca de 75 a 100% dos tatus de nove bandas (Bagagli et al., 1998; 2003; Restrepo et
al., 2000). Evidências experimentais confirmaram que o P. brasiliensis isolado de
pacientes e tatus possuem o mesmo perfil molecular, antigênico e de virulência,
sugerindo que os mesmos isolados têm a capacidade de infectar o homem e o animal
(Sano et al., 1999; Hebeler-Barbosa et al., 2003). Infecções em animais domésticos e
selvagens também já foram descritas (Ricci et al., 2004; Garcia et al., 1993; Grose e
Tramsitt, 1965; Johnson e Lang, 1977).
3
Dasypus novemcinctus
Cabassou centralis
Figura 3. Espécies de tatus de onde foi isolado P. brasiliensis.
Um amplo estudo de multilocus, para o qual foram comparadas seqüências de oito
regiões de 5 locus diferentes em 65 isolados de P. brasiliensis provenientes de diversas
áreas endêmicas da América Latina, classificou-os em 3 espécies filogeneticamente
distintas, a saber S1, PS2 e PS3 (Matute et al., 2006). O grupo S1 foi composto por 38
isolados do Brasil, Peru, Venezuela, Argentina e Paraguai, para os quais foram apontadas
diversas evidências de reprodução sexuada. O grupo filogenético PS2 compreendeu 6
isolados, 5 brasileiros e 1 venezuelano, e sua especiação ocorreu mais provavelmente
pela incapacidade da transmissão genética com o grupo S1 do que pelo isolamento
geográfico, uma vez que se situam nos mesmos países. O último grupo, PS3, constituído
por 21 isolados colombianos, foi considerado uma linhagem evolucionária independente
provavelmente causada pelo isolamento geográfico.
1.2 Paracoccidioidomicose humana
4
Durante as décadas de 30 e 40 houve um aumento de novos casos no Brasil de
PCM, principalmente com lesões cutâneas e mucosas, quando ficou conhecida como
blastomicose brasileira ou blastomicose sul-americana. Observações mais detalhadas
confirmaram que a PCM não era limitada a lesões mucocutâneas e de pele, mas sim uma
doença com amplo espectro de manifestações clínicas e patológicas (revisão em Lacaz,
1994). A PCM ocorre principalmente no Brasil, Argentina, Venezuela e Colômbia
(Figura 4), onde é endêmica em áreas rurais, com temperaturas em torno de 18 a 25 oC,
elevado índice pluviométrico (800 a 2000 mm por ano), altitude entre 50 a 1.300 metros e
predomínio de florestas nativas (Bagagli et al., 1998). Também há relatos de PCM na
Europa, nos Estados Unidos, África, Ásia e Oriente Médio, porém nesses casos todos os
pacientes viveram por anos em áreas endêmicas da América Latina. Não há registros de
casos em Belize, Nicarágua, Guiana, Guiana Francesa, Suriname, Chile ou na maioria das
ilhas caribenhas (Wanke e Londero, 1994). Estima-se que cerca de 10 milhões de pessoas
estejam infectadas e que a incidência anual da doença ativa em áreas endêmicas seja de 1
a 3 indivíduos para cada 100.000 habitantes (San-Blas et al., 2002). Entre 1980 e 1995, a
PCM destacou-se como a 8a causa de mortalidade por doenças infecciosas e parasitárias
de caráter crônico e apresentou a mais elevada taxa de morbidade entre as micoses
sistêmicas no Brasil (Coutinho et al., 2002).
5
Figura 4. Regiões endêmicas da paracoccidioidomicose (retirado de Shikanai-Yasuda et al., 2006)
A infecção do hospedeiro humano ocorre através da inalação de conídeos do
fungo que se instalam nos pulmões, onde se transformam em leveduras que causam a
infecção (McEwen et al., 1987). As lesões podem regredir com a destruição do fungo,
regredir com focos de fungos quiescentes que podem causar a doença posteriormente, ou
progredir imediatamente com o desenvolvimento dos sintomas da PCM. Na maior parte
dos casos há uma infecção sub-clínica com cura espontânea (revisão em Lacaz, 1994),
sugerindo uma predisposição genética para a progressão da doença (Brummer et al.,
1993). No Brasil sabe-se que o risco de desenvolver a PCM é 4,3 vezes maior em
indivíduos portadores de antígeno HLA-B40 (Lacerda et al., 1988); na Colômbia,
predominam pacientes com antígenos HLA-A9 e HLA-B13 (Restrepo et al., 1983).
De acordo com registros médicos, a doença ativa manifesta-se na forma aguda
(juvenil ou linfática) em 5 a 10% dos casos, ou na forma crônica (tipo adulto ou
6
pulmonar) na maioria dos indivíduos. A PCM juvenil progride rapidamente, atingindo o
sistema retículo endotelial de adultos jovens (baço, fígado, linfonodos e medula óssea). A
PCM adulta progride lentamente, é prevalente em homens de 30 – 60 anos, e pode ser uni
ou multifocal. A disseminação para outros órgãos ocorre pela via hematogênica e/ou
linfática. A baixa incidência da PCM em mulheres (13 vezes menor) está possivelmente
ligada com a presença do 17 β-estradiol, que é um inibidor da transformação de micélio
para levedura in vitro (Clemons et al., 1989).
Diferenças na evolução da PCM devem ter relação com a existência de isolados
do P. brasiliensis apresentando variação no grau de virulência e de sua interação com
fatores inerentes ao hospedeiro, como idade, sexo, raça, grau de nutrição e especialmente
o status imunológico. Modelos experimentais de camundongos isogênicos foram
selecionados quanto à susceptibilidade para infecção com P. brasiliensis isolado Pb18
(revisão em Calich et al., 1998). Em infecções intratraqueais e intraperitoniais, os animais
da linhagem A/Sn foram os mais resistentes, já que apresentaram um padrão de
imunidade predominante para o tipo Th-1. Camundongos B10.A, e em menor grau
Balb/c, foram susceptíveis à infecção, observando-se neles uma resposta imune Th-2
orientada, onde prevalesceram altos títulos de anticorpos, imunidade celular
comprometida e produção insuficiente de IFN-γ (Calich et al., 1985, 1994; Cano et al.,
1995).
A depressão da imunidade mediada por células está fortemente relacionada à
progressão da doença (Mota et al., 1985). A defesa do organismo é mediada pela
formação de granulomas compostos de células gigantes e epitelióides com a presença de
polimorfonucleares e leucócitos próximos ao fungo (Franco et al., 1987). Em pacientes
7
com infecções tênues, observam-se granulomas compactos com reduzido número de
fungos, enquanto em pacientes com sintomas graves, pode-se observar granulomas
frouxos e um influxo celular de polimorfonucleares, macrófagos e células mononucleares
com capacidade reduzida de combater o fungo (Bernard et al., 1994).
Estudos realizados in vitro com macrófagos mostraram que o P. brasiliensis é
capaz de se multiplicar intracelularmente, destruir o macrófago e liberar inúmeras células
leveduriformes (Brummer et al., 1989). Porém macrófagos ativados com IFN-γ possuem
a capacidade de inibir a replicação do fungo em 65 a 95% dos casos (Moscardi-Bacchi et
al., 1994). Estudos mais recentes mostram que macrófagos ativados com IFN-γ e TNF-α
são capazes de produzir óxido nítrico (NO), que pode levar à morte do fungo em
concentrações ideiais (Nascimento et al., 2002). Além disso, os macrófagos e monócitos
são capazes de produzir TNF-α, que é uma citocina envolvida no recrutamento e ativação
de células inflamatórias e na formação do granuloma (Kindler et al., 1989).
Recentemente foi demonstrado que monócitos ativados com essa citocina são capazes de
liberar H2O2 e causar a destruição do fungo (Carmo et al., 2006). Os polimorfonucleares
constituem outro tipo celular que tem efeito fungicida e fungistático aumentado por IFNγ e GM-CSF (Kurita et al., 2000).
A hiperreatividade da imunidade humoral em pacientes com PCM ocorre
principalmente nas formas disseminadas e severas. Cerca de 90% dos pacientes com
sintomas clínicos possuem anticorpos específicos das classes IgG, IgM e IgA em 98, 45 e
33% dos casos, respectivamente (Biagioni et al., 1984). Pacientes com PCM apresentam
ativação policlonal de células B, como ocorre normalmente em outras doenças
infecciosas (Ortiz-Ortiz et al., 1980; Galvão-Castro et al., 1994; Chequer-Bou-Habib et
8
al., 1989). O papel das imunoglobulinas na PCM ainda não está totalmente esclarecido, e
elas possivelmente não são relevantes para proteção nos estágios avançados da doença,
quando os títulos de anticorpos contra antígenos fúngicos são elevados (Restrepo et al.,
1982). Porém foi verificada uma alta disseminação do fungo em animais que produzem
baixas taxas de anticorpos, indicando que a imunidade humoral estaria participando
ativamente na contenção da doença (Carvalhaes et al., 1986). Também é importante
ressaltar que experimentos de imunização passiva utilizando anticorpos monoclonais
anti-gp70 (Mattos Grosso et al., 2003), em estágios iniciais da infeção, ou anti-gp43
(Buissa-Filho et al., manuscrito em preparação) em camundongos com a doença
estabelecida, impediram quase completamente a formação de granulomas no pulmão.
O tratamento da PCM é prolongado por anos de terapia. São utilizados os
derivados de azol, além das sulfonamidas, e anfotericina B (Mendes et al., 1994). O
primeiro derivado azólico, cetoconazol, demonstrou uma eficácia de 90% e seu
mecanismo de ação está relacionado à inibição da síntese de ergosterol de membrana ao
ligar-se em enzimas relacionadas ao citocromo P450, porém apresenta efeitos colaterais
como anorexia, náusea, vômito e diminuição dos níveis de testosterona. Outros derivados
como itraconazol ou fluconazol parecem ter uma eficácia semelhante ao cetoconazol,
porém com menos adversidades. A anfotericina B foi a droga mais utilizada durante
muitos anos. É um composto poliênico capaz de se ligar ao ergosterol da membrana
fúngica alterando sua permeabilidade, mas atualmente só é utilizado em casos graves ou
de resistência a outras drogas, porque os efeitos colaterais são muito acentuados,
notadamente nos rins e fígado. As sulfonamidas foram utilizadas pela primeira vez em
1940, quando a PCM era considerada uma doença incurável. Essa classe de antibióticos
9
age como antagonista metabólico competitivo do ácido para-aminobenzóico, interfere na
síntese do ácido fólico e consequentemente na produção de DNA. Seus derivados ainda
são utilizados quando combinados com outras drogas por causa do baixo custo e eficácia.
A cura da PCM é caracterizada pela falta de anticorpos circulantes contra antígenos
específicos de P. brasiliensis. Deve-se estender a manutenção do tratamento por um ano
após a negativação dos testes sorológicos e o paciente deve ser reavaliado
periodicamente. A remissão e as sequelas de fibrose pulmonar são frequentes na PCM
(revisão em Brummer et al., 1993).
1.3 Parede celular, transição e virulência
A transição do P. brasiliensis de micélio para levedura (Figura 5) é um
requerimento essencial para a ocorrência da PCM e de outras doenças causadas por
fungos dimórficos (Namecek et al., 2006). A diferenciação morfológica que o fungo sofre
no hospedeiro, por outro lado, é consequência de um conjunto de alterações bioquímicas
e fisiológicas desencadeadas pelo aumento de temperatura, a qual estimula uma
adaptação no padrão transcricional (Nunes et al., 2006) e/ou regulatório em geral de
genes e proteínas.
10
Figura 5. Transição do P. brasiliensis de micélio para levedura após aumento de temperatura de
incubação de 26 oC para 36 oC pelos tempos indicados (adaptado de Goldman et al., 2003).
As proteínas de choque térmico (Hsps) formam uma grande classe de moléculas
relacionadas à variação de temperatura, porém também podem eventualmente ser
induzidas por estresse nutricional, osmótico, oxidativo e por substâncias tóxicas (revisão
em Walter e Buchner, 2002). Em P. brasiliensis já foram caracterizadas algumas
proteínas de choque térmico, a saber, Hsp70 (Diez et al., 2002), Hsp60 (Izacc et al.,
2001), Hsp100 ou ClpB (Jesuíno et al., 2002), Mdj1 (Batista et al., 2006) e Lon (Barros e
Puccia, 2001).
Os eventos moleculares que podem afetar a transição são pouco conhecidos até o
momento, principalmente pela falta de um sistema padronizado de transformação do
fungo, que ocorreu apenas recentemente (Almeida et al., 2007). Contudo estudos
abrangentes tentando detectar proteínas e genes envolvidos no dimorfismo ou
relacionados à virulência estão sendo realizados na última década. Trabalhos prévios
identificaram algumas proteínas (Cunha et al., 1999; Salem-Izacc et al., 1997) e genes
(Silva et al., 1999; Venâncio et al., 2002) diferencialmente expressos nas duas fases do
fungo. Atualmente há 2 grandes bancos de sequências expressas (ESTs) de P.
11
brasiliensis. O primeiro foi desenvolvido pelo grupo paulista liderado por Goldman e
colaboradores (2003), que identificaram 4.692 genes do isolado Pb18 recém-isolado de
baço de camundongo B10.A infectado intraperitonealmente. O segundo foi produzido por
grupos do centro-oeste brasileiro liderados pelas pesquisadoras Maria Sueli Felipe e Célia
Maria A. Soares, que detectaram 6.022 genes expressos nas fases miceliana e
leveduriforme do isolado Pb01 (Felipe et al., 2003 e 2005). Uma utilização imediata das
sequências identificadas de P. brasiliensis foi detectar transcritos diferencialmente
expressos na transição de micélio para levedura, que é fundamental para o
estabelecimento da infecção. Para tal, foram utilizadas genotecas de subtração, macro e
microarray, além de comparações in silico (Marques et al., 2004; Felipe et al., 2005;
Nunes et al., 2005; Ferreira et al., 2006).
Numa tentativa de identificar genes envolvidos com o processo de infecção,
Tavares e colaboradores (2007) estudaram a transcrição gênica de 1.152 genes
selecionados após 6 horas de co-cultura do Pb01 com macrófagos peritoneais murinos.
Nesse período, 90% das células estavam internalizadas ou aderidas aos fagócitos.
Utilizando hibridização em microarray, verificou-se que 152 transcritos aumentaram pelo
menos 2 vezes quando comparados ao controle crescido in vitro. Diversos genes
relacionados ao estresse oxidativo sofreram aumento do acúmulo de mRNA, como por
exemplo a superóxido dismutase 3, Hsp60 e QCR8 (subunidade do citocromo oxidase c.)
Em outro estudo sobre a expressão diferencial de genes de P. brasiliensis em condições
de infecção, Bailão e colaboradores (2006) analisaram duas populações de cDNA por
subtração. Uma delas, proveniente de fígado de camundongos infectados, revelou que
mRNAs de genes ligados à defesa celular, produção de melanina e aquisição de ferro
12
estavam aumentados. Em outra análise, o fungo foi colocado brevemente na presença de
sangue humano, onde se observou que genes principalmente relacionados ao
remodelamento e síntese da parede foram detectados em maior abundância.
Como consequência da transformação dimórfica, um importante compartimento
celular que sofre diversas modificações, das quais algumas são conhecidas, é a parede
celular. A parede celular era considerada um compartimento estático e basicamente
estrutural, porém hoje se sabe que é uma estrutura dinâmica composta de glicoproteínas,
proteínas, lipídeos e principalmente polissacarídeos do tipo quitina e glucanas (de Groot
2005). No P. brasiliensis, grande parte das 1,3-glucanas da parede fúngica sofrem uma
alteração da conformação β, na fase miceliana, para α, na levedura. Além dessa
conversão, a levedura possui maior quantidade relativa de quitina (média de 43%), e
menor concentração de proteínas (média de 11%), quando comparada ao micélio que
contém em média de 13 e 33% respectivamente. Em ambas as formas, os lipídeos
respondem por uma média de 8% e as glucanas por média de 42% da parede celular
(Kanetsuna et al., 1969). Em termos glucanas, estima-se que a fase leveduriforme possui
cerca de 83% de α-1,3-glucana e 17% de β-1,3-glucana, enquanto a fase miceliana
contém 100% de β-1,3-glucana. As β-1,3-glucanas são receptores de dectina-1 (Brown e
Gordon, 2003) e são, portanto, inflamatórias (Silva e Fazioli, 1985; Figueiredo et al.,
1993). Em H. capsulatum, cepas contendo o gene que codifica a α-1,3-glucana sintase
mutado ou seu RNA interferido tiveram sua virulência atenuada (Rappleye et al., 2004).
Nesse fungo, foi demonstrado que a α-1,3-glucana localiza-se na camada mais externa da
parede recobrindo a camada de β-1,3-glucana, o que bloqueia a ligação da dectina-1 e
inibe em 5 vezes a produção de TNF-α por células fagocíticas, com a consequente
13
redução da eficácia da resposta imune (Rappley et al., 2007). No P. brasiliensis, pelo
menos parte da β-1,3-glucana pode estar acessível na superfície (Diniz et al., 2004).
No P. brasiliensis, além das alterações nas glucanas, foi observado um aumento
significativo de 3 vezes mais quitina na fase leveduriforme que no micélio. A quitina é
um importante componente estrutural da parede celular dos fungos. Em P. brasiliensis já
foram identificadas 4 quitinas sintases (Niño-Vega et al., 2000). Em C. albicans existem
4 genes para quitina sintase, sendo um deles essencial (Munro et al., 2001). Mutantes de
C. albicans contendo os outros 3 genes de quitina sintase deletados foram mais
susceptíveis a agentes que estressam a parede celular, como por exemplo o calcofluor, e
apresentaram divisão celular deficiente, especialmente na formação de um septo íntegro
(Munro et al., 2007). Recentemente foi demonstrado que uma lectina de P. brasiliensis,
conhecida como paracoccina, foi capaz de interagir com a quitina da parede celular.
Anticorpos reativos com essa proteína foram capazes de reduzir o crescimento do fungo
in vitro, que apresentou colônias menores e células menos agregadas quando comparadas
ao controle. A presença destes anticorpos também alterarou o padrão da distribuição da
quitina na parede celular, sugerindo que a interação da paracoccina com quitina é um
evento importante na organização da parede e consequentemente no crescimento fúngico
(Ganiko et al., 2007).
Poucas moléculas do P. brasiliensis têm sido apontadas como fatores de
virulência com alguma base experimental, a saber, a glicoproteína gp43 (Puccia et al.,
1986), melanina (Gomez et al., 2001), uma serino-tiol proteinase extracelular (Carmona
et al., 1995) e adesinas de 19 e 32 kDa (Gonzales et al., 2005), 30 kDa (Andreotti et al.,
2005) e a gliceraldeído-3-fosfato desidrogenase (GADPH) (Barbosa et al., 2006), que
14
possuem habilidade de ligação a proteínas associadas à matriz extracelular. Ensaios
utilizando células de P. brasiliensis pré-incubadas com anticorpos policlonais antiGADPH ou com GADPH recombinante inibiram a aderência e internalização do fungo
por pneumócitos.
A lacase é a enzima responsável pela síntese de melanina, a qual é um fator de
virulência reconhecido em Cryptococcus neoformans (Kwon-Chung et al., 1982) e foi
também encontrada em P. brasiliensis (Gomez et al., 2001). Estudos recentes
demonstraram que células de P. brasiliensis melanizadas são 3,5 vezes menos
internalizadas por macrófagos e possuem menor susceptibilidade a várias drogas
antifúngicas como anfotericina B, fluconazol, cetoconazol e itraconazol (da Silva et al.,
2006).
A gp43 é o antígeno majoritário do fungo (Puccia e Travassos, 1991) e protetor da
PCM murina (Taborda et al., 1998; Pinto et al., 2000). Além de induzir resposta imune
humoral a gp43 contém epitopos que suscitam uma resposta imune celular de
hipersensibilidade tardia (Rodrigues et al., 1994; Saraiva et al., 1996). Taborda et al.
(1998) mostraram que camundongos imunizados com a gp43 nativa produzem uma
resposta T-CD4+ do subtipo Th1 capaz de liberar IFN-α e IL-2, porém não IL-4 ou IL-5.
Através de ensaios de linfoproliferação com animais imunizados com a gp43, os autores
identificaram o epitopo imunodominante da resposta imune celular em um peptídeo
interno de 15 aminoácidos, denominado P10. Animais imunizados com a gp43 nativa, o
gene PbGP43 e o peptídeo P10 mono ou tetravalente (M10) foram protegidos contra
subsequente desafio intratraqueal por leveduras virulentas do P. brasiliensis (Taborda et
al., 1998 e 2003; Pinto et al., 2000), sugerindo sua utilidade como vacina. Além disso, o
15
peptídeo P10 tem utilidade terapêutica comprovada em modelo animal (Marques et al.,
2006).
A gp43 é capaz de modular a infecção experimental em camundongos. Estudos in
vivo monstraram um aumento da infecção intratesticular de hamster quando os animais
foram infectados com P. brasiliensis previamente recoberto com laminina murina
(Vicentini et al., 1994; Lopes et al., 1994). Essa ligação in vivo e in vitro foi inibida com
anticorpos monoclonais anti-gp43 (Gesztesi et al., 1996). Recentemente foi evidenciada a
interação da gp43 com a fibronectina, um outro componente asssociado à matriz
extracelular (Mendes-Giannini et al., 2006).
Nosso grupo constatou um extenso polimorfismo no exon 2 do gene PbGP43
(Morais et al., 2000), principalmente entre as posições 578 e 1160. As seqüências mais
polimórficas foram encontradas no Pb2, Pb3 e Pb4, as quais codificam uma proteína
básica com pI em torno de 8,0. Essas seqüências são filogeneticamente distantes das
demais, incluindo aquela de Pb18, o que contribuiu para a classificação desses isolados
no grupo filogenético PS2 (Matute et al., 2006). Um trabalho posterior do nosso grupo
investigou a virulência de isolados pertencentes a grupos genotípicos de PbGP43
distintos em camundongos B10.A pelas vias intraperitoneal, intratraqueal e endovenosa
(Carvalho et al., 2005). Os camundongos infectados com Pb2, Pb3 e Pb4 sofreram uma
infecção branda e regressiva, com baixo índice de mortalidade, em contraste com os
demais, inclusive Pb18; Pb12 foi o mais agressivo. Em infecções posteriores com Pb3,
Pb18 e Pb12 adaptados in vivo, verificou-se que Pb3 induziu um tipo de resposta imune
com crescente produção de IFN-γ e predomínio de IgG2a e 2b, e IgG3 anti-gp43, ao
contrário de Pb18, que induziu anti-gp43 dos tipos IgG1 e IgA, predominantemente, e
16
níveis decrescentes de IFNγ. Camundongos infectados intratraquealmente com Pb12 não
produziram IFN-γ detectável (Carvalho et al., dados não publicados).
1.4 Proteinases de P. brasiliensis
Enzimas proteolíticas podem desempenhar um importante papel na virulência de
bactérias (Finlay et al., 1989), protozoários (McKerrow et al., 1993) e fungos patogênicos
(Ogrydziak, 1993). Proteinases extracelulares de fungos saprófitas como A. niger e N.
crassa são secretadas principalmente para a nutrição. Fungos patogênicos, no entanto,
parecem ter adaptado funções especializadas para essas hidrolases durante a infecção,
como por exemplo, desestruturar a membrana celular para facilitar a adesão e invasão do
hospedeiro como demonstrado em plantas (Chaffin et al., 1998) e em insetos (Smithson
et al., 1995), ou causar danos a células e moléculas do sistema imune (Salyers et al.,
1994). A família das SAPs (aspartil proteinases secretadas), constituída de 10 genes de C.
albicans, é a melhor caracterizada entre os fungos patogênicos. Através de estudos de
diversas naturezas, demonstrou-se que as SAPs 1, 2 a 3 estão ativamente envolvidas em
infecções nas mucosas, enquanto as SAPs 4, 5 e 6 são ativas especialmente nas infecções
sistêmicas (revisão em Naglik et al., 2003).
Todas as proteinases catalisam a hidrólise de ligações peptídicas (CO-NH) de
proteínas, todavia apresentam diferentes mecanismos de ação e especificidade (Barrett et
al., 1991). As proteinases são classificadas com base no seu mecanismo catalítico e não
pela estrutura tridimensional, especificidade por substratos ou função fisiológica. De
acordo com a natureza dos aminoácidos do sítio catalítico as proteinases pedem ser
divididas em 5 classes: serino, aspartil, cisteíno, treonino e metalo proteinases, além de 9
17
proteinases com mecanismo catalítico desconhecido (Rawlings et al., 2006). Os
inibidores específicos mais comuns utilizados para classificar as proteases são: PMSF
(fluoreto de fenilmetanosulfonila) para as serino, pepstatina para as aspartil, E-64 (transepoxisuccinil-leucilamido(4-guanidino)butano) para as cisteíno e EDTA (ácido
etilenodiaminotetracético) para as metalo proteinases.
As serino proteinases são as mais abundantes, e na maioria dos casos seu
mecanismo catalítico caracteriza-se pela presença da tríade catalítica no sítio ativo da
enzima, composto por histidina, serina e ácido aspártico. O grupamento –OH da serina
age como nucleófilo e ataca o grupo carbonila na ligação peptídica do substrato. O par de
elétrons do nitrogênio da histidina faz a captação do hidrogênio do grupo –OH da serina
(Figura 6). Em conjunto, o grupo carboxila do ácido aspártico forma pontes de
hidrogênio com a histidina, fazendo o par de elétrons muito mais eletronegativo para que
ocorra a hidrólise da ligação peptídica (Hedstrom, 2002). Dentro da classe das serino
proteinases há 68 famílias, das quais a quimotripsina (S1) e a subtilisina (S8) são as mais
numerosas (Rawlings et al., 2006).
18
Figura 6. Mecanismo catalítico geral das serino proteinases (retirado de
http://employees.csbsju.edu/hjakubowski/classes/ch331/catalysis/serprotease3.gif)
As proteinases da família da quimotripsina são endopeptidases subdivididas em
tripsina-like, quimotripsina-like e elastase-like, as quais clivam os substratos após os
seguintes aminoácidos na posição P1 (nomenclatura de Schechter e Berger, 1967):
arginina/lisina, hidrofóbicos e alanina, respectivamente. A maioria dessas proteinases
possui um peptídeo sinal para exportação e uma região N-terminal que necessita ser
clivada para a enzima ficar ativa. Inúmeras funções biológicas de proteinases já foram
identificadas, como por exemplo, digestão de alimentos, resposta imune mediada por IgA
19
(enzimas como a triptase e quimase presentes em mastócitos) e até mesmo na sinalização
dorso-ventral de drosófila (Rawlings et al., 2006).
Pensava-se que as subtilisinas fariam parte da família da quimotripsina, porém
análises de sequência (Smith et al., 1966) e estruturais (Wright et al., 1969) mostraram
diversas diferenças. A maior parte das subtilisinas são endopeptidades com pH ótimo
neutro ou alcalino, termoestáveis e não-específicas. A clivagem ocorre após um
aminoácido hidrofóbico, sendo a caseína comumente utilizada como substrato em ensaios
de clivagem (Rawlings et al., 2006). Em fungos e bactérias, grande parte das subtilisinas
estão envolvidas com nutrição, porém já foram descritos membros dessa família
implicados em virulência, por exemplo a cspA de Streptococcus (Harris et al., 2003).
Dentro das subtilisinas encontra-se um grupo com uma característica peculiar,
especificamente a susceptibilidade à inibição por mercuriais. Nesse caso, as moléculas de
Hg ligam-se a um resíduo de cisteína próximo ao sítio catalítico, como é o caso da
proteinase K (Muller e Saenger, 1993) e da cerevisina (Kominami et al., 1981). Foi
demonstrado por cristalografia de raio-x que a ligação do mercúrio ao grupamento tiol da
cisteína ocasiona uma mudança estrutural no anel imidazólico da histidina do sítio
catalítico (como indicado na Figura 7) provocando alta inibição da atividade hidrolítica
(Muller e Sanger, 1993).
20
Figura 7. Mudança estrutural no anel imidazólico da histidina no sítio catalítico da proteinase K
provocado por mercuriais (Muller e Sanger, 1993).
Em P. brasiliensis, foram detectadas proteinases citosólicas descritas nas duas
fases do fungo (San-Blas et al., 1998). Verificou-se que a atividade enzimática de
extratos totais foi maior na fase miceliana, aparentemente com preponderância de serino
proteinases devido à alta inibição por PMSF e pH ótimo alcalino. Todavia a preparação
enzimática também foi parcialmente inibida por iodocetamida, EDTA e ortofenantrolina, indicando a presença de cisteíno e metalo proteinases. Em 2005, Parente e
colaboradores analisaram o transcriptoma de P. brasiliensis quanto à presença de genes
de proteinases. Foram detectadas 15 proteinases ATP-independentes, 12 ATPdependentes, 22 subunidades do proteassomo e 4 proteinases relacionadas com a
desubiquitinação. As proteinases ATP-independentes subdividiram-se em 5,6% de
aspartil proteinases, 11,3% cisteíno proteinases, 22,6% de metaloproteinases, 18,8% de
serino proteinases e 41,5% de subunidades do proteossomo. Apenas uma protease da
família S8 das subtilinas de P. brasiliensis foi sequenciada e depositada no GenBank
21
(AAP83193) pelo grupo da Professora Célia M. A. Soares. Essa proteinase chama a
atenção porque seu mRNA foi diferencialmente expresso em leveduras expostas ao soro e
em células isoladas de fígado infectado (Bailão et al., 2006). Não parece, no entanto,
corresponder à PbST (Matsuo et al., 2007, artigo 2).
Uma serino-tiol proteinase extracelular de P. brasiliensis tem sido caracterizada
em nosso laboratório em colaboração com o Departamento de Biofísica da UNIFESP,
especialmente com a Professora Adriana K. Carmona. Pensava-se que a atividade
enzimática em sobrenadante de cultura de P. brasiliensis crescido na fase leveduriforme
era proveniente da gp43 (Puccia et al., 1991), a qual é a proteína secretada mais
abundante. Porém cromatografias de afinidade em coluna contendo monoclonal anti-gp43
revelaram que a atividade não era retida na coluna (Carmona et al., 1995; Puccia et al.,
1999). Puccia e colaboradores (1998) observaram que a atividade proteolítica estava
diretamente ligada ao aumento do pH extracelular da cultura, onde o pico de atividade
ocorreu em pH 7.7 com o fungo crescendo em fase logarítmica. Para caracterizar essa
atividade, Carmona e colaboradores (1995) testaram diversos substratos peptídicos
sintéticos com supresssão intramolecular de fluorescência do tipo Abz/EDDnp (ácido
orto-aminobenzóico e 2,4-dinitrofeniletilenodiamina) (Chagas et al., 1992). O substrato
originalmente clivado foi Abz-MKRLTL-EDDnp, para o qual houve uma clivagem única
entre os aminoácidos leucina e treonina. A partir deste, outros foram sintetizados para
determinar a importância de P2 e P3 (nomenclatura de Schechter e Berger, 1967). O
melhor substrato foi Abz-MKALTL-EDDnp, para o qual a troca da arginina pela alanina
aumentou a atividade catalítica 5 vezes, com pH ótimo em torno de 9. A classificação da
proteinase foi realizada através de inibidores especificificos: houve inibição irreversível
22
por PMSF (serino proteinases) e inibição reversível utilizando agentes redutores por pHMB e acetato de mercúrio. Outras classes de inibidores como EDTA (metalo),
pepstatina (aspartil) e E-64 (cisteíno) não tiveram efeito. Por isso a atividade enzimática
foi atribuída a uma serino proteinase contendo um grupo tiol livre (Carmona et al., 1995),
a qual denominamos PbST.
Para estimar a massa molecular da proteinase, foram realizados ensaios de
zimograma com gelatina e “overlay” com gel de agarose contendo o substrato sintético
Abz-MKALTLQ-EDDnp. Em ambos os casos foi observada uma atividade hidrolítica
difusa em torno de 43 a 69 kDa, a qual foi inibida totalmente por PMSF e parcialmente
por p-HMB, provavelmente pela presença de um agente redutor no tampão de amostra
(Puccia et al., 1999). A importância desta proteinase deve-se à clivagem seletiva de
substratos proteicos. Enquanto a maioria das subtilisinas é promíscua, a PbST possui a
capacidade de clivar componentes asssociados à matriz extracelular como laminina,
fibronectina, colágeno tipo IV e proteoglicanos. Porém não foi detectada atividade sobre
colágeno tipo I, fibrinogênio bovino, imunoglobulina G humana, albumina bovina, gp43
e nem mesmo caseína (Puccia et al., 1998). Por esse motivo especula-se que a PbST
possa ser importante na disseminação do P. brasiliensis para outros órgãos durante a
infecção.
A purificação da PbST ativa tem sido impedida por ocorrer em baixas
concentrações protéicas e ter tendência à autólise e à agregação com componentes
polissacarídicos de alto peso molecular (Carmona et al., 1995). Esse componente de alta
massa molecular refere-se à peptidogalactomana cuja estrutura foi analisada em nosso
laboratório (Puccia et al., 1986). Por muito tempo a atividade proteolítica foi concentrada
23
e fracionada a partir de filtrado de cultura saturado com sulfato de amônio, por interação
hidrofóbica em coluna de Phenyl Superose (Amersham Pharmacia), porém a banda da
protease nunca foi detectada com exatidão. Também tentou-se obter informações sobre
sequências internas da PbST pois são de fundamental importância para a aplicação de
uma estratégia direta de síntese de oligonucleotídeos e obtenção de produtos de PCR
correspondentes ao seu gene. Previamente no laboratório foram obtidas informações
sobre a seqüência interna de aminoácidos e do N-terminal de um componente de 55 kDa,
supostamente a PbST (Puccia et al., 1999), enriquecida por cromatografia em
concanavalina A. O perfil peptídico e a comparação com bancos de dados indicou a
presença de sequências homólogas à catalase, além de peptídeos da gp43 e concanavalina
A (dados não publicados). Ou seja, as condições de purificação e eletroforese utilizadas
propiciaram a formação, em preparações concentradas, de um agregado de catalase, gp43
e da própria concanavalina A, migrando em 55 kDa. A atividade proteolítica estava
incluída nesse agregado ou co-migrou com ele, porém a proteinase estava aparentemente
em quantidade insuficiente para ser detectada.
Na tentativa de identificar um fragmento do gene da PbST por PCR utilizando
oligonucleotídeos degenerados para domínios conservados dos sítios catalíticos de
subtilisinas, foi clonado e sequenciado ao acaso um gene homólogo ao da proteinase Lon
(Barros e Puccia, 2001). O gene em P. brasiliensis, denominado PbLON, é constituído de
3.369 pb com 2 introns na porção 3’, e origina uma proteína predita de 1.063
aminoácidos que contem um sinal de importe mitocondrial na região N-terminal. Possui
identidade com os genes LON de S. cerevisiae (73%), Homo sapiens (62%) e E. coli
(56%). O gene completo foi clonado a partir de um fragmento genômico SmaI de
24
aproximadamente 6,5 kb, o qual contem adicionalmente um fragmento 5’ do gene
homólogo ao MDJ1 de S. cerevisiae em direção oposta. Os genes PbLON e PbMDJ1
compartilham a região 5’ intergênica, na qual Batista e colaboradores (2006) mapearam 3
regiões putativas para elementos de choque térmico e um domínio ARE ligante de AP-1,
que é um fator de transcrição envolvido na indução de genes relacionados ao estresse
oxidativo em fungos (Wu et al., 1994; Grant et al., 1996). Em P. brasiliensis, a proteinase
PbLon está localizada exclusivamente na mitocôndria (Batista et al., 2006, artigo 3), onde
as proteínas estão sendo constantemente atacadas por radicais livres devido ao
metabolismo aeróbico, drogas ou condições adversas do hospedeiro. Estudos recentes
demonstraram que a transcrição do gene PbLON aumentou 5 vezes em condições de
estresse oxidativo e provavelmente está relacionada com a homeostase mitocondrial
promovendo a degradação seletiva de proteínas danificadas (Batista et al., 2007).
A proteinase Lon está inserida em uma família própria (S16) constituída por
endopeptidases ATP-dependentes. A primeira Lon foi descoberta em E. coli por Swamy e
Goldberg (1981) e denominada proteinase La. Está presente em todos os tipos de
organismos e o sítio catalítico é formado por uma serina e uma lisina (Besche e Zwickl
2004; Botos et al., 2004). Em E. coli, Lon é formada por 6 unidades idênticas de
aproximadamente 87 kDa e em S. cerevisiae é formada por 7 unidades de 117 kDa com
localização intramitocondrial (Stahlberg et al., 1999). Cada unidade possui 4 domínios
(Figura 8): na região N-terminal há o sítio para importe mitocondrial; na região central
ocorre a ligação e hidrólise do ATP (Fischer et al., 1994) e a discriminação do substrato
pelo domínio SSD (Smith et al., 1999); na região C-terminal localiza-se o sítio catalítico
25
(Amerik et al., 1991). A proteinase é expressa constitutivamente, mas sua expressão pode
aumentar após choque térmico (Van Dyck et al., 1994).
Figura 8. Desenho esquemático dos “motifs” da proteinase Lon
A principal função da Lon é degradar proteínas de meia-vida curta que estão
envolvidas em diversos processos biológicos e proteínas anormais que agregariam na
ausência da proteinase ou do sistema de chaperoninas DnaK (revisão em Tsilibaris et al.,
2006). O mecanismo pelo qual Lon reconhece os substratos ainda não é totalmente
entendido. Apesar de reconhecer preferencialmente certos amino ácidos ou domínios
(Griffith et al., 2004; Ishii et al., 2001), nenhum “motif” consenso ou combinações de
“motifs” foram identificados. A discriminação de substratos parece ocorrer através da
estrutura, ou seja, proteínas alvo de meia-vida curta e anormais não estariam em sua
conformação globular (Jubete et al., 1996; Van Melderen et al., 1996). Em eucariotos, a
deleção ou desregulação do gene LON leva a defeitos celulares. S. cerevisiae mutante
para o gene apresenta deficiência quando crescido em condições aeróbicas e não cresce
em meio de cultura com fonte de carbono não-fermentável; apresenta inclusões elétrondensas na matriz mitocondrial devido ao acúmulo de proteínas mitocondriais e deleções
no mtDNA (Van Dyck et al., 1999). Em células de mamíferos a proteinase degrada
proteínas oxidadas e está implicada na disfunção mitocondrial relativa à idade (Bota et
al., 2002). Além disso, reduções nos níveis de Lon prejudicam a divisão celular e levam à
26
necrose, sendo que a depleção total leva à apoptose (Bota et al., 2005). A proteinase
também pode estar envolvida com regulação gênica (Langer e Neupert, 1996; Fu et al.,
1997) e virulência em bactéria (Robertson et al., 2000; Boddicker e Jones et al., 2004).
A PbMdj1 faz parte da família das proteínas com domínio J, que compartilham
identidade entre 35 a 59%. Em E. coli, a molécula típica possui um domínio J constituído
de 70 aminoácidos, seguido de um segmento ligante de zinco rico em glicina e uma
região C-terminal pouco conservada (Szabo et al., 1996). As proteínas J são subdivididas
em 3 tipos: o tipo I contém todos os domínios conservados em E. coli, o tipo II não
contém a região ligadora de zinco, e o tipo III possui apenas um domínio J em qualquer
região da proteína (Fliss et al., 1999). Seu papel principal é regular a atividade das Hsp70
cognatas, localizadas em diversos compartimentos celulares (Revisão em Walsh et al.,
2004). Em S. cerevisiae, Mdj1 é a única DnaJ mitocondrial do tipo I e é essencial na
biogênese da mitocôndria funcional (Rowley et al., 1994). O sistema Ssc1 (Hsp70
mitocondrial) / Mdj1 (Hsp 40 mitocondrial) está envolvido na manutenção da
configuração correta das proteínas mitocondriais ou da solubilidade das proteínas
desnaturadas que serão degradadas pela proteinase Lon e outras proteinases ATPdependentes (Figura 9) (Wagner et al., 1994; Savel’ev et al., 1998).
27
Figura 9. Rede de chaperones da matriz mitocôndrial de S. cerevisiae (retirado de Voos et al., 2002)
28
Objetivos
•
Em relação à proteinase serino-tiol extracelular de P. brasiliensis (PbST), os
objetivos foram purificar e identificar a proteína, estudar sua modulação com
açúcares e estudar a inibição por compostos (2,4-dinitrophenyl)ethylenediamine
(Npys) acoplados com substratos peptídicos.
•
Em relação à PbLon, objetivamos fazer a expressão heteróloga, purificação da
proteína PbLon recombinante, obtenção de anticorpos e localização celular. Um
estudo sobre a especificidade da enzima com substratos peptídicos foi previsto se
a expressão heteróloga resultasse em proteinase ativa.
•
Objetivos posteriores do projeto incluíram:
a) a caracterização preliminar de vesículas extracelulares do fungo, as quais poderiam
conter a PbST.
b) a análise proteômica da expressão diferencial de proteínas associadas à parede celular.
Uma introdução específica e um racional para esses objetivos estão apresentados
no capítulo 2 desta tese.
29
Capítulo 1
Proteinases de Paracoccidioides
brasiliensis: serino-tiol extracelular
(PbST) e PbLon mitocondrial
30
Proteinase serino-tiol extracelular
de P. brasiliensis (PbST)
31
Artigo 1: Modulation of the exocellular serine-thiol proteinase activity of
Paracoccidioides brasiliensis by neutral polysaccharides.
Alisson L. Matsuo, Ivarne L. Tersariol, Silvia I. Kobata, Luiz R. Travassos, Adriana K.
Carmona e Rosana Puccia
Microbes and Infection 2006, Vol 8 (1) 84-91
32
Racional
A serino-tiol proteinase de P. brasiliensis (PbST) tem sido caracterizada em nosso
laboratório em colaboração com o Departamento de Biofísica da UNIFESP, como
detalhado na Introdução geral. Desde as primeiras tentativas de purificação da enzima a
partir de sobrenadantes de cultura (Carmona et al., 1995), ficou nítido que a proteinase
agrega-se com componentes glicosilados secretados pelo fungo. Por outro lado, a
atividade proteolítica é retida em coluna de concanavalina A, possivelmente carregada
por componentes glicosilados com os quais se associa. No trabalho apresentado a seguir,
foi estudado o efeito modulatório de polissacarídeos sobre a PbST. Primeiramente tentouse observar um efeito por glicosaminoglicanos, visto que este composto está contido na
matriz extracelular e tem a capacidade de modular cisteíno proteinases, porém o resultado
foi negativo. Todavia quando se testou dextrana, na verdade como controle, foi observada
uma intensa modulação da afinidade da enzima pelo substrato, o que ocorreu
posteriormente também com açúcares neutros derivados de uma preparação de mannana
de S. cerevisiae e de galactomanana do sobrenadante de P. brasiliensis. Os dados de
modulação de atividade proteolítica com açúcares neutros são originais e têm uma
possível relevância no controle da atividade da PbST in vivo.
33
Microbes and Infection 8 (2006) 84–91
www.elsevier.com/locate/micinf
Original article
Modulation of the exocellular serine-thiol proteinase activity
of Paracoccidioides brasiliensis by neutral polysaccharides
Alisson L. Matsuo a,1, Ivarne I.L. Tersariol b,1, Silvia I. Kobata c, Luiz R. Travassos a,
Adriana K. Carmona c, Rosana Puccia a,*
a
Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, UNIFESP, Rua Botucatu, 862, oitavo andar,
São Paulo, SP 04023-062, Brazil
b
Centro Interdisciplinar de Investigação Bioquímica da Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil
c
Departamento de Biofísica da Universidade Federal de São Paulo, São Paulo, SP, Brazil
Received 21 March 2005; accepted 31 May 2005
Available online 08 August 2005
Abstract
Our group characterized an exocellular serine-thiol proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a
dimorphic human pathogen. The fungal proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as
human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans
(GAGs) and neutral polysaccharides upon the serine-thiol proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the proteinase, while sulfated GAGs had no effect.
Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic
parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 µM increased 6.8-fold the enzyme
affinity for the substrate. In the presence of 8 µM of purified baker’s yeast mannan, the apparent KM of the enzyme increased about 5.5-fold,
reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from
P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was
able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel
modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-thiol proteolytic activity,
which is possibly involved in fungal dissemination.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Paracoccidioides brasiliensis; Serine-thiol activity; Neutral polysaccharides; Modulation
1. Introduction
Paracoccidioidomycosis (PCM) is a systemic mycosis
caused by the dimorphic fungus Paracoccidioides brasilien-
Abbreviations: Abz, ortho-aminobenzoic acid; AUF, arbitrary units of
fluorescence; EDDnp, ethylenediaminedinitrophenyl; FPLC, fast performance liquid chromatography; GAGs, glycosaminoglycans; GalMan, galactomannan; PbST, exocellular serine-thiol activity from P. brasiliensis; PCM,
paracoccidioidomycosis; p-HMB, sodium 7-hydroxymercuribenzoate;
PMSF, phenyl-methylsulfonyl fluoride.
* Corresponding author. Tel.: +55 11 5084 2991; fax: +55 11 5571 5877.
E-mail address: [email protected] (R. Puccia).
1
Alisson L. Matsuo and Ivarne L.S. Tersariol contributed equally to this
work.
1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.micinf.2005.05.021
sis. Acute and sub-acute PCM affect both sexes, progress rapidly, and the fungi disseminate through the lymphatic system. Chronic forms are the most common; they predominantly
affect male adults, starting as a lung granulomatous infection
and eventually involving other organs [1]. The disease is
restricted to Latin America, where P. brasiliensis has also
been isolated from the soil and from organs of nine-banded
armadillos [2]. The fungus penetrates the human body through
inhalation of propagules, which can only establish infection
once they undergo phase transition to yeasts in the pulmonary alveolar epithelium [3,4]. The disease can be fatal if not
treated adequately.
We have previously characterized a subtilisin-like, SHdependent serine proteinase activity in culture fluids of P. bra-
A.L. Matsuo et al. / Microbes and Infection 8 (2006) 84–91
siliensis grown in the yeast phase [5]. The enzyme is able to
hydrolyze fluorescence resonance energy transfer (FRET)
peptides flanked by ortho-aminobenzoic acid (Abz) and ethylenediaminedinitrophenyl (EDDnp), homologous to
MKRLTL. Cleavage occurs on the Leu–Thr bond, at an optimum alkaline pH, and is irreversibly inhibited by PMSF, mercuric acetate and p-HMB (sodium 7-hydroxymercuribenzoate). Hydrolysis of synthetic peptides and proteins
involves an enzyme of high specific activity, which is however unstable and lost after several chromatographic steps aiming at its purification. Additionally, it tends to aggregate with
glycosylated extracellular fungal components [5]. Early chromatographic analysis of culture filtrates showed that the
enzyme activity was eluted in a single peak from an anionexchange Resource Q column followed by gel filtration [5].
The active fractions, however, yielded several bands in SDSPAGE stained gels. We then observed that chromatography
of culture fluid supernatants pretreated with (NH4)2SO4 at
40% saturation in Phenyl Superose resulted in concentration
of the proteolytic activity in two eluted fractions [6]. When
these fractions were analyzed by electrophoresis and in situ
hydrolysis of both gelatin (zymogram) and Abz-MKALTLQEDDnp incorporated into buffered agarose overlays, the
serine-thiol exocellular activity appeared as a diffuse and heterogeneous band in SDS-PAGE gels. It localized to a region
between 69 and 43 kDa, but could not be correlated with any
silver-stained component.
The most relevant characteristic of the serine-thiol proteinase activity of P. brasiliensis is its capacity of selectively
cleaving, in vitro, laminin, fibronectin, type IV collagen and
proteoglycans [7]. Since these components are associated with
the basal membrane, such hydrolytic activity could play a
significant role in tissue invasion by the fungus. There are a
few reports on the modulation by complex and negatively
charged carbohydrates, more specifically glycosaminoglycans (GAGs), of the activity of proteolytic enzymes [8]. In
the present work we describe a novel type of proteinase regulation by neutral polysaccharides using the Abz-MKALTLQEDDnp substrate.
2. Materials and methods
2.1. Fractionation of the serine-thiol proteinase activity
P. brasiliensis strain 339 (originally provided by Dr. Angela
Restrepo-Moreno, Medellin, Colombia) was cultivated with
shaking at 36 °C in a modified YPD medium (0.5% of dialyzed, < 10 kDa, Bacto-yeast extract, 0.5% casein peptone
and 1.5% glucose, pH 6.5). The culture supernatant was collected by paper filtration at the peak of the proteinase activity
against Abz-MKALTLQ-EDDnp, when the culture fluid pH
was about 7.0 [7]. Ammonium sulfate was gradually added
to 40% saturation (1.7 M) and the supernatant centrifuged
(16,300 × g, 30 min) to discard eventual precipitates. Aliquots of the supernatant containing ammonium sulfate (50 ml)
85
were fractionated by fast performance liquid chromatography (FPLC) (FPLC System - Pharmacia) in a Phenyl Superose HR 5/5 (Pharmacia/LKB) column equilibrated with 1.7 M
ammonium sulfate in 50 mM sodium phosphate buffer, pH
7.0 [6] and eluted with 50 mM sodium phosphate into 0.5 mlfractions. The proteolytic activity was measured (10 µlaliquots) against Abz-MKALTLQ-EDDnp, as described previously [5]. The fractions were analyzed in silver-stained SDSPAGE gels [9,10].
2.2. Purification of yeast mannan
The procedure of Gorin and Spencer [11] described in
detail by Travassos et al. [12] was used. Briefly, Saccharomyces cerevisiae cultures were autoclaved for 30 min. Supernatants were precipitated with 3 volumes of ethanol after adding 1% of sodium acetate. The precipitate and the mass of
cells were suspended in 20% aqueous KOH and extracted for
2 h at 100 °C in a water bath. The suspension was neutralized
with glacial acetic acid and the supernatant was evaporated
to 100–150 ml, then precipitated with ethanol. The solubilized precipitate was precipitated with Fehling’s solution at
4 °C. The copper complexes were treated with Amberlite
1R-120 for 1 h at room temperature and the solution pooled
with distilled water washings of the beads were precipitated
with 4 ml of ethanol with drops of HCl. The final mannan
preparation contained more than 98% carbohydrate with less
than 1% protein, and no contamination with alkali-soluble
glucan. Yeast mannan obtained by hot alkali extraction and
Fehling precipitation has a molecular size of 20–60 kDa, averaging 40 kDa [13]. Milder methods used to prepare mannoproteins provide complexes of 133 kDa. Molecular sizes are
determined in filtration columns calibrated with dextran of
different sizes. Our calculations to determine the kinetic
parameters used the average mannan size of 40 kDa.
2.3. Purification of the P. brasiliensis galactomannan
complex
Phenyl Superose fractions containing the GalMan complex previously described [14] were pooled and fractionated
by FPLC (ÄKTA purifier – Amersham Pharmacia Biotech)
through a reverse-phase Sephasil Protein C4 (Amersham Biosciences) equilibrated with 0.1% trifluoracetic acid (TFA) and
eluted with 30 ml of a 0–90% acetonitrile gradient in 0.1%
TFA into 0.5-ml fractions. Fifty-one fractions were dried in a
speed-vacuum and resuspended in 100 µl of 50 mM Tris, pH
8.0, for SDS-PAGE analysis. The GalMan complex was predominantly eluted in fraction 14, as shown by the presence of
a broad and heavily silver-stained component migrating
slowly in SDS-PAGE gels [14]. This procedure was repeated
several times, and the GalMan was pooled, lyophilized and
further purified by FPLC (ÄKTA purifier) in a Superose 12
(Amersham Biosciences) column equilibrated and eluted with
50 mM Tris–HCl, 0.15 M NaCl, pH 7.0. Total carbohydrate
contents of the peak fractions were quantified using the
phenyl-sulfuric acid method described by Dubois et al. [15].
86
2.4. Proteolytic assays
The effect of polysaccharides on the exocellular serinethiol proteinase activity of P. brasiliensis against AbzMKALTLQ-EDDnp was determined in a thermostatic Hitachi F-2000 spectrofluorometer (kex = 320 nm; kem 420 nm).
Kinetic parameters were determined by measuring the initial
rate of hydrolysis at various substrate concentrations in the
presence or absence of different polysaccharide concentrations. We tested the effect of GAGs (heparin, heparan sufate
and chondroitin) and neutral polysaccharides (dextran, yeast
mannan and P. brasiliensis GalMan complexes). The reactions were carried out at 37 °C in 10 mM Tris–HCl (pH 8.0).
Progress of the reaction was continuously monitored by fluorescence record of the released product. Analysis of the data
obtained was performed by nonlinear regression using the
GraFit 3.0 computer program (Erithacus Software Ltd.).
The modulatory effect of dextran and GalMan complexes
on the proteolytic activity of the enzyme was calculated following Eq. (1), where S is Abz-MKALTLQ-EDDnp, NP is
the polysaccharide, KS is the substrate dissociation constant,
KN is the apparent polysaccharide dissociation constant, ␣ is
the parameter of KS perturbation, and b is the parameter of
VMAX (kcat) perturbation.
v=
Ks
冉
冉
Vmáx × [S]
1+
1+
[NP ]
Kn
冊
b × [NP ]
␣ × Kn
冉
冊冉
1+
+ [S]
1+
[NP ]
␣ × Kn
冊
冊
(Eq. 1)
b × [NP ]
␣ × Kn
Since the interaction between mannan and PbST resulted
in a powerful inhibition of the enzyme activity, the modulatory effect of yeast mannan on the proteolytic activity of the
enzyme was analyzed by nonlinear regression according to
Eq. (2) and by linear regression according to Eq. (4), where
K′ is the observed substrate dissociation constant in the presence of mannan and KMan is the true mannan-protease dissociation constant; M is mannan and ␣ is the cooperative parameter for binding of the second mannan to enzyme.
v=
Vmax · [S]
冉
KS 1 +
冉
2 · [M ]
K′ = KS · 1 +
1
K′
=
1
2
␣ · KMan
+
K Man
2 · [M]
KMan
[M ]
␣ · K M an
2
+
· [M ] +
2
[M]
␣ · KMan
冊
or in the presence of 300 nM of GalMan at 50 °C in 10 mM
Tris–HCl buffer (pH 8.0). The reactions were carried out in
the presence of 0.24 µM of Abz-MKALTLQ-EDDnp, which
is 10-fold below the KS value. The progress of the reaction
was continuously monitored by fluorescence measurement
of the released product. The obtained exponential decay
curves were best fitted to the first-order relationship shown in
Eq. (5),
P = P∞共 1 − e
-kobs.t
兲
(Eq. 5)
where P and P∞ are the product concentrations at a given
time and at infinite time, respectively; kobs is the observed
first-order rate of temperature-induced enzyme inactivation.
2.6. Protein and carbohydrate contents
Protein contents were estimated by a modification of the
method described by Bradford [16], using bovine serum albumin as standard. Total carbohydrates were quantified by the
method of Dubois et al. [15], using mannan as standard.
3. Results
We studied the influence of polysaccharides upon the
endopeptidase activity of a fungal serine-thiol proteinase using
the Abz-MKALTLQ-EDDnp substrate. When culture supernatants of P. brasiliensis B-339 were fractionated in a Phenyl
Superose column, the peptidase activity capable of hydrolyzing Abz-MKALTLQ-EDDnp was predominantly eluted in
fraction 3. This fraction showed few protein/glycoprotein
silver-stained bands, as seen in the profile of Fig. 1, which
(Eq. 2)
+ [S]
冊
2
(Eq. 3)
(Eq. 4)
[M ]
2.5. Effect of GalMan on the temperature-induced
inactivation of the PbST activity
The kinetics of thermal inactivation of the PbST activity
over Abz-MKALTLQ-EDDnp was evaluated in the absence
Fig. 1. Serine-thiol proteinase preparation. Silver-stained 10% SDS-PAGE
gel showing the fractionation of a P. brasiliensis culture supernatant (sup, in
40% ammonium sulfate) through a Phenyl Superose column eluted with
50 mM sodium phosphate, pH 7.0. The hydrolytic activity of each fraction
(3–6) was measured in a 10 µl-aliquot against Abz-MKALTLQ-EDDnp and
the results are shown as arbitrary units of fluorescence (AUF). Fraction 3 of
this experiment, which contained the activity peak, was used as enzyme
source (PbST). The position of molecular mass markers (in kDa) is shown
on the left.
also shows that most of the supernatant components were
eluted in fractions 4 and 5. Therefore, we used fraction 3 as
enzyme source (PbST) in the experiments shown below. Enzymatic cleavage of Abz-MKALTLQ-EDDnp was due to the
exocellular serine-thiol proteinase activity of P. brasiliensis
contained in fraction 3, since the hydrolysis was specifically
inhibited by p-HMB and PMSF, as previously reported [5]. It
is of note that total purification of the active proteinase has
never been achieved due to aggregation to other fungal components and/or loss of enzymatic activity after several chromatographic steps.
Negatively charged GAGs, namely heparin, chondroitin
and heparan sulfate, did not affect the rate of AbzMKALTLQ-EDDnp hydrolysis when added at a concentration range of 0–100 µM (not shown). In addition, PbST did
not bind to heparin-Sepharose (not shown), suggesting that
the enzyme does not interact with GAGs.
Surprisingly, in the presence of dextran (Sigma, average
molecular mass of 100–200 kDa, where Mr = 100 kDa was
used in the calculations), a neutral polysaccharide initially
used as control, there was a significant alteration in the kinetic
parameters of PbST (Fig. 2). The VMAX value for the hydrolysis of Abz-MKALTLQ-EDDnp decreased significantly as a
function of dextran concentration (Fig. 2A). Dextran also
caused a marked increase in the enzyme affinity for this substrate, evidenced by a sharp decrease in the KS value, which
Fig. 2. Modulatory effect of dextran on the Abz-MKALTLQ-EDDnp hydrolysis by PbST. The apparent VMAX (VMAX app), recorded as AUF per min
(A), and the apparent affinity constants (Ks app) (B) were calculated for the
reactions carried out in the presence of increasing amounts of dextran.
87
dropped from 2.4 ± 0.2 to 0.35 ± 0.04 µM at the highest dextran concentration of 20 µM (Fig. 2B). The effect of dextran
on PbST can be described as a hyperbolic mixed type inhibition according to Eq. (1) (Section 2), where the efficiency of
substrate hydrolysis was estimated by changing either the KM
(parameter ␣) or the VMAX (parameter b). These data were
fitted to the equation using nonlinear regression, and the values for the constants were determined. The results revealed
that dextran bound to free PbST (E) with a dissociation constant of KDX = 2.7 ± 0.3 µM, whereas the parameter obtained
for binding to the enzyme–substrate complex (ES) was
␣KDX = 0.35 ± 0.04 µM (parameter ␣ = 0.13 ± 0.01). The
data also showed that the interaction between dextran and the
serine-thiol proteinase resulted in a 3.6-fold decrease in the
enzyme VMAX (b = 0.28 ± 0.03) and in an almost 2.2-fold
increase in its catalytic activity (b/␣ = 2.2).
We then tested the effect of purified baker’s yeast mannan, since mannan structures are commonly found in fungal
glycoconjugates. Interestingly, the interaction between mannan and PbST resulted in a powerful inhibition of the enzyme
activity. The effect of mannan upon PbST can be described
as a parabolic competitive type inhibition according to Eqs.
(2)–(4) of Section 2. The data show that two molecules of
mannan bound to the enzyme and that the first inhibitor
changed the intrinsic dissociation constant of the vacant one
by factor ␣. The influence of mannan upon the substrate dissociation constant can be observed in Fig. 3A. Basically, the
mannan–proteinase interaction did not affect the catalytic constant of Abz-MKALTLQ-EDDnp; the presence of mannan
excluded substrate binding at the active site, suggesting that
the enzyme is capable of binding two molecules of mannan
competitive inhibitor in a parabolic manner. It was observed
that binding of the first mannan induced structural changes in
the enzyme that facilitated binding of the second mannan
inhibitor (Fig. 3B). These data were fitted to Eq. (2) using
nonlinear regression, and the values for the constants were
determined. The results show that the first molecule of mannan bound to free PbST (E) with a dissociation constant of
KMan = 11.4 ± 0.9 µM. The second mannan molecule bound
to the mannan–proteinase complex (EI) with a dissociation
constant of ␣KMan = 2.1 ± 0.2 µM (␣ parameter =
0.18 ± 0.02). Therefore, binding of two molecules of mannan to the enzyme was not random and independent, but
occurred in a cooperative manner: binding of the first molecule of mannan (KMan = 11.4 ± 0.9 µM) resulted in a 5.5fold increase in the affinity of the enzyme for ligation of the
second mannan (␣KMan = 2.1 ± 0.2 µM).
It is noteworthy that high performance liquid chromatography (HPLC) and mass spectrometry analyses previously
showed that Leu–Thr is the only peptide bond cleaved in AbzMKALTLQ-EDDnp by PbST [5]. We presently observed by
HPLC analysis that the presence of either mannan or dextran
did not change the cleavage pattern of this peptide by the
proteinase activity (not shown).
The modulatory effect of dextran and mannan upon the
serine-thiol proteinase activity of P. brasiliensis prompted us
88
Fig. 3. Inhibitory effect of baker’s yeast mannan on the Abz-MKALTLQEDDnp hydrolysis by PbST. The apparent affinity constants (Ks app) (A)
and 1/K′ were calculated for the reactions carried out in the presence of
increasing amounts of dextran.
to investigate the effect of an endogenous fungal galactomannan (GalMan) complex originally described by Puccia et al.
[14]. This component is commonly found in the supernatant
fluids of P. brasiliensis cultures and migrates slowly in SDS-
PAGE gels, forming a diffuse smear due to its high degree of
glycosylation (see fractions 4 and 5, Fig. 1). In order to purify
this component, we pooled Phenyl Superose fractions 4 and
5 and fractionated them in a Sephasil C4 reversed-phase column. After this kind of chromatography (not shown), the largest peak (fraction 14) concentrated a slow-migrating, broad
component corresponding to the GalMan complex (see profile in “F14”, Fig. 4B). We then submitted a pool of fractions
14 from independent chromatographic runs to gel filtration
in a Superose 12 column (Fig. 4A) in order to isolate GalMan
from a minor 50-kDa component (Fig. 4B). The GalMan complex was mostly excluded in the void volume, which corresponded to fractions 8–11, as verified by calibration of the
column with dextran blue (Sigma) and dextran T70 (Pharmacia). GalMan was eluted from Superose 12 in fraction 10,
which also contained the total carbohydrate peak
(0.375 mg ml–1). This fraction was therefore used in the subsequent experiments, at the estimated average molecular mass
of 100 kDa, which was the value used to calculate the kinetic
parameters.
Fig. 5A shows that the P. brasiliensis GalMan complex
caused a marked increase in the PbST affinity for AbzMKALTLQ-EDDnp, which was evidenced by the decrease
in the Ks value. The presence of GalMan caused a threefold
decrease in the enzyme KM (␣ = 0.33 ± 0.03), i.e. from
2.4 ± 0.2 to 0.78 ± 0.08 µM. On the other hand, the interaction of GalMan with PbST resulted in a 3.1-fold decrease in
the enzyme VMAX (b = 0.32 ± 0.03), as seen in Fig. 5B. These
data were fitted to Eq. (1) (Section 2), and the values for the
constants were determined using nonlinear regression. The
results showed that GalMan bound to free PbST (E) with a
high affinity dissociation constant of KGM = 94 ± 7 nM, while
binding to the enzyme–substrate complex (ES) resulted in an
␣KGM = 30 ± 2 nM (␣ = 0.33 ± 0.03).
The effect of GalMan on the optimum pH of PbST was
analyzed by monitoring the enzyme-catalyzed hydrolysis of
the fluorogenic substrate Abz-MKALTLQ-EDDnp. As shown
Fig. 4. Purification of P. brasiliensis GalMan. A, Elution profile (A215) of a gel filtration chromatography in Superose 12 (FPLC) of a pool of fractions 14 (F14,
4B) recovered from a Sephasil Protein C4 reverse-phase column (details in the text). B, Silver-stained 10% SDS-PAGE profile of the peak fractions seen in A
(8–12). Total carbohydrate (Tcarb) corresponding to each fraction is indicated in mg ml–1. The position of molecular mass markers (in kDa) is shown on the left.
Fraction 10 was used in the subsequent experiments.
89
Fig. 7. Protective effect of P. brasiliensis GalMan (300 nM) on the AbzMKALTLQ-EDDnp hydrolysis by PbST at 50 °C.
4. Discussion
Fig. 5. Effect of P. brasiliensis GalMan complex on the Abz-MKALTLQEDDnp hydrolysis by PbST. The apparent VMAX (VMAX app), recorded as
AUF per min (A), and the apparent affinity constants (Ks app) (B) were calculated for the reactions carried out in the presence of increasing amounts of
dextran.
in Fig. 6, when the enzyme was assayed in the presence of
300 nM GalMan, no significant effect on the enzyme pH
response was observed. On the other hand, the presence of
GalMan caused a threefold decrease in the first-order rate of
the serine-thiol proteinase inactivation at 50 °C, since the kobs
value dropped from 11.1 ± 0.6 to 3.6 ± 0.2 ms−1 (Fig. 7).
Fig. 6. Lack of effect of P. brasiliensis GalMan preparation (300 nM) on the
Abz-MKALTLQ-EDDnp hydrolysis by PbST at different pH values.
The results of the present work show the modulation by
neutral polysaccharides of a fungal endopeptidase activity in
vitro. Binding of neutral polysaccharides to the extracellular
serine-thiol proteinase (PbST) secreted by the human pathogen P. brasiliensis [5] changed its kinetic parameters of
hydrolysis of the fluorogenic substrate Abz-MKALTLQEDDnp. We found two different effects: commercial dextran
and an endogenous galactomannan (GalMan) complex markedly increased the enzyme affinity for the substrate, while
baker’s yeast mannan inhibited the PbST activity. The GalMan component did not affect the optimum pH of the enzyme,
whereas the presence of mannan or dextran did not change
the enzyme cleavage point of the substrate Abz-MKALTLQEDDnp (L/T), suggesting that the interaction with these
polysaccharides does not alter the enzyme specificity. Any
effect on the enzyme–carbohydrate interaction caused by contaminants present in the enzyme preparation would be irrelevant due to the range of carbohydrate concentrations used in
the reactions.
Previous unpublished results from our group suggested a
possible protective role of the GalMan complex on the PbST
activity. A concanavalin A-bound preparation of the enzyme,
which also co-purified GalMan and a 55-kDa glycoprotein,
seemed to be less labile than the active fractions obtained
after anion-exchange and gel filtration chromatography [5].
The 55-kDa glycoprotein was later sequenced and found to
be homologous to catalases (unpublished results). We therefore empirically associated stability of PbST with either GalMan or catalase, or with both.
Our present data show that the P. brasiliensis GalMan component indeed protects the peptidase activity of PbST at high
temperatures. At 37 °C, GalMan modulated the enzyme at
the nanomolar range (KGM = 94 nM and aKGM = 30 nM), preserving its catalytic activity (b/␣ = 1.0) by increasing affinity
(threefold) for the substrate, but decreasing the catalysis velocity (3.1-fold). Dextran affected the PbST activity similarly,
however at the µM concentration range (KDX = 2.7 µM) and
with an increase in the catalytic activity (b/␣ = 2.2).
The GalMan component was previously characterized
among other concanavalin A-eluted components of the super-
90
natant fluids of P. brasiliensis yeast phase cultures [14]. Further analysis using a chemically defined medium to grow the
yeast phase rendered a carbohydrate rich (88%) exocellular
component containing two types of residues: a-Manp and
b-Galf (unpublished results). Nuclear magnetic resonance
(NMR) studies showed that the polysaccharide has an a-1,6linked mannopyranan main chain and a-1,2/a-1,3-linked
Manp side chains with terminal nonreducing units of a-Manp
and b-Galf (50% of the side chains), (1,3), (1,6), and (1,4)linked to mannopyranosyl units [17]. Structural differences
in the P. brasiliensis GalMan and baker’s yeast mannan might
explain the opposite, inhibitory effect on enzyme activity
caused by the latter. Baker’s yeast mannan is composed solely
of mannopyranosyl units both in the backbone and in the side
chains, which contain up to four residues linked either a-1,2 or
a-1,3. The size of the chains can vary with the glycoprotein:
high mannose short chains bear 8–14 mannose residues, while
longer chains will carry 20–200 residues [18]. The mannan
preparation used in the present work was extracted from whole
cells of S. cerevisisae and was therefore rather heterogeneous.
We have previously observed that the serine-thiol proteinase activity secreted by the human pathogen P. brasiliensis is
able to cleave tumor and cartilage decorin, aggrecan and versican. In the present work we observed that heparin, heparan
sulfate and chondroitin do not interact with this proteinase.
On the other hand, there is accumulating data on the interference of highly sulfated GAGs, especially heparin and heparinlike, in the activity of various classes of proteinases (reviewed
in [8]). They include papain [19], cathepsin B [20], cathepsin
G [21], neutrophil elastase [22], chymase [23], collagenolytic
activity of cathepsins [24,25] and tryptase [26]. The mechanisms involved in modulation of the protease activities by
GAGs have been attributed to their ability to a) change protein conformation upon binding, which leads to a change in
the catalytic activity or to a decreased rate of inhibition by
natural inhibitors, b) protect against pH inactivation or c)
expose binding regions in the target proteins (reviewed in [8]).
On the other hand, electrostatic interactions are probably
essential but not sufficient to govern binding specificity of
GAGs with proteinases and its consequences on their activity: the glycosidic linkage, type of saccharide and molecular
weight also determine the fine specificities of the interaction
[25,27].
The protection of proteins against dehydration and denaturation has been achieved in several instances through the
interaction with carbohydrates. A notorious example is that
of neutral disaccharides, mainly sucrose and trehalose. In
aqueous solution the major mechanisms may involve watertrehalose H-bond replacement, protein coating by a trapped
water layer and inhibition of conformational fluctuations [28].
A study on the trypsin interaction with trehalose showed that
protein preservation was directly related to the number of
hydrogen bonds formed [29]. Several sugars are able to prevent protein unfolding but its functional inactivation depends
on the nature of the sugar [30]. Dextran, for instance, fails to
inhibit dehydration-induced lysozime unfolding because it
does not H-bond adequately to the protein [31]. Seemingly,
the interaction of carbohydrates with enzymes can protect the
integrity of protein structure even in adverse conditions of
heat shock, dehydration and presence of chaotropic agents,
but also it may modulate their activity by restricting conformational states directly related with the catalytic reaction.
In the present work we have examined the modulation by
neutral polysaccharides of a fungal protease. At first hand,
we would expect H-bonding as the generalized interaction
between carbohydrate and protein to be quantitatively different while using dextran, yeast mannan and galactomannan.
They could either stimulate or inhibit protease activity, or even
have no effect. In principle, specific recognition of sugar units
by a lectin type interaction was not foreseen for the serinethiol protease. In the case of mannan, there are several mammalian lectins and a mannan binding protein (MBL), which
are involved in the host innate defense mechanisms (reviewed
in [32,33]). The specificity of carbohydrate recognition of
MBL relies on the identity of the monosaccharide (mannose), and on the fine structure and length (pattern) of the
oligosaccharide side chains.
The serine-thiol proteinase interacts with polysaccharides
although there is no evidence for a lectin-like recognition.
They could form H-bonding with the enzyme varying quantitatively and even involving different sites according to their
structure and hydration. In the case of the galactomannan, we
could speculate that the mechanism of secretion of the serinethiol proteinase in P. brasiliensis and its cellular trafficking
may depend on the fungal polysaccharide for presentation at
the cell surface and release into the medium. Before and after
secretion, the PbST and GalMan could aggregate for a more
stable conformation allowing the enzyme to increase its affinity for the substrate. A threefold decrease in VMAX would be
important to prevent rapid autolysis until the protease found
the specific substrate. In consequence, the enzyme could
degrade components of the membrane more successfully,
leading to a more efficient fungal dissemination.
Acknowledgments
Research and fellowships were financed by FAPESP,
PRONEX/CNPq and CNPq. The peptide substrate AbzMKALTLQ-EDDnp used in this work was produced at the
Department of Biophysics at UNIFESP and kindly provided
by Dr. Maria Aparecida Juliano.
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Artigo 2: C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a
serine-thiol proteinase activity from Paracoccidioides brasiliensis.
Alisson L. Matsuo, Adriana K. Carmona, Luiz S. Silva, Carlos E. Cunha, Ernesto S.
Nakayasu, Igor C. Almeida, Maria A. Juliano e Rosana Puccia
Biochemical and Biophysical Research Communications 2007, Vol 355 (4) 1000-1005
34
Racional
Diversas proteinases extracelulares de microorganismos patogênicos estão
relacionadas com invasão e disseminação pela degradação de componentes da matriz
extracelular. A importância no estudo da proteinase PbST de P. brasiliensis deve-se
principalmente à sua seletividade para proteínas associadas à matriz extracelular.
Determinar inibidores específicos constituiu-se em uma ferramenta importante para o
estudo de proteinases e também para uso terapêutico. Nesse sentido, a inibição da PbST
por compostos mercuriais levou-nos a testar o poder inibitório do composto C(Npys)
acoplado com o peptídeo MKRLTL, que é substrato da enzima. C(Npys) foi inicialmente
usado como protetor na síntese de peptídeos e posteriormente como inibidor de cisteíno
proteinases, pois tem a capacidade de reagir com grupos tiois livres. O trabalho que se
segue mostra que notadamente o composto Bzl-C(Npys)KRLTL-NH2 apresentou uma
elevada capacidade inibitória da PbST, a qual foi completamente revertida com
ditiotreitol. O inibidor não foi tóxico em cultura de células, oferecendo uma alternativa de
inibição in vivo, notadamente se a especificidade para PbST for comprovada.
35
Biochemical and Biophysical Research Communications 355 (2007) 1000–1005
www.elsevier.com/locate/ybbrc
C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit
a serine-thiol proteinase activity from Paracoccidioides brasiliensis
Alisson L. Matsuo a, Adriana K. Carmona b, Luiz S. Silva a, Carlos E.L. Cunha b,
Ernesto S. Nakayasu c, Igor C. Almeida c, Maria A. Juliano b, Rosana Puccia a,*
a
Department of Microbiology, Immunology and Parasitology, Cell Biology Division, Federal University of São Paulo, São Paulo, SP 04023-062, Brazil
b
Department of Biophysics, Federal University of São Paulo, São Paulo, SP 04044-062, Brazil
c
Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA
Received 30 January 2007
Available online 23 February 2007
Abstract
The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulfenyl]) derivatives over thiol-containing serine proteases has never been
tested. In the present work we used an extracellular serine-thiol proteinase activity from the fungal pathogen Paracoccidioides brasiliensis
(PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH2 and Bzl-MKRLTLC(Npys)-NH2. The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can
cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L–T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH2
(Ki = 16 nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further
position from the L–T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the
reactivity of the C(Npys) moiety with a free SH– group.
2007 Elsevier Inc. All rights reserved.
Keywords: Paracoccidioides brasiliensis; Serine-thiol proteinase; Npys
Extracellular proteinases can facilitate the process of
invasion and dissemination of microorganisms through
the human tissues by degrading extracellular-associated
proteins [1]. Our group has previously characterized an
Abbreviations: Abz, ortho-aminobenzoic acid; AUF, arbitrary units of
fluorescence; Bzl, benzoyl; Boc, tert-butyloxycarbonyl; C(Npys), S-(3nitro-2-pyridinesulfenyl); E-64, (L-trans-expoxysuccinyl-leucylamido[4guanidino butane]); EDDnp, N-(2,4-dinitrophenyl)ethylenediamine;
FRET, fluorescence resonance energy transfer; pABA, p-aminobenzamidine; PbST, exocellular serine-thiol activity from Paracoccidioides
brasiliensis; PCM, paracoccidioidomycosis; p-HMB, sodium 7-hydroxymercuribenzoate; PMSF, phenylmethylsulfonyl fluoride.
*
Corresponding author. Present address: Disciplina de Biologia Celular,
UNIFESP, Rua Botucatu, 862, oitavo andar, São Paulo, SP 04023-062,
Brazil. Fax: +55 11 5571 5877.
E-mail address: [email protected] (R. Puccia).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.02.070
extracellular subtilisin-like serine proteinase activity in
the yeast phase of the human pathogen Paracoccidioides
brasiliensis [2]. This activity is able to selectively degrade,
in vitro, murine laminin, human fibronectin, type IV-collagen, and proteoglycans, while common proteins like
bovine serum albumin (BSA) or casein are not cleavable
substrates [3]. The proteinase is able to hydrolyze fluorescence resonance energy (FRET) peptides homologous to
MKRLTL, which are flanked by Abz (ortho-aminobenzoic
acid) and EDDnp (N-[2,4-dinitrophenyl]ethylenediamine).
Cleavage occurs between the Leu–Thr bond at an optimum alkaline pH and is inhibited by PMSF, mercuric acetate, and p-HMB (sodium 7-hydroxymercuribenzoate), but
not by E-64 [2]. Therefore, the enzymatic activity has been
classified as a thiol-containing subtilisin-like serine proteinase (PbST, for P. brasiliensis serine-thiol proteinase)
A.L. Matsuo et al. / Biochemical and Biophysical Research Communications 355 (2007) 1000–1005
belonging to the group of proteinase K. In addition, we
have recently described a novel type of regulation of
the PbST activity against Abz-MKALTLQ-EDDnp by
neutral polysaccharides, including a fungal extracellular galactomannan, which might help stabilize the enzyme
[4].
Paracoccidioides brasiliensis is a dimorphic fungus that
causes paracoccidioidomycosis (PCM), a potentially lethal
systemic mycosis with multiple clinical forms that is prevalent in South America [5]. The fungus grows in the infective mycelial phase at environmental temperatures below
26 C and in the yeast pathogenic phase at body temperatures of about 37 C. PCM is a granulomatous mycosis
that starts in the lungs and tends to disseminate rapidly
through the lymphatic system in juvenile (acute and subacute) forms. In adult (chronic) forms, active disease affects
especially the lungs, however dissemination to the cutaneous, mucocutaneous tissues or other organs is likely to
occur through the blood and lymphatic route. In this
context, PbST is a potential virulence factor that could
contribute to fungal spread to surrounding and/or distant
tissues.
The S-(3-nitro-2-pyridinesulfenyl) group—C(Npys)—
has originally been used for protection in peptide synthesis
[6] and later demonstrated to be effective in the design and
synthesis of thiol proteinase irreversible inhibitors [7].
These compounds have been found to be highly specific
to cysteine proteinases due to their capacity of selectively
reacting with free thiol groups to form unsymmetrical
disulfide bonds [8] and their inability to inhibit serine or
acid proteinases [7]. A variety of cysteine proteinase inhibitors have been developed such as diazomethyl ketones,
halomethyl ketones, and epoxides [9]. However, they are
strong electrophilic reagents and may alkylate non-targeted
enzymes and other biomolecules in vivo [10], preventing any
therapeutic use. The C(Npys) compounds represent an
alternative to overcome this problem, considering that they
promote the formation of disulfide bonds without non-specific alkylation, but still keep the ability of being specific
inhibitors.
The inhibitory capacity of C(Npys) derivatives over
thiol-containing subtilisins has never been tested. In the
present work, we used PbST to describe a potent inhibitory
capacity of two peptides derived from MKRLTL coupled
with the C(Npys) group.
Materials and methods
Isolation of the serino-thiol proteinase activity. Culture supernatants
containing PbST activity against Abz-MKALTLQ-EDDnp were
obtained from P. brasiliensis B-339 as previously described [3,4] and
buffered to a final concentration of 50 mM Tris–HCl, pH 8.0, containing
1.0 M NaCl, which did not alter the initial proteolytic activity. Aliquots
(40 ml) were chromatographed, using a peristaltic pump, in an affinity
column of p-aminomethylbenzamidine (pABA)-Sepharose (Pharmacia/
LKB) previously equilibrated in the same buffer. After a washing step
with 10 volumes of the same buffer, bound compounds were eluted with
50 mM glycine, pH 3.0, into 1.0 mL fractions immediately neutralized
1001
with Tris–HCl. The fractions were analyzed for proteolytic activity
against Abz-MKALTLQ-EDDnp, as described below, and against
fibronectin (FN, 2 lg) as described by Puccia et al. [3]. Activity was
assayed in 50 mM Tris–HCl, pH 8.0. Fibronectin was purified from sera
donated from healthy individuals by chromatography in gelatinSepharose (Amersham Biosciences, GE Healthcare), as described [11].
Protein contents were visualized in Coomassie blue and/or silver-stained
SDS–PAGE gels [12].
Peptide synthesis. The FRET substrate Abz-MKALTLQ-EDDnp was
synthesized using the solid phase synthesis method [13] according to the
Fmoc procedure. An automated bench-top simultaneous multiple solidphase peptide synthesizer (PSSM 8 system; Shimadzu) was used in the
synthesis. The inhibitor peptides Bzl-MKRLTLC(Npys)-NH2 and BzlC(Npys)KRLTL-NH2 were prepared by the solid phase methodology
using Boc-amino acids according to the general procedure described
previously [14]. All the peptides were purified by semi-preparative h.p.l.c.
using an Econosil C-18 column. The molecular mass and purity were
checked by amino acid analysis and by mass spectrometry using a matrix
assisted laser-desorption ionization-time-of-flight-mass spectrometer
(MALDI-TOF-MS) TOFSpec E instrument (Micromass, Manchester,
Waters, UK).
Enzymatic assays. The proteolytic activity of PbST against AbzMKALTLQ-EDDnp was determined at 37 C in 50 mM Tris–HCl, pH 8.0,
in a Hitachi F-2000 spectrofluorometer (excitation = 320 nm; emission = 420 nm), as described previously [2]. The effect of Boc-C(Npys), BzlMKRLTLC(Npys)-NH2 and Bzl-C(Npys)KRLTL-NH2 in the enzymatic
activity was determined using the same procedure, after 5 min of pre-incubation with the enzyme preparation. Kinetic parameters were determined by
measuring the initial rate of hydrolysis at various inhibitor concentrations
and the residual activity was evaluated. Fluorescence emission was continuously measured and inhibition constant values (Ki) were obtained using the
GraFit 3.0 computer program (Erithacus Software Ltd.).
Cell detachment assays. LLC-MK2 cells were expanded overnight in
24-well culture plates (2 · 104 cells/mL/well) at 37 C, in a 5% CO2
chamber. The cells were maintained in DMEM (Dulbecco’s modified
Eagle’s medium, Gibco) containing 0.37% NaHCO3 and 10% FBS. The
plastic-attached cells were washed five times with 0.5 mL of phosphatebuffered saline (PBS), then incubated for 5, 10, 15, 20, 30, 45 or 60 min
with 200 lL of PBS/0.05 M EDTA containing 10 lL of PbST preparation (fraction 4, Fig. 1) previously inactivated or not by incubation
with C(Npy) inhibitors for 10 min at 37 C. Controls were assayed in
the absence of enzyme. Classical inhibitors were used to characterize the
activity at the following excess concentrations: 2 mM PMSF, 1 mM pHMB, 0.13 mM E-64, and 13 lM pepstatin. The tests were performed
in the presence of EDTA, which inhibits metallo proteinases. Cells were
harvested and counted in a Neubauer chamber using Trypan blue
staining for viability. The experiments were carried out in triplicates
and statistical analyses were performed using the one-tailed distribution
Student’s t-test.
In-gel protein digestion and LC–MS/MS analysis. Proteins fractionated by SDS–PAGE were silver-stained using the Vorum method [15].
In-gel protein digestion was performed as described [16]. Resulting
peptides were recovered from the gel and desalted in a Poros R2-50
(Applied Biosystems) zip-tip, dried in a vacuum centrifuge, dissolved in
30 lL of 0.1% formic acid (FA), and subjected (1 lL) to liquid chromatography-mass spectrometry (LC–MS) analysis. LC was performed
in a PepMap reversed-phase column (15 cm · 75 lm, 3-lm C18, LC
Packings, Dionex) coupled to an Ultimate (LC Packings, Dionex)
nanoHPLC. Bound peptides were eluted with 5–42.5% acetonitrile/0.1%
FA over 30 min and directly analyzed in a Q-tof 1 (Micromass, Waters)
mass spectrometer. Spectra in positive-ion mode were collected in the
400–1800 m/z range, and each peptide was fragmented (MS/MS) for 3 s
in the 50–2050 m/z range. MS data were converted into peak lists (PKL
format) and analyzed by the Phenyx (GeneBio) and Mascot (Matrix
Science) softwares through the NCBInr and P. brasiliensis protein and
nucleotide sequences from GenBank. Peptides with no match in these
databases were subjected to de novo sequencing using the MassLynx
v4.0 software (Waters).
0
10
81
75
20
0
0
(AUF/min)
0
60
1002
In
FT
2
3
4
5
6
73
47
29
200
FN
0
5’
10’
15’
30’
200
Fig. 1. Isolation of PbST by chromatography in a pABA-Sepharose
column. Upper panel: silver-stained 10% SDS–PAGE gel showing the
profile of culture supernatant (10 ll) from P. Brasiliensis yeast cells before
(In) and after (FT) chromatography. Eluted fractions are numbered. The
hydrolytic activity against Abz-MKALTLQ-EDDnp produced by a 10 llaliquot of each fraction is shown as arbitrary units of fluorescence per
minute (AUF/min). The results are not presented in terms of specific
activity due to the low protein contents. The hydrolytic activity against
fibronectin (FN) correspondent to incubation with each fraction at 37 C
for 30 min is shown in a Coomassie blue-stained 8% SDS–PAGE gel. A
time-course of FN cleavage after incubation for 5, 10, 15, and 30 min with
peak fraction 4 (10 ll) is also depicted. The positions of molecular mass
markers (kDa) and of one of the FN chain are shown on the left.
Results and discussion
In previous publications we characterized the PbST
activity using preparations of P. brasiliensis supernatants
enriched by either ion exchange/gel-filtration chromatography [2,3] or hydrophobic phenyl-Superose columns [4,17].
To date, we have tried to purify the proteinase using many
different chromatographic methods (not published), however total purification of the active proteinase has never
been achieved due to aggregation to other fungal components and/or loss of enzymatic activity after several chromatographic steps.
Presently, a cell-free supernatant from P. brasiliensis B339 yeast-cell culture bearing PbST proteolytic activity was
fractionated in a pABA-Sepharose column. The p-aminobenzamidine (pABA) compound is a commercially available competitive inhibitor of trypsin that binds to the
specificity cleavage pocket of the enzyme [18]. We observed
that the PbST activity capable of hydrolyzing the FRET
peptide substrate Abz-MKALTLQ-EDDnp was bound to
the column and predominantly eluted in fractions 4 and
5, as seen in Fig. 1. These fractions also concentrated the
proteolytic activity towards fibronectin found in total
supernatants, as suggested by extensive degradation of
the protein resulting from incubation with fractions 4 or
5 (Fig. 1). A time-course assay showed that the proteolytic
activity contained in fraction 4 (PbST-4) was so strong that
after only 5 min of incubation protein degradation was
already evident (Fig. 1, bottom). Enzymatic cleavage of
Abz-MKALTLQ-EDDnp or human fibronectin was due
to PbST, since hydrolysis was specifically inhibited by
p-HMB and PMSF (not shown), as previously reported
[2]. In our experimental conditions, the activity against
Abz-MKALTLQ-EDDnp was fully bound to the column
and was recovered in the eluted fractions at a yield of about
65%. In addition, PbST-4 was not active against casein or
BSA (not shown), as previously described [3]. The chromatographic profile in SDS–PAGE silver-stained gels
showed that fractions 4 and 5 had a predominant 55 kDa
component (Fig. 1), among others that appeared after
longer development times of silver-stained gels. Preparation PbST-4 could be kept at 4 C for about 7 days before
activity started to gradually decrease.
Previously, we have been able to detect in situ activity of
PbST in SDS–PAGE gels [17]. The PbST activity was
revealed as a broad smeared band migrating between 43
and 68 kDa in gelatin zymograms or using an AbzMKALTLQ-EDDnp-agarose overlay. It is uncertain
whether the proteinase molecular mass is around 50 kDa
or if migrates as such due to aggregation in our electrophoretic conditions, where the samples were denatured at low
SDS concentration and without boiling. Presently, pABA
preparation PbST-4 was tested in gelatin zymograms, but
a proteolysis band has not been detected, possibly because
pABA-isolated PbST was unstable after electrophoresis.
We then chose to carry out an LC–MS/MS analysis of
the whole PbST-4 preparation and also of individual main
gel bands present in overloaded gels. We consistently identified NADP-dependent glutamate dehydrogenase (correspondent to the 55-kDa band), LPD1 (lipoamide
dehydrogenase), and chitinase based on the high number
of tryptic peptides bearing over 10 amino acids identical
to peptides found in the NCBInr and GenBank database
containing fungal proteins. The identified proteins should
contain motifs that were recognized by pABA or they were
unspecifically bound to the resin as aggregates. Although a
couple of peptides would match those of subtilisins, the
number of identical amino acids was not enough to guarantee reliable protein identification. One difficulty to analyze
generated peptides is the lack of complete genome information for P. brasiliensis, although there are two GenBank
databases of partial translated sequences [19,20]. It is
important to point out that there is a 481-amino acid
P. brasiliensis subtilase of the S8 family deposited in the
GenBank (Accession No. AAP83193). However, our present analysis suggests that PbST does not correspond to this
proteinase, since identical peptides have not been
identified.
We used pABA-eluted PbST-4 to evaluate the inhibitory
effect of peptides derived from MKRLTL coupled with
C(Npys). A general mechanism of action of C(Npys)-peptides is schematically represented in Fig. 2, which shows
that a peptide sequence containing C(Npys) can block a
free thiol group, liberating Npys. We presently synthesized
two analogous peptides, namely Bzl-MKRLTLC(Npys)NH2 and Bzl-C(Npys)KRLTL-NH2, which bear the
C(Npys) group, respectively, at the C- and N-terminus. A
third compound, Boc-Cys(Npys), was used to test the
inhibitory capacity of C(Npys) devoid of a peptide
sequence.
We observed that the three compounds inhibited the
enzymatic activity against Abz-MKALTLQ-EDDnp [17]
at different levels (Table 1). The best inhibitory activity
was seen with Bzl-C(Npys)KRLTL-NH2 (Ki = 16 nM),
where the C(Npys) group was placed at the N-terminus
substituting the original methionine residue. The peptide
bearing C(Npys) at the C-terminus was one order of magnitude less effective (Ki = 160 nM), whereas the BocC(Npys) compound was a weaker inhibitor (Ki = 1.7 lM).
These results indicated that the peptide sequence promotes
a favorable interaction increasing the magnitude of inhibition, especially when C(Npys) was placed at a further position from L–T, at the N-terminus. The inhibitory effect of
all tested peptides was completely reverted with 5 mM dithioerythritol, indicating that it was due to the reactivity of
the C(Npys) moiety with a free SH– group of the proteinase (Fig. 2). It is noteworthy that the inhibitor peptides
have not been cleaved by the proteinase activity, as
revealed by h.l.p.c. analysis (not shown).
We have previously reported on the PbST capacity to
cleave components associated with the basement membrane [3]. Presently, we have not been able to test the inhibitory capacity of C(Npys) peptides on the cleavage of
fibronectin visualized in SDS–PAGE gels (as in Fig. 1)
because they altered fibronectin resolution. Therefore, we
developed a cell detachment assay for this purpose. We
tested PbST ability to detach cells of the LLC-MK2 lineage
from plastic dishes, which is generally achieved using trypsin. Cell detachment depends on cleavage of extracellular
matrix components or of surface integrins that directly
interact with them. Fig. 3A shows that PbST-4 was able
to detach LLC-MK2 at increased rates over time, which
reached a plateau corresponding to approximately 50%
of total cells detached after 20 min. Consequently, this
incubation time was chosen for the inhibition experiments
shown below. All experiments were carried out in triplicates and harvested cells were 70–80% viable. It is worth
mentioning for comparison that 2.5 mg/mL of trypsin
1003
Table 1
Inhibition of PbST activity by Boc-C(Npys) and its derivatives bound to
the sequence MKRLTL
Compound
Ki (M)
Boc-C(Npys)
Bzl-MKRLTLC(Npys)-NH2
Bzl-C(Npys)KRLTL-NH2
1.7 · 10
1.6 · 10
1.6 · 10
6
7
8
can detach all the cells (90–95% viable) in 5 min (not
shown). Fig. 3B shows that the detachment activity was
due to PbST, considering that pre-incubations with E-64
and pepstatin (or EDTA contained in the PBS) did not
affect the activity, whereas p-HMB and PMSF totally abolished it (Fig. 3B).
In order to evaluate the inhibitory capacity of C(Npys)
peptides in the cell detachment activity of PbST-4, the
experiments were performed with the enzyme preparation
previously incubated or not (control) with 6 or 12 lM of
each peptide for 20 min at 37 C. Fig. 3C shows that both
Bzl-C(Npys)KRLTL-NH2 and Bzl-MKRLTLC(Npys)NH2 exhibited statistically significant inhibitory effects, in
a dose-dependent fashion. Bzl-C(Npys)KRLTL-NH2 was
a better inhibitor than Bzl-MKRLTLC(Npys)-NH2, in
agreement with the Ki results seen in Table 1. BocCys(Npys) showed no statistically significant effect at the
concentrations tested, probably due to its lower binding
affinity, as suggested from the results in Table 1.
This is the first time C(Npys) compounds are used to
inhibit a thiol-containing subtilisin. It remains to be tested
whether these compounds would effectively decrease P.
brasiliensis virulence in vivo. For that purpose, C(Npys)
might be bound to peptides of the D-anomericity, for
e.g., to avoid proteolysis by local endopeptidases.
The results presented here elegantly show that PbST
activity can be blocked with compounds interfering with
a free cysteine, confirming that it belongs to the subtilisin
S8 family of serine proteinases, from which proteinase K
from Tritirachium album is the best studied member [21].
By analogy with the members of this group [22], we speculate that there is a thiol group lying near the imidazole of
the histidine from the Ser-His-Asp catalytic triad. The presence of such a residue does not change the catalytic activity
of subtilisin Savinase, although it makes it susceptible to
mercurials [23]. In proteinase K, the thiol group (Cys78)
is buried in the molecule and covalently binds to the first
Hg+ molecule, which disrupts the catalytic triad by changing the imidazole conformation [24]. A second Hg+ complexes non-covalently with His72, Cys78 and Thr76,
Fig. 2. Schematic representation of the mechanism of inhibition of thiol proteinases by Cys(Npys).
1004
Cells/field
A
10
9
8
7
6
5
4
3
2
1
0
0
10
20
30
40
50
60
70
time (min)
B
120
100
*
80
60
40
*
20
*
0
Control
*
*
PMSF p-HMB
*
E-64 pepstatin
6
12
C-
6
12
N-
6
12
µM
Boc-
Fig. 3. Evaluation of PbST inhibition in a cell detachment assay. (A) Time-course of cell detachment when incubated with PbST-4 (10 lL, fraction 4,
Fig. 1). (B) Percentage of cell detachment when compared to the control (100%), as counted after 20 min of incubation with PbST-4 in the presence of
either standard proteinase inhibitors or Bzl-MKRLTLC(Npys)-NH2 (C-), Bzl-C(Npys)KRLTL-NH2 (N-) and Boc-Cys(Npys) (Boc-) at the indicated
concentrations. Incubation of cell cultures with equivalent concentrations of the inhibitors alone has not resulted in cell detachment or loss of cell viability
(not shown). Statistically significant values (p < 0.05) are indicated with an asterisk.
decreasing substrate-binding affinity. However, 100% inhibition by mercurials is never achieved, possibly due to flexibility of the molecule [24]. In contrast to proteinase K,
PbST is selective regarding substrate specificity and is
inhibited 100% by p-HMB [2]. On the other hand, proximity of the thiol group to the catalytic triad would explain
why the C(Npys)-peptide derivatives are better inhibitors
than Boc-C(Npys), considering that the peptide used here
fits in the substrate pocket to target the inhibitor.
[4]
[5]
[6]
Acknowledgments
[7]
We thank Dr. Ivarne Tersariol for critical review of the
manuscript. This work and fellowships involved were supported fully or partially by grants from FAPESP, PRONEX/CNPq, CNPq, and NIH/NCRR (Grant No.
5G12RR008124).
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Proteinase Lon mitocondrial
36
Artigo 3: The PbMDJ1 gene belongs to a conserved MDJ1/LON locus in
thermodimorphic pathogenic fungi and encodes a heat shock protein that localizes to both
the mitochondria and cell wall of Paracoccidioides brasiliensis.
Wagner L. Batista, Alisson L. Matsuo, Luciane Ganiko,Tânia F. Barros, Thiago R.
Veiga, Edna Freymuller e Rosana Puccia
Eukaryotic Cell 2006, Vol 5 (2) 379-390
37
Racional
Em nosso laboratório, o gene PbLON foi clonado acidentalmente quando
buscávamos clonar o gene da PbST. Durante a caracterização do seu locus, foi
encontrado em direção oposta o gene da PbMDJ1 compartilhando com PbLON a região
5’ intergênica. Ambos os genes codificam proteínas com direcionamento mitocondrial, as
quais em leveduras têm uma relação funcional, já que a Mdj1 participa do complexo
formado pela Lon e a proteína desnaturada alvo de proteólise. Neste trabalho verificou-se
que o locus LON/MDJ1 é conservado entre os Ascomicetos e que a PbMdj1 tem uma
localização extramitocondrial na parede celular de leveduras do P. brasiliensis, enquanto
a PbLon fica restrita à mitocôndria.
38
EUKARYOTIC CELL, Feb. 2006, p. 379–390
1535-9778/06/$08.00⫹0 doi:10.1128/EC.5.2.379–390.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 5, No. 2
The PbMDJ1 Gene Belongs to a Conserved MDJ1/LON Locus
in Thermodimorphic Pathogenic Fungi and Encodes a Heat
Shock Protein That Localizes to both the Mitochondria
and Cell Wall of Paracoccidioides brasiliensis
Wagner L. Batista,1 Alisson L. Matsuo,1 Luciane Ganiko,1† Tânia F. Barros,1‡
Thiago R. Veiga,1 Edna Freymüller,2 and Rosana Puccia1*
Department of Microbiology, Immunology and Parasitology1 and Center of Electron Microscopy,2
Federal University of São Paulo, Rua Botucatu 862, 04023-062 São Paulo, SP, Brazil
Received 30 September 2005/Accepted 18 November 2005
J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones,
besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1,
a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell
wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The antirecombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial
and cell wall (alkaline ␤-mercaptoethanol extract) component, compatible with the predicted size of the protein
devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in
the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro,
possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood
preferentially close to the plasma membrane, and gold particles were detected in the thin space between them,
toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock
proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and
Aspergillus, where the genes are adjacent and have a common 5ⴕ region. This is the first time a DnaJ member
has been observed on the cell surface, where its function is speculative.
brasiliensis yeast versus mycelium, or undergoing phase transition in vitro, making use of microarrays, suppression subtraction hybridization, real-time reverse transcriptase (RT) PCR,
and in silico analyses (14, 15, 27, 33). Considering that these
analyses have been undertaken with P. brasiliensis transforming
to the yeast phase upon temperature increase, differentially
regulated heat shock protein homologues have indeed been
detected, especially in the initial hours of temperature change
(15, 33). Some of them, however, seem to be necessary for
growth in the yeast phase, e.g., the HSP70 and HSP60 genes in
P. brasiliensis that have been previously characterized (18, 41).
We have characterized a LON gene homologue from P.
brasiliensis (PbLON) (2). Lon proteins are conserved ATPbinding, heat-inducible serine proteinases. They form highmolecular-mass complexes of homo-oligomers, which in
Saccharomyces cerevisiae contain the stoichiometry of seven
subunits of 117 kDa (46). The yeast Lon, also called PIM1, is
synthesized as a pre-proenzyme precursor in the cytosol and
then is sorted to the mitochondrial matrix where it is processed
(54). It controls proteolysis by mediating cleavage of misfolded
or unassembled matrix proteins and has an essential role in the
maintenance of mitochondrial DNA integrity and mitochondrial homeostasis (51). Lon is also a DNA-binding protein (26)
and is essential for cell survival in humans (4).
In P. brasiliensis, PbLon consists of 1,063 residues containing
a mitochondrial import signal, a conserved ATP-binding site,
and a serine catalytic motif (2). Its open reading frame (ORF)
is within a 3,369-bp fragment interrupted by two introns lo-
Paracoccidioides brasiliensis is the fungal species responsible
for paracoccidioidomycosis (PCM), which is endemic in certain areas of Latin America. PCM is one the four deep-seated
granulomatous mycoses caused by thermodimorphic fungi that
also include Histoplasma capsulatum, Blastomyces dematitidis,
and Coccidioides immitis. These fungal species are phylogenetically related, according to sequence analysis of the ribosomal RNA gene locus (3, 36), and have been classified as
Ascomycetes of the Onigenaceae family. P. brasiliensis grows as
a multibudding yeast when in parasitism or cultivated at 37°C
and as a mycelium when incubated at temperatures below
28°C. The fungus is multinucleated, and its sexual form is still
unknown. Genetic manipulation in this species is only starting
to be attempted (23).
The human host most frequently acquires the fungus by
inhalation of mycelial particles, but infection can be established only after transition to the yeast parasitic phase takes
place (28). A number of biochemical processes are associated
with fungal adaptation to the host’s environment and temperature, and many of them are probably responsible for phase
transition and/or phase maintenance. Different groups have
recently addressed the overall scenario of gene expression in P.
* Corresponding author. Mailing address: Rua Botucatu 862, oitavo
andar, 04023-062 São Paulo, SP, Brazil. Phone: (55) 11 5084 2991.
Fax: (55) 11 5571 5877. E-mail: [email protected].
† Present address: University of Texas at El Paso, El Paso, Tex.
‡ Present address: Department of Clinical and Toxicological Analyses, Federal University of Bahia, Bahia, BA, Brazil.
379
380
BATISTA ET AL.
cated in the 3⬘ segment; an MDJ1-like gene was partially sequenced in the opposite direction but sharing with PbLON a
common 5⬘ untranslated region (2). This chromosomal organization might be functionally relevant, since Mdj1p is a type I
DnaJ molecule located in the yeast mitochondrial matrix and is
essential for substrate degradation by Lon and other stressinducible ATP-dependent proteinases (39, 53). In bacteria,
DnaJ alone and/or the DnaJ/DnaK complex determine the fate
of nonnative substrates to be cleaved by Lon (17).
DnaJ members (more recently referred to as J-domain proteins) belong to the Hsp40 family of molecular chaperones and
localize to various cellular compartments, where their primary
role is to regulate the activity of their cognate Hsp70 homologues (55). In the bacterial DnaK (Hsp70)/DnaJ/GrpE complex, a transient association of DnaJ stimulates the ATPase
activity of DnaK, prompting substrate binding, while GrpE
exchanges nucleotides (ADP/ATP), with the consequent substrate dissociation (12). This complex prevents aggregation of
nonnative proteins, thus facilitating their folding by Hsp60
(22). In S. cerevisiae, Mdj1 is essential for the biogenesis of
functional mitochondria, but it is not involved in protein translocation into the matrix (reviewed in reference 52). Typically,
DnaJ members are constituted of a structurally conserved J
domain located near the N-terminal end, followed by a glycine/
phenylalanine-rich segment (G/F domain) and four repetitions
of a zinc-binding CXXCXGXG motif (55). The J domain and
the G/F segment are essential for interaction with DnaK, while
the zinc finger-like motif contains elements for binding nonnative substrates (49) and also displays thiol-disulfide oxidoreductase activity (50). In S. cerevisiae, 22 J-domain proteins
(types I, II, and III) have been found in the genome; their
subcellular localizations are attributed to the cytoplasm, nucleoplasm, mitochondria, and endoplasmic reticulum (55).
We have presently characterized the P. brasiliensis PbMDJ1
gene and verified that the PbLON/PbMDJ1 locus is conserved
among the dimorphic pathogenic fungi besides Aspergillus nidulans and A. fumigatus. We show that PbLON and PbMDJ1 encode
mitochondrial heat shock proteins; however, PbMdj1 has also
been found in the cell wall of P. brasiliensis. The finding of a
DnaJ member on the cell surface is a novel observation.
MATERIALS AND METHODS
Fungal strains and culture conditions. We used P. brasiliensis isolates B-339
and 18, provided by Angela Restrepo (Colombia) and Zoilo P. Camargo (Brazil),
respectively. More details about the fungal isolates can be found elsewhere (29).
Cultures were maintained at 36°C (yeast phase) in solid modified YPD (0.5%
casein peptone, 0.5% yeast extract, 1.5% glucose, pH 6.3).
Isolation of total DNA and RNA. For DNA extraction, strain B-339 was
expanded for 10 days at 36°C in liquid modified YPD under agitation. Total
DNA was extracted from a 10-ml pellet of nitrogen-frozen cells that were disrupted in a mortar and then briefly in a pestle, as described previously (11). Total
RNA was isolated from fresh cells mechanically disrupted by vortexing with glass
beads for 10 min in the presence of TRIzol reagent (Invitrogen), according to the
instructions provided by the manufacturer. Contaminating DNA in these preparations was digested with RNase-free DNase I (Promega), as described previously (15). The efficiency of hydrolysis was tested by PCR with primers of the
PbGP43 gene (11). For analysis of gene expression in cells undergoing heat shock
at 42°C, yeast cells growing logarithmically at 36°C under shaking in modified
YPD were transferred to 42°C for 30 and 60 min.
Cloning of the 3ⴕ end of PbMDJ1. We followed the strategy of 3⬘ rapid
amplification of cDNA ends to clone the 3⬘ end of the PbMDJ1 cDNA, using the
SuperScript II RNase H RT kit (Invitrogen), because at that time the PbMDJ1
expressed sequence tag (EST) had not yet been found in the P. brasiliensis EST
EUKARYOT. CELL
bank (15). The positions of the primers can be seen in Fig. 1A. The first strand
of the total cDNA pool was obtained from total RNA (2.5 ␮g) extracted from
strain B-339 yeast cells previously heat shocked for 60 min at 42°C. The template
was incubated (10 ␮l, final volume) with a 1 ␮M concentration of a poly(T)containing cassette primer (511, 5⬘-GACTCGAGTCGACATCGT17-3⬘) at 65°C
(5 min), cooled in ice, mixed with synthesis buffer (final concentrations of 0.05 M
Tris-HCl [pH 8.3], 0.075 M KCl, 3 mM MgCl2, 0.01 M DDT, 1 mM [each]
deoxynucleoside triphosphate, 2 U/␮l of RNAseOUT, and 10 U/␮l of SuperScript II RT), and incubated at 55°C (50 min) and then at 85°C (5 min).
Template RNA was removed with 0.1 U/␮l of RNase for 20 min at 37°C.
Negative controls were incubated in the absence of RT. The second strand was
obtained from total cDNA (1 ␮1 of the above reaction product) with an internal
sense primer (B4, 5⬘-TGGTGGTGGGTTCTCTGC-3⬘) under standard PCR
conditions at an annealing temperature of 52°C. We used Platinum Taq polymerase (Invitrogen) and an antisense cassette primer (512, 5⬘-GACTCGAGT
CGACATCG-3⬘) for amplification of the double-stranded PbMDJ1 fragment.
The final product was used as a template in a second round of PCR primed with
antisense primer 512 and nested sense primer G5 (5⬘-TGCCTTTGCGGGTGG
TTC-3⬘). Sequencing information from the resulting 1,161-bp amplicon was used
to design primers located at the 3⬘ end (antisense 2A7, 5⬘-TACCATTTTTTG
CCTGCC-3⬘), which enabled PCR amplification of the whole genomic PbMDJ1
gene using genomic DNA as a template. A cDNA segment of the 5⬘ region, used
later for heterologous expression, was amplified by RT-PCR carried out with
total RNA (1 ␮g) and the ThermoScript RT-PCR System (Gibco BRL). The first
cDNA strand was amplified with the internal antisense primer E4 (5⬘-GCCGCA
CGAGGAACAGG-3⬘), according to the supplier’s recommendations. A PCR
product of 757 bp was then obtained with primers B3 (sense, 5⬘-CTCCCG
GCTCGTCTCCTG-3⬘) and A7 (antisense, 5⬘-CCGCTTTGTACCCTGTTT-3⬘),
with the first-strand reaction product as a template. Sequence comparison between DNA and cDNA fragments enabled the precise localization of two introns.
Negative controls for 3⬘ rapid amplification of cDNA ends and RT-PCRs were
incubated in the absence of RT. DNA fragments were recovered from agarose
gels using either the Nucleiclean kit (Sigma) or the Concert gel extraction system
(Gibco BRL). Purified amplicons were cloned into the pGEM-T easy vector
(Promega) for maintenance and sequencing. Nucleotide sequencing was carried
out in the facilities of the Center of Human Genome at the São Paulo University.
Sequences were compared with those of the National Center for Biotechnology
Information through the GenBank database and the BLAST network service and
analyzed with the EditSeq, Seqman, Megalign, and Protean programs of the
Lasergene System (DNAstar Inc.).
Northern blotting. Standard conditions were used for electrophoresis and
Northern blotting (38), carried out with 25 ␮g of total RNA per lane. Hybridizations with [␣-32P]dCTP-labeled probes were done in nylon Hybond-N membranes (Amersham) under high-stringency conditions with a B3/2A7 DNA fragment of PbMDJ1 (Fig. 1A) and a PbLON amplicon of 850 bp (from nucleotide
[nt] 536 to nt 1386). The probes were labeled using the RadPrime DNA labeling
system (Gibco BRL) as specified by the manufacturer and separated from free
nucleotides in a molecular biology-grade Sephadex G-50 column (Pharmacia).
The membranes were hybridized with the labeled probes (1 ⫻ 106 cpm/ml) at
42°C overnight in 50% formamide, 6⫻ SSC (1⫻ SSC is 0.15 M NaCl, 0.015 M
sodium citrate), 5⫻ Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS),
and 100 ␮g/ml of salmon sperm DNA; washed one time with SSC–0.1% SDS (20
min, 42°C) and three times with 0.2⫻ SSC–0.1% SDS (20 min, 68°C); covered
with Vitafilm (Goodyear); and exposed to an X-ray film (X-Omat; Kodak) at
⫺70°C with an intensifying screen.
Protein extraction. Total cell extracts of P. brasiliensis were obtained through
glass bead disruption (8). For isolation of total mitochondrial fractions, we adapted
for P. brasiliensis the method described by Suzuki et al. (48) for S. cerevisiae. In order
to accomplish cell lysis under mild conditions, nitrogen-frozen yeast cells were
mechanically disrupted in a mortar until they formed a fine white powder, which was
subsequently thawed in BB buffer (0.6 M sorbitol, 20 mM HEPES, pH 7.4) and
sonicated for 5 min, alternating 15-s pulses with 15 s in ice. Cell debris were pelleted
by centrifugation (1,500 ⫻ g, 5 min) and the ice-cooled supernatant was centrifuged
at 1,200 ⫻ g for 10 min to precipitate the mitochondrial fraction (brownish pellet),
which was then rinsed with BB and centrifuged (1,500 ⫻ g, 5 min). The supernatant
was subjected to the same procedure two times, the resulting brownish pellets were
pooled and washed two times with BB and suspended in 0.2 ml of BB, and the
protein concentration was estimated by spectrometry in an aliquot diluted 100 times
in 0.6% SDS (an A280 of 0.2 corresponds to 10 mg/ml of mitochondrial proteins).
Total mitochondrial protein preparations were kept at ⫺70o in 20% glycerol–1
mg/ml bovine serum albumin (BSA).
A ␤-mercaptoethanol cell wall extract was obtained from intact cells as described previously (9). P. brasiliensis yeast cells grown under shaking for 10 days
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PbMdj1 LOCALIZES TO THE MITOCHONDRIA AND CELL WALL
381
FIG. 1. (A) Schematic representation of PbMDJ1, highlighting the localization of the primers and probes used in this work. Introns were
localized from nt 205 to 313 and nt 1552 to 1683. (B) Schematic representation of PbMdj1 showing the localization of conserved domains.
Kyte-Doolittle hydrophilicity plots (Protean module; DNAstar Inc.) of fungal Mdj1-like sequences are as follows: Pb, P. brasiliensis (AF334811);
Bd, B. dermatitidis (http://genome.wustl.edu/BLAST/blasto_client.cgi); Hc, H. capsulatum (http://genome.wustl.edu/BLAST/histo_client.cgi); Ci, C.
immitis (http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis); An, A. nidulans (EAA57980); Cn, C. neoformans (EAL21819); Ca, C.
albicans (EAK92195); Sc, S. cerevisiae (CAA82189); Ec, Escherichia coli (BA000007). The boxes indicate the predicted mitochondrial target
sequence (MT) and the J domain. The dots localize the Zn2⫹ finger domains. Horizontal bars indicate the location of mouse T-cell epitopes with
a minimum of 12 amino acids, as predicted by the Sette major histocompatibility complex motif (Protean module; DNAstar Inc). Percentages of
identity with PbMdj1, as calculated with the MegAlign module of the DNAstar program, are given at right.
(cell viability, ⬎85%) in modified YPD were pelleted by centrifugation (1,500 ⫻
g, 15 min) and rinsed three times with an excess volume of phosphate-buffered
saline (PBS). Washed cells (12 ml of wet pellet) were suspended in 30 ml of 20
mM ammonium carbonate (pH 8.6)–1% ␤-mercaptoethanol–1 mM phenylmethylsulfonyl fluoride and agitated for 1 h at 36°C. The released soluble components
were separated from the cells by centrifugation, dialyzed in H2O, and concentrated to 1.5 ml by lyophilization (about 8 mg/ml of total protein). The treatment
did not affect cell integrity, as verified by microscopy of the treated cells. Protein
contents were estimated using BSA as a standard (5).
Expression of PbMdj1 and PbLon in bacteria. Expression of PbMdj1 and
PbLon in bacteria was achieved with the pHIS1 and pHIS3 vectors (40). A
757-bp cDNA fragment of the 5⬘ region of PbMDJ1, obtained by RT-PCR with
primers B3/A7 (Fig. 1A; see description above), was excised from pGEM-T with
EcoRI/SpeI and subcloned in the same sites of pHIS3 in frame with the sequence
encoding a His6 tag. The resulting construct was called pHISMdj1. For expression of PbLon, an 845-bp BamHI/HindIII intron-free 5⬘ gene fragment was
excised from a pUCSmaI plasmid containing the entire PbLON genomic sequence (2) and subcloned into the same sites of pHIS1 to generate a pHISLon
expression plasmid. Vector pHIS1 expresses heterologous proteins bearing His6
in both the N and C termini. Plasmids pHISMdj1 and pHISLon were used to
transform Escherichia coli DH5␣ for maintenance and amplification, where positive clones were selected for resistance to ampicillin. Correct in-frame ligation
was checked by endonuclease restriction and sequencing. Selected plasmids were
inserted into E. coli BL21/pLysS (Novagen) for IPTG (isopropyl-␤-D-thiogalactopyranoside)-induced protein expression. For purification of recombinant
PbMdj1 (rPbMdj) and rPbLon, individual bacterial clones were cultivated overnight at 37°C under shaking in LB medium containing 100 ␮g/ml of ampicillin
and 37 ␮g/ml of chloramphenicol. An aliquot of this initial culture was diluted
1:100 in fresh medium and grown until it reached an A600 of 0.6. At this point,
protein expression was induced for 3 h with 0.5 M IPTG. Bacterial cell pellets
obtained by centrifugation (10,000 ⫻ g, 5 min) were suspended in 50 mM
Tris-HCl (pH 7.0)–0.2 M NaCl–10% sucrose–1 mM phenylmethylsulfonyl fluoride and disrupted by sonication for 5 min (15-s pulses alternated with 15 s in
ice). Precipitates were pelleted by centrifugation (10,000 ⫻ g, 5 min), suspended
in 100 mM NaH2PO4–10 mM Tris-HCl, pH 8.0, containing 8 M urea, and
centrifuged, and the supernatant was chromatographed in Ni-nitrilotriacetic acid
(NTA) columns (QIAGEN), according to the manufacturer’s instructions.
rPbMdj1 and rPbLon fractions eluted at pH 4.5 were pooled for further use.
Fractions eluted at pH 6.3 and 5.9 were discarded.
Rabbit immunization, antibody purification, and Western immunoblotting.
Approximately 300 ␮g of Ni-NTA-eluted rPbMdj1 and rPbLon were fractionated in preparative SDS-polyacrylamide gel electrophoresis (PAGE) gels (21)
and stained briefly with Coomassie blue. The correspondent bands were cut off
the polyacrylamide gels, homogenized in PBS, and injected subcutaneously at
several spots on the shaved backs of rabbits. The same procedure was followed
40 days later; 25 days after this booster, the rabbits were bled and the serum was
separated, tested, and aliquoted at ⫺20°C. The rabbits were also bled before
immunization to obtain preimmune sera. Polyclonal rabbit immunoglobulin G
(IgG) anti-rPbMdj1 and anti-rPbLon were purified from both preimmune and
immune sera in protein A-Sepharose (Pharmacia), checked for purity in SDSPAGE gels, pooled, dialyzed in PBS, aliquoted, and frozen at ⫺20°C. The IgG
concentration was estimated by spectrometry at A280. For purification of affinitypurified antibodies, rPbMdj or rPbLon eluted from Ni-NTA columns (150 ␮g, as
estimated in SDS-PAGE gels) were immobilized onto nitrocellulose membranes
(0.45 ␮m) (Amersham) over an area of 12 cm2, which was then quenched with
5% skim milk in PBS (overnight at 4°C) and rinsed with PBS for 10 min under
shaking. The membranes were incubated for 1 h in ice with total immune sera
(1 ml) and/or immune IgG diluted 1:1 (vol/vol) in PBS. The membranes were
382
BATISTA ET AL.
thoroughly rinsed with PBS–0.5 M NaCl, and the bound antibodies were eluted
with 50 mM glycine, pH 3.0 (500 ␮l), which was immediately neutralized with 1
M Tris-HCl, pH 9.0. The protein profile of the eluted material in silver-stained
SDS-PAGE gels indicated the presence of a major IgG band. Preimmune sera
and/or IgG fractions were processed similarly. IgG and affinity purified aliquots
were kept at ⫺20°C.
Western immunoblot reactions with sera from hyperimmune rabbits or patients with PCM were carried out for 3 h at room temperature under shaking,
after which the membranes were washed six times with 0.1% Tween 20 in PBS
and incubated for 1 h at room temperature with goat anti-rabbit or horse
anti-human Ig conjugated to peroxidase (Sigma). The reactions were developed
with an enhanced chemiluminescence kit (Amersham Pharmacia).
Confocal analysis. P. brasiliensis yeast cells growing in aerated liquid cultures
in logarithmic phase (viability, ⬎90%) were collected, washed twice in modified
YPD, and adjusted to a concentration of 2 ⫻ 106 to 3 ⫻ 106 viable cells/ml.
Washed cells were initially incubated with a 20 nM concentration of MitoTracker
Red CMXRos probe (Molecular Probes) for 20 min at 36°C, in order to stain for
active mitochondria, and then rinsed three times with PBS and fixed in cold
methanol for 30 min in the dark. The MitoTracker Red-labeled cells were
incubated with 3% (wt/vol) BSA in PBS (blocking buffer) for 16 h at 4°C, washed
three times with PBS, and then incubated with either 100 ␮g/ml of IgG or 60
␮g/ml of affinity-purified antibodies from polyclonal rabbit anti-rPbMdj and
anti-rPbLon for 4 h at room temperature. Control reactions used preimmune
IgG. The cells were then incubated for 1 h in the dark with 7.5 ␮g/ml of
fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch, West Grove, PA) in blocking buffer. Between each incubation
step with antibodies, the fungal cells were washed six times with PBS. A drop of
10 ␮l of each suspension was mounted on glass slides with Vectashield mounting
medium (VECTOR Laboratories, Inc., Burlingame, CA), and the slides were
sealed with nail polish. Labeling was analyzed using a laser scanning confocal
microscope, LSM-510 NLO (Carl Zeiss, Jena, Germany).
Electron microscopy. P. brasiliensis yeast cells growing in logarithmic phase
(viability, ⬎90%) under aerated conditions were collected by centrifugation,
washed twice with 0.1 M sodium cacodylate buffer, pH 7.2, and fixed with 2%
(wt/vol) formaldehyde and 2.5% (vol/vol) glutaraldehyde in cacodylate buffer for
3.5 h at room temperature under agitation (47). Yeast cells were stored in fixative
at 4°C until use. Cell pellets were solidified in 2.5% (wt/vol) agarose and sliced
at a thickness of approximately 1 to 2 mm. Cell slices were postfixed with 1%
potassium ferrocyanide-reduced osmium tetroxide (1%) in cacodylate buffer for
1 h at room temperature under shaking (56). After two washes with ultrapure
water, the cell slices were transferred to 1% aqueous uranyl acetate for 1 h in the
dark. Finally, the cell pellets were washed twice in water, dehydrated, and
embedded in Spurr resin (Electron Microscopy Sciences). Ultrathin sections (70
to 120 nm) were collected on Formvar/carbon-coated nickel grids, treated for 15
min with saturated sodium meta-periodate, freshly prepared, and washed five
times with ultrapure water. The preparation was quenched with 5% (vol/vol)
acetylated BSA (cBSA) (Aurion) in PBS for 1 h and incubated for 30 min with
a drop of 0.1 M glycine in PBS. The grids were washed three times with PBS
containing 1% cBSA and 0.5% Tween 20 (buffer A) and incubated overnight
with 60 ␮g/ml of rabbit anti-rPbMdj1 IgG in 1% cBSA-PBS (buffer B) in a moist
chamber at 4°C. After five washes with buffer A, the grids were incubated
with 12-nm colloidal gold–AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch) at 1:30 in buffer B for 1 h. The washing step was repeated, followed
by three rinses in PBS and a fixing step with 2.5% glutaraldehyde in PBS for
5 min. The grids were washed five times with ultrapure water and dried. The
ultrathin sections were then contrasted with 2% uranyl acetate and lead citrate.
Control grids were incubated with rabbit preimmune IgG (60 ␮g/ml). The sections were examined in a JEOL 1200 EX-II electron microscope.
Flow cytometry (FACS) analysis. For fluorescence-activated cell sorter
(FACS) analysis, P. brasiliensis yeast cells were prepared as described by Soares
et al. (43), with modifications. Fungal cells were cultivated for 5 days at 36°C in
modified YPD under shaking (viability, ⬎90%), collected by centrifugation,
rinsed, suspended in PBS, and left to rest for 3 min, when clumped cells tend to
sediment by gravity. The supernatant was collected, adjusted to 1 ⫻ 106 cells/ml,
and fixed for 1 h at room temperature with an equal volume of 4% (vol/vol)
paraformaldehyde in PBS, pH 7.2. Fixed cells were precipitated by centrifugation
(1 min at 5,600 ⫻ g), rinsed three times with filtered PBS, and incubated for 30
min in 150 mM NH4Cl to block free amine groups. Quenching was carried out
for 1 h at room temperature, with shaking, in PBS containing 1% BSA (blocking
buffer). The cells were then incubated overnight at 4°C with rabbit IgG antirPbMdj1 (200 ␮g/ml in blocking buffer) or with the same concentration of rabbit
preimmune IgG. Following five rinses with PBS, the fungal cells were incubated
in the dark for 1 h at room temperature in FITC–polyclonal anti-rabbit IgG
EUKARYOT. CELL
(Jackson ImmunoResearch) at 15 ␮g/ml (1:100) in blocking buffer. Labeled cells
were rinsed five times in PBS, suspended in the same buffer (1 ml), and analyzed
in a Calibur FACS (Becton Dickinson, Mountain View, CA). A total of 10,000
cells were analyzed for fluorescence at 492 to 520 nm. Unlabeled control cells
were previously analyzed for autofluorescence, relative cell size, and granularity.
Effect of anti-rPbMdj1 on in vitro growth of P. brasiliensis. The effect of
polyclonal anti-rPbMdj1 IgG on in vitro growth of P. brasiliensis yeasts was tested
in strain 18 cells growing at 36°C for 5 days (viability, ⬎90%) in modified YPD
under agitation. The cells were collected, rinsed in culture medium, and individuated by being passed through a 25-mm needle. The assay was performed as
described by Rodrigues et al. (37), with modifications, in a 96-well culture plate
using 100 to 500 viable yeast cells per well in modified YPD containing 0.5 mg/ml
of ampicillin. The cells were incubated for 48 to 72 h at 36°C in the presence or
absence of different concentrations (25 to 200 ␮g/ml) of either anti-rPbMdj1 or
preimmune IgG. After this incubation period, the same concentration of antibodies was added again and the cultures were further incubated for 3 to 4 days.
The number of CFU and the cell morphology were observed in an inverted
microscope (Olympus CK40).
RESULTS
The PbMDJ1 ORF lies within a 1,897-bp fragment (GenBank
accession number AF334811), where two introns were localized (Fig. 1A). The deduced PbMdj1 protein contains 551
amino acids and has a deduced molecular mass of 58.7 kDa
and a basic isoelectric point of 8.9. In the sequence we can
recognize the J domain and the glycine/phenylalanine-rich and
zinc finger domains (Fig. 1B), which are characteristic of type
I J-domain proteins. Although we found six potential glycosylation sites (Asp-X-Ser/Thr) in PbMdj1, apparently none is
substituted with endoglycosidase H-sensitive oligosaccharide
chains (not shown), suggesting that the molecule does not
go through the secretory pathway. The computer program
TargetP (http://www.cbs.dtu.dk/services/TargetP/ [30]) predicted a mitochondrial targeting peptide within the 28 first
amino acid residues (2.94 kDa). A comparison among fungal
Mdj1 homologues indicated a high percentage of identity
(85%) with sequences from the dimorphs B. dermatitidis and
H. capsulatum, which can be visualized by the similarities in the
hydrophilicity profiles (Fig. 1B). The J domain is the most
conserved region among the Mdj1 homologues analyzed. It
contains four characteristic helixes (reviewed in reference 20)
and a highly conserved HPD tripeptide between helixes II and
III, which is essential for the DnaJ/Hsp interaction (35). In the
C-terminal half of PbMdj1 there is one predicted T-cell mouse
epitope with a minimum of 12 residues (LYTAQIPLTTALL)
between amino acids 379 and 391 (Fig. 1B), which is conserved
in the homologues from B. dermatitidis and H. capsulatum.
We observed an extremely conserved gene organization of
MDJ1 and LON among members of the Eurotiomycetes family,
i.e., P. brasiliensis, B. dermatitidis, H. capsulatum, C. immitis, A.
nidulans, and A. fumigatus. The number and position of the
introns are conserved, although their sizes and sequences may
differ (Fig. 2). MDJ1 from Neurospora crassa and Fusarium
graminearum have the same intron numbers and positions as in
P. brasiliensis, while their LON gene has only one 3⬘ intron. In
Cryptococcus neoformans, both genes bear several small introns. The most interesting finding of our comparative analysis
was the fact that the whole MDJ1/LON locus is conserved in
Eurotiomycetes species, where the genes are adjacent, inversely
oriented, and separated by a common 5⬘ region ranging between 400 and 485 nt (Fig. 2). In these species, a BroA (Bro1)
gene homologue is found adjacent to MDJ1, in the same di-
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383
FIG. 2. Phylogenetic tree of fungal Mdj1 sequences according to the Clustal W program. Besides the fungal sequences indicated in the legend
to Fig. 1, we included those of A. fumigatus (EAL93469), N. crassa (EAA32959), F. graminearum (EAA76137), S. pombe (CAB09769), C. glabrata
(CAG60838), and Homo sapiens (AF061749). A phylogenetic tree of the correspondent Lon sequences showed similar distributions. On the right,
schematic representations of the chromosomal organization and gene direction in the MDJ1/LON locus of the species that bear these genes in the
same chromosome. The distances separating the genes (interrupted line) in N. crassa, F. graminearum, and C. glabrata are, respectively, 236,960
kb, 1,982,719 kb, and 19,091 kb.
rection. In S. cerevisiae, BroA encodes a cytoplasmic protein
involved in the sorting of membrane proteins into the lumenal
vesicles of endosomal multivesicular bodies (34).
We expressed a His-tagged N-terminal region of PbMdj1
(252 amino acids, 25.8 kDa), which included the entire J domain, the Gly/Phe-rich region, and two zinc finger domains.
This fragment contains a number of hydrophilic regions (Fig.
1B) and antibody epitopes, as predicted by the Jameson-Wolf
antigenic index of the Protean module of the Lasergene program (DNAstar Inc.). We additionally expressed a 282-aminoacid His-tagged N-terminal fragment of PbLon (31.7 kDa, between residues 179 and 463), which also contains several peaks
of predicted antibody epitopes. Both truncated recombinant
proteins (rPbMdj1 and rPbLon) were expressed as major insoluble cytoplasmic proteins of calculated molecular masses of
approximately 31 kDa for rPbMdj1 and 36 kDa for PbLon,
including their vector sequences. These values are compatible
with their SDS-PAGE mobilities (Fig. 3A). The correspondent
pH 4.5-eluted SDS-PAGE bands were cut off the gels to immunize rabbits. Anti-rPbMdj1 and anti-rPbLon rabbit immune
sera had high immunoblot titers, of 1:8,000, when tested with
small amounts of the correspondent recombinant proteins, as
seen in Fig. 3B. We therefore used these antibodies in subcellular
localization experiments.
Immunoblot reactions were carried out with both cell lysates
and total mitochondrial proteins from P. brasiliensis yeast cells
growing at 36°C or heat shocked for 60 min at 42°C (Fig. 3C).
Anti-rPbMdj1 immune serum specifically reacted with a single
mitochondrial component of approximately 55 kDa (Fig. 3D),
which is comparable to the molecular mass predicted for the
processed protein lacking the mitochondrial targeting region.
Expression of this component increased in heat-shocked cells,
demonstrating that the PbMDJ1 gene encodes a heat shock
protein in P. brasiliensis. A single 55-kDa band was also seen
when total mitochondrial extracts were captured with immo-
bilized anti-PbMdj1 IgG and then tested by immunoblotting
(not shown). When the same strategy was applied with wholecell extracts, the reaction was negative (not shown), showing
that these antibodies were not cross-reacting with other intracellular components. The anti-rPbLon immune serum recognized a mitochondrial component of approximately 110 kDa,
whose expression also increased in heat-shocked cells, suggesting that PbLON encodes a high-molecular-weight heat shock
mitochondrial proteinase. The estimated molecular mass corresponds well to the PbLon monomer (117.6 kDa) deprived of
the 6.1-kDa mitochondrial targeting region of 54 amino acids.
In nonreducing SDS-PAGE gels (not shown), anti-rPbLon reacted with a mitochondrial component migrating slowly near
the gel top, which is in accordance with the polymeric nature of
the Lon-like proteases. The migration of the component recognized by anti-rPbMdj1 was not changed in the absence of a
reducing agent (not shown). Neither PbLon nor PbMdj1 could
be detected when 20 mg of total cell extracts was tested, possibly because of their small relative concentration in the cytoplasm. The heat shock nature of the mitochondrial proteins
was confirmed at the transcription level. PbMDJ1 and PbLON
RNA bands could be seen in Northern blots with total RNA
extracted from P. brasiliensis yeast cells only after heat shock of
30 or 60 min at 42°C, but not with genetic material isolated
from the starting culture growing at 36°C (Fig. 3E). The estimated sizes of the transcripts (2.05 kb for PbMDJ1 and 3.4 kb
for PbLON) are in agreement with the correspondent genes
and protein monomers labeled with anti-PbMdj1 and antiPbLon (Fig. 3D).
Using confocal microscopy of P. brasiliensis yeast cells growing under aerobic conditions, we observed that anti-rPbMdj1
and anti-rPbLon antibodies reacted with the correspondent
proteins in the mitochondria, as suggested by colocalization
with MitoTracker Red (Fig. 4). Both antibodies (green) and
MitoTracker (red) stained the cytoplasmic compartment with a
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BATISTA ET AL.
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FIG. 3. (A) Coomassie blue-stained 10% SDS-PAGE gels showing insoluble total bacterial extracts from recombinant bacteria expressing
PbMdj1 or PbLon (extract) and the respective proteins eluted from Ni-NTA columns with pH 4.5 buffer. (B) Titration of anti-rPbMdj1 and
anti-rPbLon rabbit immune sera (1:500 to 1:16,000) in immunoblotting. As a substrate, 100 ng of rPbMdj1 (left) or rPbLon (right), as shown in
panel A, was used. NC, negative control with preimmune serum. The same lack of reactivity was observed when testing insoluble bacterial
components from recombinant bacteria with vector alone eluted at pH 4.5 from a Ni-NTA column. (C) Coomassie blue-stained 10% SDS-PAGE
gel profiles of total cell (T) and mitochondrial (M) extracts (20 ␮g per lane) from a P. brasiliensis yeast culture growing at 36°C before and after
heat shock at 42°C for 60 min. (D) Western blot of the gels shown in panel B, probed with the indicated rabbit sera at a concentration of 1:1,500.
SDS-PAGE molecular markers are indicated (in kilodaltons) in panels A through D. (E) Northern blot analysis of total RNA isolated from P.
brasiliensis cells growing at 36°C before and after heat shock at 42°C for 30 and 60 min, using PbMDJ1 and PbLON probes (upper panels). The
band sizes are indicated (in kilobases). Lower panels show ethidium bromide-stained agarose gel replicas of the blotted gels in which the 18S and
28S rRNA components are visible.
punctuated pattern, which merged in tones of yellow and orange. Surprisingly, however, anti-rPbMdj1 antibodies also labeled the whole cell surface, where patches of stronger green
staining could be seen at the budding sites (Fig. 4A). This is
particularly evident in the images of Fig. 4D, where the bud
neck is intensely bright. The longitudinal slice of this image
facilitated a view of a large fluorescent green surface on the
daughter cell. Control images obtained with preimmune antibodies showed only background fluorescence (Fig. 4C). We
tried to label S. cerevisiae, C. albicans, and C. neoformans with
anti-PbMdj1 immune serum, but the reactions were negative,
as were immunoblotting reactions with S. cerevisiae cell extracts (not shown).
Details of the cellular localization of PbMdj1 were further
exploited by transmission electron microscopy using immunogold labeling (Fig. 5). Our preparations had preserved cell
surface and organelle structures (Fig. 5A), and the images show
intense labeling with anti-PbMdj1 antibodies of the doublelayered cell wall. A large number of mitochondria are seen
surrounding the cell membrane (Fig. 5A) yet are not in contact
with it (Fig. 5B and C). The immunogold particles seem to be
clustered inside the mitochondria, but in Fig. 5C they can also
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385
FIG. 4. Localization of PbMdj1 and PbLon by confocal microscopy of P. brasiliensis yeast cells. Red images are active mitochondria stained with
MitoTracker Red. Green images are reactions with affinity-purified anti-rPbMdj1 or anti-rPbLon conjugated with FITC. Control reactions were
carried out with preimmune IgG (panel C, same result for both preimmune sera). Merged images show colocalization of the labels (yellow-orange).
Panel D shows two inner sections of budding cells reacting only with anti-rPbMdj1 (lower panel) or as a merged image (upper panel). Similar
results were obtained with anti-rPbMdj1 IgG or affinity-purified antibodies, while only affinity-purified anti-PbLon showed interpretable images.
be seen near/at the mitochondrial surface and within the thin
space between the mitochondrial surface and the cell membrane. Mitochondria that were not near the cell wall were less
labeled, and the gold particles in the cytoplasm (far from the
cell wall) were scarce. The bud neck in Fig. 5D has a great
concentration of gold particles, in accordance with the intense
fluorescence of this region seen in Fig. 4D. Nonspecific labeling with preimmune rabbit antibodies was reduced to scattered
particles in the cytoplasm and the cell wall (Fig. 5E).
Surface labeling of P. brasiliensis yeast cells was also suggested by FACS. We observed a typical dose-response curve
upon incubation of paraformaldehyde-fixed cells with 50, 100,
or 200 ␮g/ml of anti-rPbMdj1 IgG. At a concentration of 200
␮g/ml, over 50% of the cells were labeled, against 7% of
preimmune IgG (Fig. 6A). In order to characterize the cell wall
component that was reacting with anti-rPbMdj1 antibodies,
cell wall components were released under mild conditions with
␤-mercaptoethanol at an alkaline pH (Fig. 6B). When this
extract was assayed by immunoblotting with anti-rPbMdj1, a
55-kDa component was revealed, which was comparable in size
to the mitochondrial band recognized by the same serum (Fig.
6B). This result adds evidence to the dual mitochondrial and
cell wall localization of PbMdj1 in P. brasiliensis. It is of note
that we have not detected measurable amounts of PbMdj1 in
culture supernatants.
Since the structure of PbMdj1 predicts several antibody
epitopes and one potential T-cell epitope, we questioned
whether paracoccidioidomycosis patients produced antibodies
recognizing the molecule and whether anti-rPbMdj1 would be
able to interfere with fungal growth. We addressed the first
question by testing the reactivity of rPbMdj1 with three patients’ sera by immunoblotting. As seen in Fig. 7, two of them
recognized rPbMdj1 at a 1:200 dilution. The effect of polyclonal anti-rPbMdj1 IgG on in vitro growth of P. brasiliensis
yeasts was tested in strain 18 cells, as described in Materials
and Methods. Under these conditions, there was no inhibitory
effect on the growth pattern or CFU counts compared with
controls (Table 1).
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BATISTA ET AL.
EUKARYOT. CELL
FIG. 5. Ultrastructural localization of PbMdj1 in P. brasiliensis yeast cells incubated with IgG fractions (60 ␮g/ml) of either anti-rPbMdj1
(A through D) or preimmune control (E). Each panel shows a different cell. (A) Panoramic photomicrograph. (B and C) Details. Note labeling
inside the mitochondria (M, arrows) and in the cell wall (CW). In panel C, labeling is also visible in the region between the cell membrane and
the mitochondrion. (D) The bud neck is intensely labeled.
DISCUSSION
The present work demonstrates the presence of a mitochondrial member of the J-domain protein family, Mdj1, not only in
the mitochondria but also in the cell wall of the pathogenic
dimorphic fungus P. brasiliensis. To our knowledge, this is the
first description of a DnaJ member on the cell surface. The
unexpected cell wall localization of PbMdj1 was visualized in
the fungal yeast cells using both confocal and electron microscopy and was confirmed by FACS analyses. The protein was
labeled with rabbit polyclonal antibodies (IgG and affinitypurified fractions) raised against a recombinant PbMdj1 containing the N-terminal half of the protein. Evidence for the specificity of the immunological reactions with PbMdj1 comes from
the following observations: (i) the anti-rPbMdj1 antibodies specifically recognized in immunoblotting, before or after antibody capture of soluble proteins, a single 55-kDa component
from total P. brasiliensis mitochondrial extracts; (ii) the antirPbMdj1 antibodies specifically recognized a single 55-kDa
immunoblotted component from a cell wall extract obtained
from yeast cells of P. brasiliensis with mildly alkaline ␤-mercaptoethanol treatment; and (iii) confocal reactions carried
out with affinity-purified antibodies resulted in a labeling pattern of both the cell wall and mitochondria.
Although the presence of surface J-domain proteins has not
been reported before, the occurrence of extramitochondrial
DnaJ homologues had been predicted by Soltys and Gupta
VOL. 5, 2006
FIG. 6. (A) FACS analysis of P. brasiliensis yeast cells incubated
with 200 ␮g/ml of either anti-rPbMdj1 or preimmune IgG. (B) Coomassie blue-stained SDS-PAGE profile of an alkaline ␤-mercaptoethanol cell wall extract (␤-ME, 40 ␮g), which was also Western blotted
and assayed with anti-rPbMdj1, anti-PbLon, and preimmune (PI) IgG
at a dilution of 1:1,500. A mitochondrial extract (Mito) was run in
parallel and assayed with anti-rPbMdj1. Molecular masses are indicated (in kilodaltons).
(45). The presence of discrete amounts of mitochondrially
sorted Hsp70 and Hsp60 on the plasma membrane, cytoplasmic vesicles, and cytoplasmic granules of mammalian cells has
been well documented (42, 44; reviewed in reference 45).
387
Hsp60 has been detected in small clusters at discrete points on
the H. capsulatum cell wall, and it has been shown to mediate
attachment of the fungus to macrophages via CD11/CD18 receptors (24). Both Hsp70 and Hsp60 need specific cochaperones for optimum chaperone function, which is thus likely to
occur extramitochondrially. Therefore, outside the mitochondria Mdj1 could be assisting Ssc1 (mitochondrial Hsp70), but it
could also be functioning as an independent chaperone. Although an Ssc1 homologue has not been described in fungal
cell walls, an Hsp70-like protein has been detected at the outer
surface of the plasma membrane, throughout the cell wall, and
at the cell surface of C. albicans (25). It will be interesting to
find out if the P. brasiliensis Ssc1, whose gene homologue has
been identified in the database, will also be found in the cell
wall and if it colocalizes with Mdj1.
The fungal cell wall is a dynamic compartment that determines and maintains cell shape but is also actively involved in
many other biological events. Although glucans and chitin are
the main scaffold components of the cell wall, it has a fascinating, complex structure with a number of other constituents
(glycoproteins, proteins, and lipids), such as enzymes, adhesins, and structural and heat shock proteins, whose features
depend on multiple intrinsic and external factors (reviewed for
C. albicans in reference 10). Some of these components from
pathogenic fungi are promising targets for drugs and immunotherapy (31). The interactions among these molecules seem to
involve not only hydrogen and hydrophobic bonds but also
covalent and phosphodiester (mediated by glycosyl phosphatidylinositol) linkages with polysaccharides. In the present study,
we detected abundant labeling of the cell wall with antirPbMdj1 antibodies; however, low yields of the reactive 55kDa protein were obtained upon alkaline extraction with
␤-mercaptoethanol, suggesting that the molecule is not loosely
bound to this compartment. An additional observation was
that when yeast cells were first treated with ␤-mercaptoethanol
and then labeled for FACS analysis, the percentage of fluorescent cells increased instead of going down to background lev-
TABLE 1. Lack of inhibition of P. brasiliensis growth by
anti-rPbMdj1 IgG
Exptl condition
(␮g/ml)a
Culture medium .......................................................................
No. of CFUb
37 ⫾ 5.2
Anti-rPbMdj1
200..........................................................................................38.33 ⫾ 4.7
100..........................................................................................46.75 ⫾ 9.0
50............................................................................................37.75 ⫾ 2.7
25............................................................................................ 38 ⫾ 0.8
12.5......................................................................................... 39 ⫾ 0.8
Preimmune serum
200.......................................................................................... 46.5 ⫾ 3.5
100..........................................................................................41.75 ⫾ 4.9
50............................................................................................42.25 ⫾ 5.7
25............................................................................................43.25 ⫾ 3.1
12.5......................................................................................... 41.6 ⫾ 5.8
FIG. 7. Immunoblot reactivity of three PCM patients’ sera with
rPbMdj1 (note visible bands for patients 1 and 2). Controls were serum
from a healthy individual (⫺) and anti-rPbMdj1 (⫹ [at 1:16,000]).
Human sera were tested at a 1:200 dilution. The PCM sera reacted
with gp43 with immunodiffusion titers of 1:64 and 1:32.
a
Yeast cells (102) were cultivated in the presence of 200, 100, 50, 25, or 12.5
␮g/ml of IgG. After incubation for 48 or 72 h at 36°C, the cultures were supplemented with the same amount of IgG.
b
Numbers of CFU were determined on day 6 of culture. Differences in
numbers of CFU were not statistically significant (P ⬎ 0.05).
388
BATISTA ET AL.
els, as would be expected if PbMdj1 had been totally extracted
(not shown). Therefore, it is likely that the initial peeling
caused by mild alkaline and ␤-mercaptoethanol treatment improved the access of anti-rPbMdj1 antibodies to formerly inaccessible PbMdj1 target molecules. That might also explain
why, although PbMdj1 seems to be actively participating in
yeast cell budding, anti-rPbMdj1 antibodies could not interfere
with fungal growth in vitro within the range of concentrations
tested: other cell components might be blocking antibody access to their target molecules. An example of such a blockage
mechanism has been recently detected in Fonsecaea pedrosoi,
for which anti-monohexosylceramide can react with the cell
wall only when melanin is absent (32). Another possible explanation is that the antibodies cannot recognize PbMdj1 well in
growing cells. On the other hand, genetic manipulation in P.
brasiliensis is still an obstacle to research of P. brasiliensis; until
we have mutants for both Lon and Mdj1, one can only speculate about their role in the fungal cell wall. Another aspect to
be investigated is the functionality of the potential T-cell receptor that is conserved among dimorphs.
A Southern blot of P. brasiliensis DNA restricted with endonucleases and assayed with an E5/E4 PbMDJ1 probe (Fig. 1A)
resulted in a pattern of single bands (not shown), which suggests the presence of a single copy of the PbMDJ1 gene in the
genome, as seen before with the adjacent PbLON gene (2).
This is supported by the previous observations that PbMDJ1
and PbLON were mapped to a unique chromosomal band in 12
individual P. brasiliensis isolates (13). In other fungi analyzed here
for which the genome is completely sequenced (Aspergillus and
Candida), both LON and MDJ1 are present in single copies,
reinforcing those observations. We have not found any evidence for the occurrence of alternative splicing in PbMDJ1: we
visualized only one PbMDJ1 RNA band in Northern blots.
Besides, RT-PCRs using primers designed to elongate the
full-length ORF consistently resulted in a single band of the
same size when we tested different RNA preparations of yeast
cells either growing at 36°C or heat shocked at 42°C (not
shown). Therefore, the components recognized by anti-rPbMDJ1
both in mitochondria and in the cell wall apparently derive
from the same gene and are not a product of differentially
sorted molecules bearing particular targeting sequences due
to alternative splicing. The gene encoding alanine-glyoxylate
aminotransferase from amphibian livers has two alternative
transcription start sites at either side of the 24-nt-long mitochondrial targeting signal, so that the protein encoded by the
long transcript localizes to the mitochondria, while the product
of the shorter transcript is cytosolic (16). We have not been
able to determine the transcription initiation site of PbMDJ1;
however, computer analysis does not predict any internal
translation start site that would result in an ORF lacking the
mitochondrial matrix-targeting signal. Moreover, the similar
SDS-PAGE gel migration of the protein labeled in mitochondria and cell wall extracts with anti-rPbMdj1 suggests that the
molecule was first sorted to that organelle, where it was probably processed by a matrix-processing peptidase into a mature
55-kDa form and then migrated to the cell wall. A matrixprocessing peptidase homologue has been found in the P.
brasiliensis database.
Recent years have seen a growing number of examples of
proteins that are sorted to the mitochondrial matrix and then
EUKARYOT. CELL
apparently exported to an additional compartment(s) of the
cell (extensively reviewed in reference 45). The extramitochondrial destination varies with the molecules, which include antigens, enzymes, receptors, hormones, and chaperones, and
with their specific roles in the destination sites. The mechanism(s) involved in mitochondrial export and its control are so
far speculative, but the endosymbiotic origin of mitochondria
suggests that some ancient bacterial secretory pathways must
have been retained and modified by the organelle (45). It is
noticeable that in a recent proteomics study of plasma membrane lipid rafts, 24% of the 196 identified proteins were
mitochondrial; however, rafts have not been found in that
compartment (1). In our electron microscopic images, mitochondria formed a ring near the cell surface. This pattern has
been mentioned previously for P. brasiliensis yeast cells (19),
and they can be seen in electron microscopic pictures of recently transformed yeasts (7), although in hyphae that pattern
was not evident (6). The image shown in Fig. 5C suggests that
some gold particles, in small clusters, are leaving the mitochondrion and moving toward the cell surface, but it is not clear
whether these clusters are inside vesicles. Thus, the peripheral
localization of mitochondria might not be fortuitous but rather
may suggest that active trafficking of molecules to the cell
surface might be taking place.
We have previously characterized the P. brasiliensis LON
homologue (2). Here we show that PbLON is a heat shock
gene that encodes a 110-kDa mitochondrial heat shock protein. In our confocal analyses, PbLon localized to the mitochondria, showing that the presence of PbMdj1 in the cell wall
does not include helping protein degradation by PbLon. We
observed that the deduced Lon and Mdj1 sequences are remarkably homologous among dimorphic fungi, especially P.
brasiliensis, H. capsulatum, and B. dermatitidis. It will be interesting to find out if Mdj1 localizes to the cell wall of these
microorganisms. We found that the LON/MDJ1 locus organization is comparable among Eurotiomycetes species. The entire
homologous locus probably also includes Bro1, CreA, a little
farther from BroA, and a hypothetical protein adjacent to LON
so far found in A. nidulans, A. fumigatus, and H. capsulatum. In
terms of comparative genomics, this is a relevant finding that
might have evolutionary significance. This is the first conserved
chromosomal organization reported for Ascomycetes, for which
genome sequencing of several species will soon be finished. We
are presently studying the regulatory elements contained in the
5⬘ untranslated region shared by LON and MDJ1, and we hope
to find conserved mechanisms of regulation among fungal heat
shock proteins.
ACKNOWLEDGMENTS
We are thankful to Caroline Z. Romera, Elizabeth N. Kanashiro,
Márcia F. A. Tanakai, André H. Aguillera, and Ronni R. Novaes e
Brito for generous and competent technical assistance. We are grateful
to Soraya S. Smaili for the use of confocal microscopy facilities. We are
also grateful to Márcio L. Rodrigues and Gustavo H. Goldman for
critical reading of the manuscript and to Luiz R. Travassos for always
reminding us that the cell wall is a fascinating dynamic compartment
where one can find any kind of molecule.
This work was supported by FAPESP, CNPq, and Pronex/CNPq.
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Proteinases de P. brasiliensis: conclusões
A serino-tiol proteinase extracelular de P. brasiliensis (PbST) é a enzima do
fungo melhor caracterizada até o momento. O desenvolvimento desta tese ampliou o
conhecimento sobre essa PbST com os seguintes dados:
1. A atividade da PbST sobre o peptídeo Abz-MKALTLQ-EDDnp sofre uma intensa
modulação por açúcares neutros do tipo dextrana, manana de levedura e galactomanana
de P. brasiliensis. A manana de levedura inibiu a interação da proteinase com o substrato,
enquanto a dextrana e a galactomanana de P. brasiliensis aumentaram a afinidade da
enzima pelo substrato. Adicionalmente, a galactomanana fúngica foi capaz de estabilizar
a atividade hidrolítica em temperaturas elevadas e diminuir a Vmáx, o que provavelmente
previne sua autólise antes de atingir o substrato in vivo (artigo 1).
2. Foram desenvolvidos compostos com intenso poder inibitório da PbST sobre AbzMKALTLQ-EDDnp e em ensaios de inibição da aderência celular de LLC-MK2. O
inibidores foram gerados a partir de um substrato peptídico da enzima (MKRLTL)
acoplado com o composto (C)Npys, o qual é capaz de reagir com grupos tiois livre. BzlC(Npys)KRLTL-NH2 apresentou um Ki de 16 nM, Bzl-MKRLTLC(Npys)-NH2 alcançou
160 nM, enquanto (C)Npys inibiu na ordem de 1,7 µM. Os inibidores não foram tóxicos
em ensaios com culturas de células, o que oferece possibilidade de utilização in vivo. A
inibição foi completamente revertida com ditiotreitol, mostrando ser devida à interação
do (C)Npys com um grupo tiol livre posicionado próximo ao sítio ativo, como sugerido
pelo aumento da eficiência de inibição com substrato peptídico (artigo 2).
39
3. Apesar do conhecimento acumulado sobre as características enzimáticas da PbST, sua
identidade continua um mistério. Sua purificação total a partir de sobrenadante de cultura
parece inviável. Utilizamos várias estratégias com o intuito de enriquecer a proteinase e
realizar análises de seqüência por espectrometria de massas ou sequenciamento de Nterminal. Todavia, nenhuma informação conclusiva foi obtida (artigo 2 e anexo 1).
Talvez os inúmeros peptídeos seqüenciados possam ser identificados em futuro breve,
visto que a análise do genoma completo do Paracoccidioides brasiliensis está em
andamento.
O gene da serino proteinase mitocondrial ATP-dependente Lon de P. brasiliensis
(PbST) foi clonado fortuitamente durante as tentativas de clonar a PbST usando
oligonucleotídeos degenerados correspondentes aos sítios ativos de subtilisinas. Sua
caracterização levou à identificação, no mesmo lócus, do gene MDJ1, codante de uma
chaperone mitocondrial que potencialmente participa na seleção de substratos da Lon. O
desenvolvimento desta tese ampliou o conhecimento sobre a PbLon com os seguintes
dados:
1. A PbLon localiza-se somente na matriz da mitocôndria de leveduras do P.
brasiliensis, enquanto a PbMdj1 foi detectada em grandes quantidades também na parede
celular do fungo. A localização em microscopia confocal foi possível a partir de
anticorpos policlonais de coelho anti-PbLon recombinante truncada. A marcação da
PbLon na mitocôndria foi intensa por microscopia confocal (artigo 3) e discreta nos
cortes de microscopia eletrônica de transmissão (anexo 2).
2. O objetivo de expressar a PbLon ativa em sistema heterólogo e com ela selecionar
substratos peptídicos específicos para a enzima foi abandonado. As inúmeras tentativas
40
de expressão da proteinase recombinante ativa foram em vão (anexo 2), possivelmente
por ser tóxica à célula quando superexpressada.
41
Capítulo 2
Moléculas secretadas de Paracoccidioides
brasiliensis: proteínas associadas à parede
celular diferencialmente expressas e
resultados preliminares sobre vesículas
extracelulares
42
Introdução
A produção intracelular de pequenas vesículas contendo proteínas da membrana
plasmática foi descrita há mais de 25 anos na maturação de reticulócitos de carneiro (Pan
et al., 1983, 1985). Sabe-se que durante a maturação de reticulócito para eritrócito há o
desaparecimento de diversas proteínas da membrana plasmática e o principal mecanismo
parece estar relacionado à formação de vesículas lipidoproteicas no endossomo (Pan et
al., 1985; Harding et al., 1984), seguido de fusão com a membrana plasmática e liberação
dos exossomos na corrente sanguínea (Johnstone et al., 1987). Porém ainda não há
descrição sobre a função fisiológica desses exosomos (revisão em Johnstone, 2006). As
proteínas eliminadas pelos exossomos irão depender do tipo celular, porém o mesmo tipo
celular pode apresentar diferenças, como eritrócitos de carneiros que irão reter a maioria
dos seus transportadores de glucose, enquanto em porcos ficam retidos transportadores de
nucleosídeos. Em células de mamíferos, já foi descrita a liberação de exossomos em
células do sistema imune, epitélio intestinal, epidídimo e túbulos do rim (revisão em
Johnstone, 2006). Atualmente os exossomos são definidos como pequenas vesículas
membranosas lipido-proteicas originadas intracelularmente pelo brotamento de
membranas que formam pequenas vesículas (vesículas intraluminais), as quais se
acumulam em corpos multivesiculados. Sua liberação como exossomos ocorre a partir da
fusão da membrana contendo os corpos multivesiculados com a membrana plasmática e
seu tamanho varia entre 20 e 150 nm (revisões em Février e Raposo, 2004; Johnstone,
2006; Li et al., 2006).
Os exossomos contém citosol e expõem o domínio intracelular de receptores.
Classicamente, podem ser obtidos através de filtrações e centrifugações diferenciais de
43
sobrenadantes de cultura e são precipitados por uma centrifugação final a 100.000 x g por
1 hora. Em gradientes de sacarose, os exossomos alojam-se na densidade entre 1,13 e
1,21 g/mL e esse passo pode ser introduzido para eliminar vesículas associadas não
especificamente a proteínas e/ou lipídeos extracelulares. Sua visualização somente pode
ser feita através de microscopia eletrônica (revisão em Johnstone, 2006). Recentemente,
foi proposto que os exossomos constituem um novo mecanismo de secreção para o
exporte de Hsp70, a qual é desprovida de peptídeo sinal e tem, como outras Hsp,
propriedades imunorregulatórias (Lancaster e Febbraio, 2005).
Além de eliminar proteínas do citosol, os exossomos desempenham importantes
funções imunológicas. Exossomos de células dendríticas maduras são 2 ordens de
grandeza mais efetivos que exossomos de células dentríticas imaturas em indução de
células-T antígeno específicas (Segura et al., 2005). Verificou-se também que exossomos
de células dendríticas estimuladas com antígenos de tumor possuem uma resposta
antitumoral in vivo (Quah et al., 2005), porém esse efeito não foi específico para o tipo
de tumor. Sprent (2005) mostrou que exossomos podem ser imunogênicos para células T
CD8+ mesmo na ausência de células apresentadoras de antígeno. Recentemente
exossomos têm sido usados no tratamento de pacientes com melanoma, pois se sabe que
eles possuem um complexo funcional MHC classe II – peptídeo capaz de promover
resposta imune e rejeição de tumor (revisão em Chaput et al., 2005).
Em bactérias gram-negativas também foi observada a liberação de vesículas
extracelulares de 50 a 250 nm formadas diretamente a partir da membrana externa. A
secreção de produtos em bactérias patogênicas gram-negativas é o principal meio de
comunicação e intoxicação das células do hospedeiro. As vesículas são produzidas em
44
tecidos infectados e participam na colonização, carregando fatores de virulência e
modulando a defesa e resposta imune do hospedeiro. Também já foram detectadas
vesículas em fluidos corpóreos demonstrando sua capacidade de disseminação para
diversos pontos. Algumas bactérias utilizam vesículas para destruir outras bactérias em
situação de competição por nutrientes ou até mesmo transferir material benéfico, como
enzimas de resistência a antibióticos. Por participar de tantas funções é considerado um
potencial fator de virulência em bactérias gram-negativas (revisão em Kuehn e Kesty,
2005).
Em nosso laboratório foi especulada a possibilidade da serino-tiol proteinase
(PbST) ou algum modulador positivo da enzima ser veiculado ao meio extracelular por
vesículas secretadas pelo fungo. Em algumas oportunidades foi observado aumento na
atividade enzimática após congelamentos e degelos sucessivos de sobrenadante, o que
poderia levar à lise de estruturas lipídicas. Estruturas semelhantes aos exossomos
observadas em Trypanosoma cruzi foram capazes de provocar uma intensa resposta próinflamatória de macrófagos murinos via receptor Toll-like 2 (Torrecilhas et al., em
revisão). Em fungos, vesículas extracelulares foram recentemente detectadas em
Cryptococcus neoformans (Yoneda e Doering, 2006; Rodrigues et al., 2007). Além da
parede celular, esse fungo possui uma cápsula composta polissacarídica que é essencial
para a virulência (Chang e Kwon-Chung, 1994), porém o mecanismo de tranporte dessas
macromoléculas nunca foi esclarecido. Recentemente Rodrigues e colaboradores (2007)
evidenciaram e caracterizaram vesículas extracelulares com 50 a 400 nm de diâmetro
contendo alta quantidade de glucoronoxilomanana (GXM), o principal polissacarídeo da
cápsula de C. neoformans. Essas vesículas também foram purificadas a partir de
45
macrófagos infectados com o fungo, demonstrando que sua produção pode ocorrer “in
vivo”. É importante ressaltar que em ensaios com células mortas não houve liberação de
vesículas para o meio extracelular, descartando a possibilidade dessas estruturas serem
meros artefatos de células lisadas. Em fungos, as vesículas possivelmente estão
relacionadas com a secreção de proteínas secretadas que não possuem um sistema
clássico de secreção. Sabe-se que há diversas proteínas citosólicas localizadas na parede
celular de fungos que não possuem peptídeo sinal (revisão em Chaffin et al., 1998).
A parede celular de fungos é uma estrutura dinâmica formada principalmente por
polissacarídeos de quitina e glucana, mas contem também proteínas, glicoproteínas e
lipídeos. Diversas funções são atribuídas à parede celular, entre elas manter o balanço
osmótico, o formato celular, promover proteção mecânica e morfogênese (revisão em de
Groot et al., 2005). O estudo detalhado desta estrutura é importante por 2 motivos: é o
primeiro compartimento em contato com estruturas e com o sistema imune do hospedeiro
e está ausente de células de mamíferos, tornando-se um ótimo alvo farmacológico. Em C.
albicans os dois assuntos mais abordados na literatura em relação à parede celular são a
presença de adesinas, pois a adesão é um pré-requisito para o estabelecimento da infecção
e seu potencial de modular a resposta imune (revisão em Chaffin, 1998).
Em Ascomycetos a parede celular é formada por uma bicamada constituída
principalmente por polímeros de carboidratos e em menor concentração por proteínas,
glicoproteínas e lipídeos. Em Neurospora crassa, a camada externa é alcali-solúvel
formada de fibrilas e glicoproteínas, enquanto a interna é formada por polímeros de β1,3-glucana e quitina (Mahadevan e Tatum, 1967). Em C. albicans, a camada
polissacarídica interna é formada pela ligação covalente de β-1,3-glucana à β-1,6-glucana
46
e quitina. Classicamente as proteínas de parede celular estão covalentemente ligadas às
pequenas cadeias laterais de β-1,6-glucana via âncora de glisosilfosfatidilinositol (GPI)
ou diretamente às cadeias de β-1,3-glucana, como é o caso das proteínas Pir (“protein
with internal repeats”). As proteínas da família Pir, que são O-glicosiladas, podem ser
extraídas com NaOH em condições brandas, assim como as proteínas ALS (“alkali
linkage sensitive”). Manoproteínas ligadas covalentemente por pontes dissulfeto podem
ser extraídas por tratamento com agente redutor. Outras proteínas, que são glicosiladas,
possuem peptídeo sinal e não são covalentemente ligadas, podem ser extraídas com água
quente ou detergentes iônicos como SDS. Nos últimos anos diversas proteínas
citoplasmáticas resistentes a esses tratamentos têm sido detectadas na parede celular, e
diversas não possuem peptídeo sinal. Especula-se que são exportadas por uma via não
convencional (revisões em Chaffin et al., 1998; de Groot et al., 2005; Ruiz-Herrera et al.,
2006, Nombela et al., 2006).
As proteínas de parede celular estão relacionadas com diversas funções como
porosidade, retenção de água, proteção contra estresse, manutenção, adesão, formação de
biofilme, antigenicidade, retenção de ions de ferro, hidrofobicidade e virulência. Yun e
colaboradores (1997) observaram que a superexpressão das proteínas Pir em S. cerevisiae
produz um efeito protetor contra a osmotina, uma proteína de defesa em plantas. O
proteoma de parede celular de C. albicans também revelou 3 proteínas GPI-ancoradas
que possivelmente contribuem com a virulência: uma delas é uma proteína ligadora de
grupo heme que poderia facilitar a sobrevivência em condições limitantes de íons ferro e
duas superóxido dismutases que agiriam contra ataques oxidantes de macrófagos (de
Groot et al., 2004; Fradin et al., 2005). Além das proteínas covalentemente ligadas, há
47
importantes proteínas que estão associadas à parede celular como a lacase presente na
camada externa da parede celular de C. neoformans, que é capaz de transformar
substratos fenólicos em melanina (Zhu et al., 2001).
Em P. brasiliensis, poucas proteínas associadas à parede cellular foram descritas,
incluindo duas adesinas (Gonzalez et al., 2005), uma gliceraldeído-3-fosfato
desidrogenase (Barbosa et al., 2006) e gp43 (Vicentini et al., 1994), todas com
capacidade de ligação com componentes da matriz extracelular, além de uma chaperone
mitocondrial PbDnaJ1 (Batista et al., 2006), e melanina (Silva et al., 2006).
48
Proteomic analysis of differentially expressed cell wall-associated
proteins from the human pathogen Paracoccidioides brasiliensis
Alisson L. Matsuoa, Ernesto S. Nakayasub, Luciane Ganikob , Kátia C. Carvalhoa,
Igor C. Almeidab, Rosana Pucciaa,*
a
Department of Microbiology, Immunology and Parasitology, Cell Biology Division, Federal
University of São Paulo, 04023-62, São Paulo, Brazil
b
Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA
49
Racional
O estudo de proteínas ligadas covalentemente ou associadas à parede de P.
brasiliensis é pobre. Até o momento foram descritos apenas um homólogo da chaperone
Mdj1 (Batista et al., 2006), duas adesinas, uma de 32 kDa (Gonzales et al., 2005) e a
gliceraldeído-3-fosfato desidrogenase (Barbosa et al., 2006) com capacidade de ligar
proteínas da matriz extracelular, além de melanina (Gomez et al., 2001). Algumas das
proteínas encontradas na parede poderiam estar transientemente localizadas nesse
compartimento inseridas em vesículas com destino extracelular. O estudo de vesículas
extracelulares no fungo é inexistente. Neste capítulo apresentamos um manuscrito
contendo as análises de proteoma de frações da parede celular de dois isolados de P.
brasiliensis e o estudo preliminar de vesículas extracelulares do fungo.
50
Abstract
This work aimed at identifying differentially expressed cell wall-associated
proteins (CWAPs) in the dimorphic human pathogen P. brasiliensis. We compared
isolates Pb18 and Pb3, yeast phase, cultivated in F12/glu supplemented or not fetal
bovine serum (FBS). These isolates belong to distinct phylogenetic groups and evoke
different types of immune response and disease progression in the B.10A mouse model.
To obtain cell wall preparations, debris from mechanically disrupted cells were
extensively washed with salt to remove contaminants and a fraction of proteins attached
by disulfides bonds was removed with dithiotreitol. CWAPs were chemically extracted
with NaOH or HF-pyridine, and digested with trypsin. The resulting peptides were
identified by mass spectrometry (LC-MS/MS), validated by Phenyx and Mascot
softwares, and quantified using an exponentially modified protein abundance index. Only
6 serum proteins were detected. We analyzed sixty fungal CWAPs that were identified by
a minimum of 4 peptides in at least one sample. The most abundant were translation
elongation factor 1, hsp70 and histone H2B. The amount of ribosomal proteins, hsp70,
hsp90, catalase, calmodulin, actin and several metabolic proteins increased when both
isolates were cultivated in FBS. Among the CWAPs prevalent in Pb18, thiol-specific
oxidant and glyceraldehyde-3-phosphate dehydrogenase might help explain Pb18 higher
virulence. Prevalence of gp43 in Pb3 cell wall, on the other hand, might contribute to its
faster elimination and decreased virulence. When compared to Pb18, the Pb3 change of
protein profile in serum suggested that it might be less adapted to host conditions.
51
Introduction
Paracoccidioides brasiliensis is a thermodimorphic fungus responsible for
paracoccidioidomycosis (PCM). PCM is a systemic granulomatous mycosis prevalent in
Latin America, where at least 10 million individuals might be infected by inhalation of
conidia (San-Blas et al., 2002). Within the pulmonary alveolar epithelium, the infectious
particles can establish infection once they transform into the parasitic yeast form. The
dimorphic transition appears to be a general requirement for pathogenicity of dimorphic
fungi (Nemecek et al., 2006). In P. brasiliensis, the transition from mycelium to yeast is
accompanied by several changes in the cell wall, such as an increase in chitin content and
a shift in the anomeric structure of the β-1,3- (alkali insoluble) to α-1,3-glucan (alkali
soluble), as reviewed by San-Blas et al. (1993), besides reorganization of membrane
glycosphingolipids (Levery et al., 1998).
The gp43 is a dominant immunodiagnostic antigen from P. brasiliensis found
predominantly in the culture supernatant (Puccia and Travassos, 1991), but also partly
associated to the cell wall (Straus et al., 1996). This glycoprotein is capable of binding to
extracellular matrix-associated components, such as murine laminin (Vicentini et al.,
1994; Gesztesi et al., 1996) and fibronectin (Mendes-Giannini et al., 2006), being
therefore a possible virulence factor. On the other hand, it bears protective T-cell epitopes
that are vaccine candidates (Taborda et al., 1998) and might be used in immunotherapy
associated with chemotherapy (Marques et al., 2006). The PbGP43 gene has up to 6
distinct genotypes (Morais et al., 2000), where the most polymorphic are characteristic of
52
a small group of isolates recently classified as phylogenetic criptic species PS2,
according to a multilocus study conducted by Matute et al. (2006). So far this group
includes Brazilian clinical isolates Pb3, Pb4, BT84, Uberlândia, Venezuelan Pb2 and
armadillo T10B1 (Matute et al., 2006). In a B10.A mouse model, isolates Pb2, Pb3 and
Pb4 evoked regressive PCM, while isolates from the main species S1, where Pb18 is
included, were consistently more virulent (Carvalho et al., 2005). Isolates from the
Colombian phylogenetic group PS3 have not yet been compared to the others in terms of
virulence.
The cell wall of most fungal pathogens is the outermost layer of contact with the
immune system and other host structures; hence its contents are fundamental to determine
the faith of the infection. The cell wall is a dynamic compartment composed mainly of
structural polysaccharides (chitin and glucans), with fewer percentages of proteins,
glycoproteins and lipids. It is responsible for osmotic balance, cell shape, mechanical
protection and morphogenesis. In Candida albicans, an internal layer of β-1,3-glucans is
covalently linked to β-1,6-glucan and chitin. Cell wall proteins (CWPs) are responsible
for remodeling, adhesion, biofilm formation and antigenicity. Classical CWPs are
covalently
linked
either
to
β-1,6-glucan
short
side
chains
via
a
glycosilphosphatidylinusitol (GPI) anchor or directly to the long β-1,3 glucan chain. This
is the case of a family of proteins with internal repeats (Pir) that are highly Oglycosylated. Pir proteins can be extracted with weak NaOH treatment, which also
extracts other alkali-sensitive proteins (ASL). GPI-anchored proteins are sensitive to HFpyridine, while mannoproteins, covalently linked by disulfide bonds, are extracted with
reducing agents. Other proteins, which are glycosylated and bear a signal peptide, are
53
non-covalently linked to the cell wall and are sensitive to extraction with hot water,
chaotropic agents or ionic detergents. A growing number of proteins have been reported
that are covalently linked, and hence resistant to these treatments, do not possess known
signal peptides or sugar moieties and are originally cytoplasmic proteins. The type of
association to or function at the cell wall is not clear, and it has been speculated that they
are transported through a non-conventional export pathway (reviews in Ruiz-Herrera et
al., 2006; Nombela et al., 2006).
In P. brasiliensis, few cell wall-associated proteins have been identified,
including two adhesins (Gonzalez et al., 2005), a glyceraldehyde-3-phosphate
dehydrogenase (Barbosa et al., 2006), and gp43 (Vicentini et al., 1994), all capable of
binding extracellular matrix-associated proteins. Another important protein associated
with the cell wall is the enzyme laccase, responsible for melanin synthesis (Silva et al.,
2006). In addition, our group has recently identified a mitochondrial member of the J
family co-localized to the cell wall (Batista et al., 2006).
In this study, we focused on the proteome of cell wall-associated proteins
(CWPAs). We evaluated by mass spectrometry total CWPAs from fractions extracted
with mild alkali and HF-pyridine of the isolates Pb18 and Pb3 grown in the presence and
in the absence of bovine fetal serum.
54
Materials and methods
Fungal isolates and culture conditions. We analyzed P. brasiliensis isolates 18 (Pb18)
and Pb3 (as in Morais et al., 2000; original name 608; B26 in Matute et al., 2006). Fungal
yeast cells were recovered from the lungs of mouse infected intratracheally, as described
(Carvalho et al., 2005), and maintained short-term at 36oC in solid modified YPD (0.5%
bacto-yeast extract, 0.5% casein peptone and 1.5% dextrose, pH 6.5) before use. For cell
wall extraction, yeast cells were recovered from liquid cultures (900 mL) grown for 7
days (late log phase) in F12 defined medium (Difco)/1.5% glucose (F12/glu)
supplemented or not with 5% fetal bovine serum (FBS), at 36oC, with agitation at 120
rpm in a rotary shaker. For adaptation to the culture medium, fungal cells from solid
medium were first grown in the same conditions (100 mL, 6 days) as pre-inocula before
being transferred to a larger volume of fresh medium.
Cell wall purification. Yeast cells grown in liquid F12/glu supplemented or not with 5%
FBS were microscopically analyzed for purity and viability (> 95%) with Tripan blue.
Fungal cells (10 ml of wet cells) were collected by centrifugation (1,300 x g for 5 min)
and washed 5 times with an excess amount of ice-cold buffer A (PBS, 0.1 M EDTA, 1
mM PMSF, pH 7.5). Washed cells were suspended in the same buffer and mechanically
disrupted with glass beads (425-600 µm, Sigma), at a proportion of 1:2:1 (v:v:v, wet
cells: buffer: glass beads), using a vortex (10 times for 30 sec, alternating with 30-sec
intervals in ice). Cells debris containing the wall fraction were separated from
cytoplasmatic contents (supernatant) by centrifugation at 1,300 x g for 5 min. Cell walls
55
were then washed with ice-cold buffer A another 47 times (1 x 1,300 x g for 5 min; 1 x
1,300 x g for 10 min; 15 x 16,000 x g for 10 min and 30 x 16,000 x g for 15 min),
according to a standard protocol for fungal cell wall isolation (Previato et al., 1979).
Washed cell walls were incubated for 1 hour at 37oC with buffer A containing 50 mM
DTT (dithio-threitol) to release proteins associated or aggregated by S-S bonds. Purified
cell walls were washed 3 extra times in ice-cold milliQ water (16,000 x g, 15 min) and
then lyophilized.
Cell wall protein extraction and fractionation. Cell wall-associated proteins (CWAPs)
sensitive to mild alkali extraction, including covalently linked Pir proteins, were released
by incubating purified cell walls (100 mg) with 30 mM NaOH (5 mL) at 4oC for 16 h.
The reaction was interrupted by adding acetic acid (8 µl), then the suspension was
centrifuged (16,000 x g) and filtrated through a sterile 0.22-micron filter. Samples were
dried in a speed vac apparatus and stored at -20oC prior to trypsin digestion. CWAPs
sensitive to acid treatment were released by HF-pyridine extraction. Purified cell walls
(100 mg) were mixed in a vortex with 500 µL of pure HF-pyridine (70% HF in 30%
pyridine, Fluka) in ice, and incubated for 3h in ice. The reactions were diluted with 9,5
mL of milliQ water and immediately desalted in a POROS R1 cartridges. Briefly, the
cartridges were prepared with 1 mL 40mg/mL POROS 50 R1 resin (Applied Biosystems,
Foster City, CA) in a solid-phase extraction support, washed with methanol and npropanol and equilibrated with 0.05%. Then the sample was loaded and washed with
0.05% TFA, before elution with 80% acetonitrile (ACN) containing 0.05% TFA. Proteins
were dried in a speed vac apparatus and stored at -20oC until trypsin digestion.
56
Sample preparation for mass spectrometry (MS) analysis. Protein digestion was
carried out as described by Stone & Williams (1996). Extracted cell wall-associated
proteins were dissolved in 40 µL 400 mM NH4HCO3, pH 8.0, containing 8 M urea and
the disulfide bonds were reduced by adding 10 µL 50 mM DTT and incubating for 15
min at 50oC. Then the cysteine residues were alkylated using 10 µL 100 mM
iodoacetoamide (IA) for 30 min, at room temperature, in the dark. Then the reaction was
diluted with HPLC-grade water to obtain a final concentration of 1 M urea. The digestion
was performed with 4 µg trypsin, for 24 h at 37oC. The reaction was terminated by
adding 1 µL pure formic acid (FA) and the peptides were desalted in a POROS R2
ziptips. The ziptips were prepared using 50 µL of a 40 mg/mL POROS 50 R2 resin
(Applied Biosystems, Foster City, CA) suspension in 2-propanol into 200-µL
micropipette tips. After equilibrating the ziptips with 0.05% TFA, peptide samples were
loaded and washed with 0.05% TFA before eluting with 80% ACN/0.05% TFA.
Peptides were 2-fold diluted in strong cation-exchange (SCX) buffer (25%
ACN/0.5% FA) and fractionated using a POROS 50 HS ion exchange ziptip. The ziptips
were prepared as the POROS R2, except that 20 µL of POROS 50 HS resin (50%
suspension in 20% ethanol) was added instead. The ziptips were equilibrated with SCX
buffer prior to loading the samples. Then the samples were washed with the same buffer
and eluted with increasing NaCl concentrations dissolved in SCX buffer (25, 50, 100, 200
and 500 mM). All fractions were dried in speed vac, desalted in POROS R2 ziptips, dried
in speed vac again and stored at -20oC until analysis.
57
Mass spectrometry analysis and peptide validation. Tryptic peptides were
dissolved in 30 µL of 0.1% F, and subjected (1 µL) to liquid chromatography-mass
spectrometry (LC-MS) analysis. LC was performed in a PepMap reversed-phase column
(15 cm x 75 µm, 3-µm C18, LC Packings, Dionex) coupled to an Ultimate (LC Packings,
Dionex) nanoHPLC. Bound peptides were eluted with 5-35% ACN/0.1% FA over 100
min and directly analyzed in a Q-tof 1 (Micromass, Waters) mass spectrometer. Spectra
in positive-ion mode were collected in the 400–1800 m/z range, and each peptide was
fragmented (MS/MS) for 3 seconds in the 50–2050 m/z range. MS/MS data were
converted into peak lists (PKL format) using ProteinLynx 2.0 (Waters). MS peaks were
smoothed twice with 5 channels using Savitzky-Golay method, and centered with 4
channels in top 80%. For the MS/MS peaks a threshold of 5% was set. After smoothing
twice with 3 channels in Savitzky-Golay method, the peaks were centered with 4
channels in top 80% and converted into monoisotopic ions. Converted peak lists were
submitted to database search analysis using Phenyx (GeneBio) and Mascot (Matrix
Science) softwares against the NCBInr and NCBInr fungi databases. The following
parameters were used: (i) enzyme: trypsin, (ii) one missed cleavage allowed, (iii) fixed
modification: carbamidomethylation of cysteines, (iv) variable modification: oxidation of
methionine, (v) peptide tolerance: 1000 ppm, (vi) MS/MS tolerance: 0.5 Da.
The
validated Mascot peptides had a minimum ion score of 48 when using the entire NCBInr
database or 37 and above using the NCBInr fungi database. Peptides validated by Phenyx
had a minimum ion score of 7.0.
58
Relative quantification of proteins. In order to estimate absolute protein
contents in a complex mixture we used the exponentially modified protein abundance
index (emPAI) as described by Ishihama et al. (2005). PAI is an index defined as the
number of observed peptides for an individual protein divided by the number of
observable (theoretical) peptides for that protein upon digestion with a proteinase of
known cleavage site. The number of observable peptides upon trypsin digestion was
estimated with the PepitideMass software (http://ca.expasy.org/tools/peptide-mass.html),
setting as parameters the same conditions used in our experiments. For absolute
abundance, PAI was converted to exponentially modified PAI (emPAI) following the
equation: 10PAI – 1.
Results
Identification of cell wall-associated proteins (CWAPs). We isolated, fractionated and
identified trypsin-cleaved peptides from total P. brasiliensis CWAPs that were sensitive
to individual extractions with NaOH or HF-pyridine. We compared isolates Pb18 and
Pb3 grown in defined F-12/glucose medium supplemented or not with 5% FBS.
Considering that P. brasiliensis cell wall preparations have not been boiled in SDS before
fractionation, our peptide analysis identified not only integral cell wall proteins, but also
those associated with other proteins or carbohydrate moieties, thus justifying the term
“cell wall-associated proteins”. The peptide sequences revealed by mass spectrometry
were scored and validated using Phenyx or Mascot softwares, which generated a list of
proteins containing identical peptides. Many peptides were detected in both NaOH and
59
HF-pyridine fractions. Hence, we merged peptide data from these fractions in order
simplify the analysis of the most abundant CWAPs in each isolate and growth condition.
In addition, we only took into consideration proteins identified by four or more peptides
in at least one of the four samples analyzed (Pb18 or Pb3 in F12/glu; Pb18 or Pb3 in
FBS). The number of peptides identifying each protein can be seen in Table 1 (in
parenthesis). Most of the proteins have identical peptides to fungal homologues, and 17
derive from the P. brasiliensis databases.
Under those stringent parameters, we have identified a total of sixty CWAPs
(Table 1), from which thirty-five were detected in both Pb18 and Pb3 irrespective of
growth condition (Fig. 1). Eight CWAPs were found exclusively in cells cultivated in the
presence of serum. Among them, 3 were common to both isolates (cytochrome c oxidase
subunit VIa, initiation factor 4A, mitochondrial carrier protein), 3 were detected only in
Pb18 (fungal transcription factor, polyketide synthase-related protein, hypothetical
protein) and 2 only in Pb3 (avidin, hypothetical protein). No exclusive protein was found
when the isolates grew only in F12/glu (Figure 1).
Quantitative analysis of CWAPs. The results of emPAI abundance, calculated as
detailed in Materials and Methods, can be seen in Table 1. According to these
calculations, about 75% of total abundance (from the four samples) scored 0.5 or lower,
24% were between 0.5 and 1.0 and only 1% were higher than 1.0.
The most abundant CWAP was by far a homologue of translation elongation
factor 1, followed by heat shock 70 and histone H2B. Two hsp70 family members were
CWAPs in P. brasiliensis, including a mitochondrial one (gi3124921), which was not the
60
most abundant. Other mitochondrial proteins have been identified, such as, putative
cytochrome c oxidase (subunits IV and VIa), carrier protein Ggc1, hsp60, probable
mitochondrial F1 ATPase (subunit alpha) and cpn10. Mitochondrial and ribosomal
proteins have previously been shown to be cross-linked to cell wall components
(Bowman et al., 2006).
It is relevant to point out that we detected only 6 FBS proteins in our samples,
specifically, gamma globin, hemoglobin alpha-1 subunit, alpha-2-HS-glycoprotein,
vitronectin, inter-alpha-trypsin inhibitor and apolipoprotein A-I (Table 2). The small
number and differential distribution between isolates suggests that they might be
specifically bound to cell wall components, but can be displaced under relatively mild
conditions.
Effect of FBS on in vitro expression of CWAP. We used the emPAI values of
abundance (Table 1) to calculate relative values that reflected how growth in 5% FBS
changed the pattern of CWAP expression comparatively in Pb18 and Pb3. The results
were then used to build a color-coded graph (FBS/F12), as explained in the legend of Fig.
2, where each protein of Table 1 is represented. Among the 60 CWAPs analyzed, 22 were
more abundant (green pallet) in both isolates cultivated in FBS, including a series of
ribosomal proteins, hsp60, hsp70 and hsp90, catalase, calmodulin, actin and several
metabolic proteins. The summation of emPAI values from all CWAPs of P. brasiliensis
(both isolates) grown in FBS was 65.48, while the values calculated for cells grown in
F12/glu totalized 38.36, suggesting that serum components evoke general overexpression
of proteins. Total emPAI was considerably higher for Pb18 than PB3 (28.69 x 17.69)
61
when both isolates were cultivated in the absence of serum. When growing FBS,
although Pb18 and PB3 contribute equally well to the total emPAI of 65.48, the number
of individual proteins whose abundance increased was higher in Pb3 (42) than in Pb18
(36). Additionally, more proteins had decrease abundance (red pallet) in Pb18 (17) than
in Pb3 (4), suggesting that these isolates have distinct regulatory mechanisms at the
transcription and/or translation/post translation levels, which ultimately might account for
a distinct host/parasite relationship.
Differentially expressed CWAPs in Pb8 and Pb3. In order to identify more clearly the
CWAPs differentially expressed in Pb18 and Pb3 in terms of abundance, we analyzed
Table 1 data comparing the values according to the isolate (Pb18/Pb3, in FBS or F12), as
seen in Fig.2. When both culture conditions are considered, 20 proteins were more
abundant in Pb18 (green pallet), including some ribosomal proteins, Hsp90, actin,
glyceraldehyde-3-phosphate dehydrogenase, thiol-specific antioxidant and catalase. Only
4 CWAPs were more abundant in Pb3 (red pallet) in both culture conditions, specifically,
Hsp70, triosephosphate isomerase, aconitase and mannitol-1-phosphate dehydrogenase.
When yeast cells cultured in FBS are analyzed, 21 CWAPs were relatively more
abundant in Pb3 than in Pb18 (e.g. gp43, enolase, calmodulin, polyubiquitin, plasma
membrane ATPase), while 33 CWAPs were prevalent in Pb18. Some of these proteins
are discussed below.
62
Discussion
The present work aimed at identifying cell wall-associated proteins (CWAPs)
differentially expressed in two P. brasiliensis isolates (Pb18 and Pb3) grown in the
presence or in the absence of fetal bovine serum (FBS). Pb18 and Pb3 have distinct
phylogenetic background (Matute et al., 2006) and prompt opposite types of disease
progression in a B.10A mouse model (Carvalho et al., 2005). To remove contaminants,
isolated cell walls were extensively washed with salt (Previato et al., 1979), and a
fraction of proteins attached by disulfide bonds was removed with dithiotreitol. We
analyzed CWAPs that have been removed under mild NaOH and HF-pyridine extraction
and blended the results because many of them were found in both fractions. Since
previous hot water and/or detergent removal (de Groot et al., 2004) have not been
undertaken, we assume that the detected proteins are bonded either covalently or not.
That could also justify why our list of proteins does not contain many previously reported
covalently-linked CPWs (de Groot et al., 2004), whose accessibility might be hampered
under our experimental conditions. Alternatively, P. brasiliensis yeast cells do not
necessarily contain the same C. albicans proteins on the cell wall. On the other hand, the
lack of complete genome information for P. brasiliensis does not presently allow for a
complete analysis of the data.
Among the CWAPs prevalent in Pb18, thiol-specific oxidant (TSA) and
glyceraldehyde-3-phosphate dehydrogenase (GADPH) were 2-fold more abundant in
Pb18 than in Pb3 grown in FBS, and that might be relevant for the Pb18 increased
virulence. P. brasiliensis GADPH has previously been detected at the cell wall by
63
immunoelectron microscopy, and found to be involved in the initial colonization and
fungal dissemination due to its capacity to bind fibronectin, type I collagen and laminin
(Barbosa et al., 2005). Assays using P. brasiliensis previously incubated with polyclonal
anti-GADPH or recombinant GADPH promoted an inhibition of adherence and
internalization by pneumocytes in vitro. In C. albicans, TSA was differentially detected
on the cell surface of the pathogenic mycelial form (Urban et al., 2003), and it seems to
be indispensable for the yeast-mycelial transition under oxidative stress (Shin et al.,
2005). In P. brasiliensis, the TSA1 gene was preferentially expressed in the pathogenic
yeast phase (Marques et al., 2004) and presently found associated with the cell wall. TSA
might play a key role in P. brasiliensis survival under an unfavorable oxidative
environment within phagocytes, especially for Pb18, which would then evoke a more
successful infection.
We interestingly detected a polyketide synthase-related protein only in Pb18
grown in serum. This enzyme is involved in the dihydroxynaphtalene (DHN)-melanin
pathway, whose precursor is acetate (Bell e Wheeler, 1986). Melanin is an important
fungal virulence factor (reviewed by Nosanchuk and Casadevall, 2006), and was also
found in P. brasiliensis, where melanized cells were poorly phagocytosed by
macrophages and were less sensitive to antifungal drugs (Silva et al., 2006). Although a
homologue for the laccase gene has not yet been found in P. brasiliensis EST databases,
yeast cells are able to synthesize a melanin-like pigment in the presence of a L-3,4dihydroxyphenilalanine (L-DOPA) precursor. Conidia, on the other hand, can produce it
also in the absence of L-DOPA, suggesting the existence of an alternative pathway that
could take acetate as precursor. Two members of a putative kinase regulator family, 14-
64
3-3-like protein and protein 2 were 2-fold more abundant in Pb18 than in Pb3 cultivated
in FSB. The relevance of this difference will be further investigated.
Cytoskeleton proteins have seldom been described associated with the cell wall.
We presently detected β-tubulina only in Pb18 (alkaline fraction), and actin abundantly
distributed in all samples. Both had increased amounts when the isolates were cultivated
in FSB, especially β-tubulina (3.5-fold). Actin is a structural protein intimately related
with S. pombe cell wall synthesis. Reverting protoplasts revealed that new cell wall
formation co-localizes with spots of actin, and treatment with cytochalasin D depleted
cell wall formation (Kobori et al., 1989). A structure called filasome was found to be
composed by a vesicle of 35-70 nm that is surrounded by fine filaments containing actin
(Takagi et al., 2003). The filasome follows an actin filament pathway and fuses with the
plasma membrane releasing the vesicle that probably carries cell wall material. That
could explain the finding of actin in our cell wall preparations, and increase of actin and
tubulin on the cell wall of P. brasiliensis cultivated in serum could reflect increased
protein transportation. Tubulins are the main constituents of microtubules, which are
responsible for many functions including segregation of genetic material, maintenance of
cell shape and intracellular transport of proteins, lipids and organelles (review in Palmer
et al., 2005).
In addition to the surface Pb18 proteins that could aid this isolate to establish a
more successful infection than Pb3, two FBS proteins were differentially bound more
abundantly to Pb18 surface, specifically, vitronectin and apolipoprotein. Vitronectin,
which is involved in the regulation of blood coagulation (Dahlbäck et al., 1987), has also
been found on cell wall extracts of C. albicans (Jakab et al., 1993), possibly bound
65
through a specific cell wall receptor (revision in Chaffin et al., 1998). Considering that in
C. albicans interaction with vitronectin increased binding to and phagocytosis by
macrophages, it might help the microorganism to establish infection (Limper et al.,
1994). The main function of lipoproteins is to transport lipids and lipid-soluble
molecules. There is no previous report on cell wall-bound apolipoprotein A-I, however
bovine apolipoprotein A-II presents an antimicrobial effect against E. coli and S.
cerevisiae (Motizuki et al., 1998). The serum apolipoprotein was detected only in Pb18
cell wall fraction extracted with HF-pyridine, but its relevance is obscure.
Two serum proteinase inhibitors, inter-alpha-trypsin (a serpin) and alpha-2-HSglycoprotein (cystatin), were found associated to the P. brasiliensis cell wall. These
inhibitors could play a role in neutralization of fungal proteinases potentially important
for fungal infection, dissemination or nutrition. Presently, we have not found any cell
wall-associated proteinases in our experimental conditions; however inter-alpha-trypsin
was detected associated with only Pb3 cell wall, possibly inhibiting a proteinase not
secreted by Pb18.
Gp43 has been detected in small amounts in the acid-extracted cell wall fraction.
In Pb18, it has been detected only in the sample grown in F12/gluc, while its contents
increase 4-fold in Pb3 cultivated in FSB. We can speculate that its presence in Pb3 cell
wall in vivo might contribute to its more effective clearance by the immune system,
resulting in regressive PCM in B10.A, as opposed to Pb18 (Carvalho et al., 2005).
Polyubiquitin was detected in comparable amounts in the alkali-extracted cell wall
fraction of both Pb18 and Pb3 grown in the absence of serum, but only Pb3 had
detectable amounts when cultivated in serum-supplemented medium, at a 2-fold increase.
66
In the cytoplasm, polyubiquitin classically tags proteins for proteolysis by proteasomes,
but it is now understood that polyubiquitin is involved in many other cellular processes,
which include tagging of membrane proteins before internalization (reviewed in Aguilar
and Wendland, 2003). Its finding on the cell wall remains to be elucidated.
Calmodulin was 3-fold more abundant in Pb3-FBS than in Pb18-FSB.
Calmodulin is a small calcium-binding protein that plays an important role as modulator
of intracellular calcium signaling, and participates in the regulation of gene expression in
response to external stimuli, including stress (Kraus and Heitman, 2003). In P.
brasiliensis, calmodulin seems to be essential for the mycelial-to-yeast-transformation
(Carvalho et al., 2003). Overall, Pb3 seemed to expose at the cell wall fewer proteins than
Pb18 when growing in defined medium and then respond more fiercely to the presence of
serum. That could explain why a potent mediator such as calmodulin prevailed in Pb3.
We have recently shown that, when compared to Pb18, Pb3 has a slower transcriptional
response of some HSP genes to heat shock (Batista et al., 2007). It is possible that a
slower transcriptional response results in a crippled adaptation of Pb3 to the host, which
will in turn more readily eliminate it.
We have detected in P. brasiliensis many cell wall-associated proteins lacking
known secretion signal, which have previously been reported in C. albicans and S.
cerevisiae (reviewed by Nombela et al., 2006). They include glycolysis proteins like
enolase, GADPH, fructose 1,6-bisphosphate aldolase, stress proteins like Hsp70, Hsp60,
Hsp90, and others like catalase, TSA, cytochrome c, 14-3-3-like protein, translation
elongation factors. We presently detected proteins from different intracellular
compartments. Histones, typically nuclear, have previously been microscopically
67
localized to H. capsulatum cell wall and anti-histone monoclonal antibodies protected
against experimental histoplasmosis (Nosanchuk et al., 2003). We found several
mitochondrial proteins, including Hsp 60. In H. capsulatum, cell wall hsp60 was shown
to bind to macrophage complement receptor type 3 (Long et al., 2003) and it protects
vaccinated BALB/C and C57BL/6 (Deepe et al., 1996; Deepe and Gibbons, 2001). Antirecombinant Hsp60 have been found in the sera from several paracoccidioidomycosis
patients (Izacc et al., 2001).
Two hsp70 members were abundantly found associated with the P. brasiliensis
cell wall from both Pb3 and Pb18. Recently, Batista et al. (2006) detected an Mdj1
homologue both in cell wall and mitochondria, where it is originally localized. It was the
first time that a protein from the J family was found on the cell wall but its role in this
compartment is speculative. Since it is responsible for mitochondrial hsp70 regulation in
yeast, finding of mitochondrial hsp70 on the cell wall suggests that the chaperone
Mdj1/hsp70 complex could be active also at the cell wall. Presently we have not found
Mdj1 in alkali or acid cell wall fractions, probably because this molecule was washed out
during DTT extraction, as seen to occur with whole cells (Batista et al., 2006).
In conclusion, by using a proteomics approach we identified cell wall-associated
proteins that are differentially expressed in Pb18 and Pb3 growing in the presence of
serum that might help explain their distinct degree of virulence in the mouse model.
68
Legends
Figure 1: Graphic representation of the 60 CWAPs analyzed in this work.
Figure 2. Color representation of the relative abundance of each protein shown in Table
1, calculated as follows: a) FBS/F12 - influence of serum in the change of abundance for
Pb18 and Pb3. Values for growth in FBS were divided by the correspondent control
values in the absence of serum (F12); b) Pb18/Pb3 - comparison between Pb18 and Pb3
cultivated both in the presence or in the absence (F12) of FBS. Red to dark red (values
between 0.5 and 0.99) indicate quantitative decrease in FBS or lower abundance in Pb18,
while dark green to green (values between 1.0 and 2.0) indicate serum-related increase in
the amount of CWAPs or higher abundance in Pb18. Unaltered abundance (1.0) is
represented in black. When peptides for a particular protein were absent (emPAI = 0) the
correspondent protein was considered 2 times repressed or abundant (0.5 or 2.0,
respectively).
69
Figure 1.
Matsuo et al., 2007
70
Figure 2
Matsuo et al., 2007
71
AC
Pb 18
emPAI
Pb18
Serum
Pb 3
Pb3
Serum
Protein
similar to eukaryotic translation elongation factor 1
alpha 1
gi|82995146 8.33 (32) 7.11 (30) 4.72 (25) 5.58 (27)
heat shock protein 70 [Paracoccidioides brasiliensis]
gi|14538021 1.12 (28) 2.16 (43) 2.71 (49) 3.25 (54)
histone H2B [Rosellinia necatrix]
gi|27531291 2.16 (12) 1.87 (11) 0.96 (7) 2.48 (13)
HHF2p [Candida glabrata]/histone 4
gi|21668067
actin
gi|66804921 0.85 (12) 1.64 (19) 0.76 (11) 1.27 (16)
ATP synthase F1, beta subunit
gi|77685559 0.80 (13) 1.25 (18) 0.80 (13) 0.80 (13)
Grp1p [Exophiala dermatitidis]
gi|82571189 0.75 (10) 1.08 (13) 0.06 (1) 0.85 (11)
1.93 (7) 1.15 (5) 1.15 (5) 1.15 (5)
calmodulin
gi|168777
0.13 (1) 0.44 (3) 0.13 (1) 1.98 (9)
bipA [Aspergillus awamori]
ribosomal protein S15, putative [Aspergillus
fumigatus Af293]
hypothetical protein MG00221.4 [Magnaporthe grisea
70-15]/ 40S RIBOSOMAL PROTEIN S7
histone H3 [Fusarium proliferatum]
40S ribosomal subunit protein S9 [Cryptosporidium
hominis]
putative cytochrome c oxidase subunit VIa
[Paracoccidioides brasiliensis]
70 kDa heat shock protein [Paracoccidioides
brasiliensis]
gi|2582633
0.69 (19) 0.56 (16) 0.40 (12) 0.95 (24)
gi|66852980
0.62 (4) 0.62 (4) 0.27 (2) 0.83 (5)
gi|39975265
gi|5918753
0.65 (7) 0.78 (8) 0.15 (2) 0.54 (6)
1.51 (6) 0.00 (0) 0.00 (0) 0.58 (3)
gi|54656881
0.12 (1) 0.78 (5) 0.41 (3) 0.58 (4)
gi|50428882
0.00 (0) 1.40 (8) 0.00 (0) 0.39 (3)
gi|31324921 0.51 (19) 0.44 (17) 0.21 (9) 0.51 (19)
thiol-specific antioxidant [Ajellomyces capsulatus]
gi|14161441
0.41 (4) 0.67 (6) 0.29 (3) 0.29 (3)
malate dehydrogenase [Paracoccidioides brasiliensis]
gi|47119068 0.54 (10) 0.36 (7) 0.14 (3) 0.54 (10)
14-3-3-like protein 2 [Paracoccidioides brasiliensis]
putative cytochrome c oxidase subunit IV
hypothetical protein [Neurospora crassa
OR74A]/Chaperonin 10 Kd subunit
hypothetical protein AN5800.2 [Aspergillus nidulans
/eukaryotic ribosomal protein L18
gi|38569374
0.18 (3) 0.73 (10) 0.32 (5) 0.32 (5)
gi|50428890 0.37 (3) 0.23 (2) 0.37 (3) 0.52 (4)
gi|3088571 0.39 (14) 0.46 (16) 0.12 (5) 0.46 (16)
gi|85079266
0.58 (4) 0.41 (3) 0.00 (0) 0.41 (3)
gi|67539260
0.45(5)
0.45 (5) 0.00 (0) 0.45 (5)
polyubiquitin [Cicer arietinum]
glyceraldehyde-3-phosphate dehydrogenase
gi|24817262
0.31 (2) 0.00 (0) 0.31 (2) 0.72 (4)
gi|30995493
0.37 (7) 0.50 (9) 0.25 (5) 0.20 (4)
gi|60656557 0.24 (11) 0.49 (20) 0.10 (5) 0.43 (18)
14-3-3-like protein [Paracoccidioides brasiliensis]
gi|30351156
0.51 (7) 0.43 (6) 0.13 (2) 0.19 (3)
thioredoxin [Paracoccidioides brasiliensis]
ADT_NEUCR ADP,ATP CARRIER PROTEIN
(ADP/ATP TRANSLOCASE)
gi|34980254
0.15 (1) 0.15 (1) 0.78 (4) 0.15 (1)
gi|42551288
0.48 (7) 0.32 (5) 0.12 (2) 0.25 (4)
72
SconC [Paracoccidioides brasiliensis]
peptidyl-prolyl cis/trans isomerase [Paracoccidioides
brasiliensis]
gi|61608602
0.29 (3) 0.29 (3) 0.09 (1) 0.41 (4)
gi|34979129
0.28 (3) 0.51 (5) 0.28 (3) 0.00 (0)
hypothetical protein MG04489.4 [Magnaporthe grisea gi|39945014
0.00 (0) 0.00 (0) 0.00 (0) 1.05 (5)
ribosomal L10 protein [Paracoccidioides brasiliensis]
unnamed protein product [Aspergillus
oryzae]/mitochondrial F1 ATPase subunit alpha
gi|28797723
0.17 (3) 0.37 (6) 0.23 (4) 0.17 (3)
gi|83765516
0.21 (7) 0.35 (11) 0.15 (5) 0.18 (6)
catR [Aspergillus niger] Catalase
gi|840716
Conserved ribosomal protein P0 similar to rat P0,
human P0, and E. coli L10e; shown to be phosphory
gi|6323371
unnamed protein product [Aspergillus
oryzae]/Ribosomal_L7
gi|83774731
fructose 1,6-biphosphate aldolase 1 [Paracoccidioides
brasiliensis]
gi|29826036
0.13 (3) 0.39 (8) 0.00 (0) 0.33 (7)
Plasma membrane ATPase (Proton pump)
Triosephosphate isomerase [Clostridium
acetobutylicum ATCC 824]
0.17 (2) 0.37 (4) 0.08 (1) 0.17 (2)
0.30 (4) 0.22 (3) 0.14 (2) 0.14 (2)
0.13 (2) 0.19 (3) 0.06 (1) 0.34 (5)
gi|728908
0.23 (7) 0.13 (4) 0.06 (2) 0.27 (8)
gi|15893999
0.00 (0) 0.13 (2) 0.19 (3) 0.34 (5)
avidin
40S ribosomal protein S3 [Chaetomium globosum
CBS 148.51]
gi|451889
0.00 (0) 0.00 (0) 0.00 (0) 0.65 (5)
gi|88183746
0.00 (0) 0.28 (5) 0.16 (3) 0.16 (3)
enolase [Cryphonectria parasitica]
gi|40806812
0.25 (5) 0.05 (1) 0.14 (3) 0.14 (3)
gi|2293
0.12 (2) 0.47 (7) 0.00 (0) 0.00 (0)
gi|71000467
0.29 (5) 0.29 (5) 0.00 (0) 0.00 (0)
40S ribosomal protein S18 [Coccidioides immitis RS] gi|90307829
0.21 (3) 0.29 (4) 0.00 (0) 0.07 (1)
hypothetical protein [Yarrowia lipolytica]
ribosomal protein S0.e.B, cytosolic [Aspergillus
fumigatus Af293]
gi|50556244
0.12 (2) 0.25 (4) 0.00 (0) 0.18 (3)
gi|70998726
0.05 (1) 0.23 (4) 0.05 (1) 0.17 (3)
elongation factor 2 [Neurospora crassa]
COG0515: Serine/threonine protein kinase [Nostoc
punctiforme PCC 73102]
gi|13925370
0.04 (2) 0.17 (8) 0.06 (3) 0.15 (7)
gi|23124801
0.05 (1) 0.21 (4) 0.05 (1) 0.10 (2)
glycoprotein gp43
electron transfer flavoprotein, subunit beta imported
[Aspergillus fumigatus Af293]
gi|1588394
0.15 (3) 0.00 (0) 0.05 (1) 0.20 (4)
gi|70998364
0.06 (1) 0.27 (4) 0.00 (0) 0.06 (1)
gi|3661614
0.00 (0) 0.05 (2) 0.15 (6) 0.15 (6)
gi|83952823
0.07 (2) 0.04 (1) 0.04 (1) 0.19 (5)
gi|28797565
0.00 (0) 0.00 (0) 0.04 (1) 0.27 (6)
gi|83767463
0.20 (4) 0.09 (2) 0.00 (0) 0.00 (0)
gi|32414453
0.00 (0) 0.17 (4) 0.00 (0) 0.08 (2)
beta-tubulin [Neotyphodium coenophialum]
cytosolic small ribosomal subunit S4 [Aspergillus
fumigatus Af293]
aconitase [Aspergillus terreus]
ATP synthase subunit A [Roseovarius nubinhibens
ISM]
mannitol-1-phosphate dehydrogenase
unnamed protein product [Aspergillus oryzae]/60S
ribosomal protein L8
EUKARYOTIC INITIATION FACTOR 4A (EIF4A) (EIF4A) [Neurospora crassa]
73
putative mitochondrial carrier protein Ggc1 [Candida
albicans SC5314]
fungal specific transcription factor [Aspergillus
fumigatus Af293]
hypothetical protein [Neurospora crassa N150]
COG3321: Polyketide synthase modules and related
proteins [Burkholderia mallei GB8 horse 4]
gi|68484497
0.00 (0) 0.20 (4) 0.00 (0) 0.05 (1)
gi|70981502
0.00 (0) 0.11 (5) 0.00 (0) 0.00 (0)
gi|85082883
0.00 (0) 0.08 (4) 0.00 (0) 0.00 (0)
gi|67640326
0.00 (0) 0.08 (6) 0.00 (0) 0.00 (0)
Table 1. Associated CWPs identified by LC-MS/MS of isolates Pb18 and Pb3 grown in
F12/glu supplemented or not with FCS. The relative abundance of proteins was recorded
as emPAI based on the number of peptides (in parenthesis) detected for each protein. The
listed proteins are organized from the most to the least abundant protein.
74
Serum Proteins
AC
gi|393
Pb 18
NaOH
8
Pb 18
HF
11
Pb 3
NaOH
4
Pb 3
HF
10
gamma globin
Hemoglobin alpha-1 subunit
gi|122272
0
4
0
5
Vitronectin
gi|73587073
5
3
3
0
inter-alpha-trypsin inhibitor
gi|27806743
0
0
4
0
apolipoprotein A-I, apoA-1
gi|245563
0
8
0
0
alpha-2-HS-glycoprotein
gi|27806751
6
2
5
2
Table 2. FBS proteins bound to cell wall of isolates Pb18 and Pb3. The number of
peptides detected by LC-MS/MS is displayed for each protein.
75
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79
Moléculas secretadas de Paracoccidioides
brasiliensis: resultados preliminares sobre
vesículas extracelulares
80
Materiais e métodos
Gel filtração em coluna com Sepharose CL-4B
Uma coluna de gel filtração (16 mm de diâmetro por 40 cm de altura da
Amersham) contendo 70 mL de Sepharose CL-4B (separação entre 20.000 kDa a 60 kDa
- Amersham) foi previamente equilibrada a 4oC com 5 volumes de tampão acetato de
amônio 100 mM, pH 7,3, em fluxo constante de 4 mL/hora, utilizando uma bomba
peristáltica. Amostras de 2 mL de sobrenadantes de cultura de P. brasiliensis concentrado
eram aplicadas na coluna e frações de 1 mL recolhidas com um coletador automático para
posterior análise.
ELISA
A detecção de componentes de alto peso molecular no sobrenadante de cultura de
P. brasiliensis foi realizada através de ELISA, em placas de 96 cavidades (Nunc,
MaxiSorpe surface). Os poços eram sensibilizados com as vesículas adicionados de 50
µL de tampão carbonato/bicarbonato 200 mM pH 9,6 e incubados a 4°C por uma noite
(o/n). Em alguns experimentos as vesículas foram tratadas com metaperiodato de sódio
(reagente que oxida epitopos de carboidratos) em 3 concentrações diferentes (5, 10 e 20
mM). As placas eram lavadas 5 vezes com PBS acrescido de 0,1% de Tween-20 (PBS-T)
e incubadas com PBS-Molico 5% (100 µl/poço) por 3 h à temperatura ambiente. Após 3
lavagens com PBS-T, os antígenos eram incubados por 2 horas a 37oC com 100 µl de
PBS-Molico 5% contendo soro de paciente com PCM (1:1000). Após 5 lavagens com
PBS-T, eram adicionados 100 µl de PBS-Molico 5% contendo anticorpo de cabra anti-
81
IgG de humano (1:1000) ou anticorpo de macaco anti-IgG de coelho (1:1000), ambos
conjugados com HRP (Amersham), e incubados por 1h a 37°C. Depois de 5 lavagens
com PBS-T, era feita uma única lavagem com 200 µL de tampão bicarbonato 50 mM, pH
9,6. A revelação era feita por quimioluminescência, com o uso de luminol (ECL reagent,
Pierce) diluído 1:20 em tampão bicarbonato 50 mM, pH 9,6, preparado de acordo com
instruções do fabricante. A leitura era feita em luminômetro de microplacas (Cambridge
Technology, Watertown, MA, Modelo 7710) e expressa como unidades de relativas de
luminescência (URL).
Purificação de vesículas do sobrenadante de P. brasiliensis
P. brasiliensis isolados Pb3 (nome original: 608), Pb12 (nome original:
Argentina), Pb18 (nome original: 18) ou Pb339 (nome original: B-339) eram crescidos a
36º C, sob agitação, por 7 a 10 dias em meio definido F12 (Invitrogen) acrescido de 1,5%
de glucose (F12glu), e em alguns experimentos complementado com 5% de soro fetal
bovino (SFB), previamente ultracentrifugado a 200.000 g por 4 horas para retirada das
vesículas do soro. Os sobrenadantes de cultura eram filtrados em membrana estéril de
0,22 µm com a ajuda de uma bomba à vácuo e concentrados em sistema AMICON
(Millipore) utilizando as membranas YM10 com limite de exclusão de 10.000 Da ou
BIOMAX (PBMK 076 10 – Millipore) com limite de exclusão de 300.000 Da. A amostra
era lavada com 20 mL de tampão contendo 100 mM de acetato de amônio pH 7,3,
concentrada até um volume final de 2 mL e aplicada na coluna de gel filtração de CL-4B.
Alternativamente, o concentrado era equilibrado em 200 mL de PBS, concentrado a um
volume final de 10 mL e submetido a uma ultracentrifugação por 4 horas a 100.000 x g, a
82
4º C. O precipitado contendo vesículas era liofilizado (para análise de proteínas) ou
estocado em geladeira por período curto (para análises funcionais). Toda a manipulação
era feita a frio. A viabilidade das culturas, recolhidas em fase logarítmica ou logarítmica
tardia de crescimento, era estimada em >90% pelo método de exclusão com Tripan blue
em microscópio ótico, com o qual adicionalmente se determinou a pureza das
preparações.
Gradiente de sacarose para purificação de vesículas de P. brasiliensis
Um precipitado de 100.000 x g conservado a 4oC foi dissolvido em 5 mL de uma
solução 2,6 M de sacarose em 20 mM de Tris pH 7,2 e colocado em um tubo de
ultracentrífuga (12 mL) específico para o rotor SW41 (Sorvall). Acima da amostra foi
adicionado um gradiente linear de 2,0 a 0,25 M de sacarose em 20 mM de Tris pH 7,2, e
a separação foi realizada através de uma ultracentrifugação de 270.000 x g por 16 horas.
As amostras foram recolhidas a partir do fundo do tubo em frações de 1 mL, adaptado-se
uma bomba peristáltica com uma pipeta Pasteur de vidro em um fluxo de 1 mL/min. As
frações recolhidas foram estocadas em geladeira.
Precipitação com ácido tricloroacético (TCA)
Para precipitação de proteínas com TCA, as amostras eram adicionadas de 10%
de TCA e conservadas por 20 min no gelo. Após centrifugar por 20 min a 4º C em
microcentrífuga (14.500 rpm), o sobrenadante era descartado e o precipitado lavado com
1 mL de acetona gelada (estocada a – 20º C). A amostra era novamente centrifugada e o
precipitado seco em speed vac.
83
Digestão de proteínas em solução aquosa/orgânica para análise em espectrometria
de massas (LC-MS/MS)
A digestão de proteínas em solução foi realizada conforme Russel (2001) e Goshe
(2003), com modificações. Proteínas em quantidade de até 100 µg eram dissolvidas em
100 µL de solução contendo metanol e 50 mM de bicarbonato de amônio pH 8,0 (v/v,
60/40) em tubo de rosca. A amostra era incubada por 5 min a 95oC e deixada resfriar à
temperatura ambiente. A redução das proteínas era realizada com 5 mM de dithiotreitol
(DTT) a 37oC por 30 min, seguida de alquilação utilizando 10 mM de iodoacetamida por
90 min no escuro. A amostra era diluída 5x no mesmo tampão e as proteínas digeridas
com tripsina em uma razão de 1/50 (wt/wt) de enzima/substrato por 16 h a 37oC. A
reação era interrompida com uma concentração final de 0,05% de ácido trifluoroacético
(TFA), a amostra seca em “speed vac” e dessalinizada em “ziptip” de fase reversa.
Dessalinização em “ziptip” de fase reversa para análise em LC-MS/MS
Um pequena quantidade de fibra de vidro era adicionada a uma ponteira de 200
µL para evitar vazamento da resina de fase reversa POROS 50 R2 (40 mg/mL em
isopropanol). A coluna era ativada com 5 volumes de metanol, equilibrada com 5
volumes de TFA (0,046%) e a amostra equilibrada na mesma solução era aplicada. A
coluna era lavada com 5 volumes de TFA 0,046%, e eluída com 5 volumes de
acetonitrila 80% contendo 0,046% de TFA. As amostras eram secas em speed vac e
analisadas em LC-MS/MS.
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Fracionamento de peptídeos em ziptip de troca-iônica
Fibra de vidro era adicionada a uma ponteira de 200 µL para evitar vazamento da
resina POROS HR50 (interação catiônica forte). Após o empacotamento, a resina era
equilibrada com 5 volumes de tampão (EB) contendo 0,5% de ácido fórmico e 25% de
acetonitrila e a amosta em EB era aplicada. Os peptídeos não ligados eram lavados com 5
volumes de EB e a eluição realizada com as concentrações de 25, 50, 100, 200 e 500 mM
de NaCl em EB. A dessalinização era realizada em “ziptip” de fase reversa.
Análise de peptídeos por LC-MS/MS
As amostras previamente secas em speed vac eram ressuspendidas em 30 µL de
ácido fórmico (AF) 0,1%, dos quais 1 µL era aplicado para análise em LC-MS. A
cromatografia dos peptídeos era realizada na coluna de fase reversa PepMap (15 cm x 75
µm, 3µm, LC Packings) em um nanoHPLC Ultimate (LC Packings), com um
autosampler Famos (LC Packings). O gradiente para eluição dos peptídeos era de 0 - 40%
de solvente B (80% acetonitrila/AF 0,1%) em 100 min, ou de 5 - 50% de solvente B em
30 min (para amostras provenientes de digestão em gel).
Os peptídeos eluídos da coluna eram analisados diretamente no espectrômetro de
massas (Q-tof 1, Micromass, Waters). Os espectros no modo MS eram coletados a cada 2
segundos, em uma faixa de 400-1800 m/z e cada peptídeo era submetido a uma
fragmentação (MS/MS) por 3 s, em uma faixa de 50-2050 m/z. Os espectros MS/MS
coletados eram convertidos em peak lists (formato PKL) e analisados com os softwares
Mascot (Matrix Science) and Phenyx (GeneBio) nos bancos NCBInr, P. brasiliensis,
Neurospora e S. cerevisiae.
85
Microscopia eletrônica
Os procedimentos de inclusão, ultramicrotomia e reações de imunocitoquímica
em leveduras de P. brasiliensis foram realizados no Centro de Microscopia Eletrônica
(UNIFESP), sob a coordenação da Dra. Edna F. Haapalainen e em colaboração com a
pós-doutoranda Luciane Ganiko do nosso laboratório na época.
Preparações de vesículas ou leveduras do isolado Pb339 crescido em meio F12glu
em fase logarítmica de crescimento eram lavadas em tampão cacodilato de sódio 0.1 M,
pH 7.2 (TC) e fixadas em solução de Karnowisk modificado (glutaraldeído 2.5%, v/v,
paraformaldeído 2%, m/v, em TC) durante 3½ h à temperatura ambiente, sob agitação
constante. As preparações permaneceram no fixador a 4oC até o momento do
processamento. O precipitado era lavado em TC e incluído em ágar 2.5% (m/v) para
obtenção de fragmentos de ~ 2 mm de espessura. A etapa de pós-fixação era feita com
solução de tetróxido de ósmio 1% (v/v) em TC durante 1 h à temperatura ambiente, sob
agitação constante. Alternativamente, as preparações eram pós-fixadas na presença de
ferricianeto de potássio 1% (v/v) para melhor preservação e resolução dos sistemas de
membranas (Forbes et al., 1977). Parte das amostras era lavada em água ultra-pura (2 x
10 min) para contrastação in bloc com acetato de uranila 0.5% (m/v) durante 30 min,
protegida da luz. As amostras não contrastadas com acetato de uranila eram lavadas em
TC (2 x 10 min).
Posteriormente, as amostras eram desidratadas em concentrações crescentes de
etanol (Merck) 70%, 90% e 100% durante 30 min cada, transferidas para óxido de
propileno (EMS), com 2 trocas de 30 min. Os fragmentos de precipitado celular eram
86
infiltrados com resina Epon (EMS) ou Spurr (EMS), com aumento progressivo da razão
de resina para óxido de propileno (1:1 durante 2 h, 2:1 durante 16 h). Para infiltração com
resina pura, eram realizadas 2 trocas de 2 - 3 h e, posteriormente, os fragmentos eram
transferidos para cápsulas contendo resina. As cápsulas permaneceram na estufa a 65oC
durante 48 h para polimerização da resina. As amostras infiltradas com Epon eram pósfixadas em tetróxido de ósmio sem ferricianeto de potássio.
Seções ultra-finas (70 – 120 nm) obtidas no ultramicrótomo Leica eram coletadas
em grades de níquel resvestidas com formvar e carbono, lavadas, fixadas com
glutaraldeído 2.5% (v/v) e enxaguadas em água ultra-pura (MilliQ). As análises eram
feitas em microscópio eletrônico Jeol JEM 1200EX model II.
87
Resultados e Discussão
•
Análise da preparação de vesículas por “Western blot” e microscopia eletrônica
Amostras de sobrenadante de cultura do P. brasiliensis Pb339 cultivado em meio
F12/glu complementado com SFB depletado de vesículas foram concentradas em
Amicon e ultracentrifugadas a 100.000 x g por 4 horas a 4oC. O precipitado foi
ressuspendido em tampão de amostra para SDS-PAGE, fervido e analisado por “Western
blot”. Na figura 1 mostramos o padrão de bandas reconhecido por soro de paciente com
PCM (1:1000), como revelado por quimioluminescência. Os componentes do precipitado
100.000 x g reconhecidos pelo soro de paciente concentraram-se na faixa de peso
molecular entre 75 e 45 kDa, aproximadamente e foram específicos, como sugerido pela
comparação com a reação negativa obtida com soro de indivíduo normal.
88
Figura 1. Análise por “Western blot” da antigenicidade das moléculas do precipitado a 100,000 x g com
soro de paciente com PCM.
Uma fração semelhante de amostra foi analisada através de microscopia eletrônica
de transmissão. Na Figura 2A podemos observar a presença neste precipitado de diversas
estruturas delimitadas por membranas, aparentemente vesículas, com tamanhos entre 20 e
100 nm, coerentes com o tamanho dos exossomos descritos na literatura. Revendo
algumas fotos de leveduras de P. brasiliensis analisadas por microscopia eletrônica,
pudemos observar também algumas células aparentemente liberando vesículas para o
meio extracelular (Figura 2B - setas). Coincidentemente estas vesículas têm tamanho de
20 a 100 nm de diâmetro, sugerindo que o fungo realmente libera vesículas para o meio
extracelular.
89
Figura 2. Detecção por microscopia eletrônica de transmissão de vesículas liberadas pelo P. brasiliensis no
meio extracelular. A) Sobrenadante de cultura concentrado e ultracentrifugado a 100.000 x g. B) Célula de
P. brasiliensis aparentemente liberando vesículas para o meio extracelular (setas).
•
Cromatografia da preparação de vesículas extracelulares de P. brasiliensis
90
Outra estratégia utilizada para o reconhecimento de estruturas vesiculares
extracelulares foi o fracionamento de sobrenadantes concentrados de cultura de fungo em
uma coluna de Sepharose CL-4B. Previamente, a coluna foi calibrada com 2 marcadores
de peso molecular, a saber, tireoglobulina (~700 kDa) e BSA (dímero com ~135 kDa),
nas mesmas condições descritas em Materiais e Métodos, e as frações analisadas em
espectrofotômetro (Abs280). Na figura 3 podemos observar que o pico de tireoglobulina
está na fração 43, enquanto o dímero de BSA ficou predominantemente na fração 60,
coerente com o resultado esperado.
Figura 3. Calibração de uma coluna de Sepharose CL-4B. Perfil de eluição (A280) de 200 µg de
tireoglobulina (~700 kDa) ou BSA (dímero ~135 kDa) em frações de 1 mL (fluxo de 4 mL/hora).
Cerca de 50 mL de sobrenadante de P. brasiliensis B-339 crescido por 10 dias em
meio F12/glu contendo 5% de SFB foi concentrado em sistema Amicon com membrana
YM10, lavado com 8 mL de tampão contendo 100 mM de acetato de amônio pH 7,3 e
concentrado até um volume final de 2 mL. A amostra concentrada foi fracionada em
91
coluna de Sepharose CL-4B ou ultracentrifugada a 100.000 x g por 4 horas a 4oC, e
estocada.
O fracionamento de sobrenadante concentrado de P. brasiliensis em coluna de
Sepharose CL-4B calibrada pode ser visto na Figura 3. Alíquotas das frações (200 µL)
foram analisadas por ELISA quanto à reatividade com soro de paciente (1:1000) com
PCM. O perfil de reatividade pode ser visto na Figura 4, onde o controle da reação foi
feito na ausência de fungo, ou seja, somente com meio F12/glu contendo ou não SFB,
concentrado e fracionado na mesma coluna. Observa-se que houve uma alta reatividade
do soro com as frações 26 a 33 eluídas a partir de sobrenadante de cultura e, portanto,
específicas para componentes fúngicos. Para uma coluna de 70 mL, o Vo estaria em
torno de 24 mL (fração 24), sugerindo que as frações entre 26 e 33 estão incluídas na
coluna. Essas frações apresentam uma massa molecular acima de 700 kDa (fração 43 –
tireoglobulina), compatíveis com a massa de vesículas (entre 1.000 e 20.000 kDa). Note
que houve uma reatividade de imunoglobulinas de paciente com componentes do soro
(perfil F12+SFB). Sobrenadantes de cultura do fungo cultivado em meio sem soro
apresentaram reatividade específica com componentes do fungo eluídos a partir da
fração 40, os quais podem representar agregados com componentes antigênicos.
92
Figura 4. CL-ELISA das frações eluídas da coluna de Sepharose CL-4B. Os gráficos mostram o perfil da
reatividade, com soro de paciente com PCM, das frações eluídas a partir do sobrenadante de cultura de P.
brasiliensis B-339.
•
Análise de vesículas purificadas em gradiente de sacarose
Um dos nossos objetivos em relação às vesículas extracelulares de P. brasiliensis
é verificar se elas carregam moléculas imunomodulatórias, como já evidenciado em
organismos patogênicos como o T. cruzi (Torrecilhas et al., 2007, em revisão). Com o
93
objetivo de testar frações purificadas do pellet de 100.000 x g de sobrenadante de cultura
do fungo, após ultracentrifugação as vesículas foram fracionadas em gradiente linear
entre 2,6 e 0,25 M de sacarose em Tris pH 7,2 (Wubbolts et al., 2003). Estávamos
particularmente interessados em separar a malha que apareceu entremeada nas vesículas,
como visto na Figura 2A. As frações ímpares foram enviadas para análise no laboratório
do Prof. Carlos P. Taborda, USP. As frações foram usadas para estimular macrófagos de
cultura J774 e no sobrenadante foram dosadas as citocinas IL-10, IL-12 e óxido nítrico
(NO). Observou-se que as frações 7 e 9 foram as mais ativas na indução de NO (Figura
5), enquanto IL-10 e IL-12 não foram detectadas em nenhuma fração (resultado não
mostrado). A constatação preliminar de que vesículas extracelulares do P. brasiliensis
podem ter uma importância imunológica é altamente relevante ao nosso estudo.
94
Figura 5. Detecção da liberação de NO por macrófagos após 24 horas de incubação com as frações
vesiculares provenientes da separação por gradiente linear de sacarose.
•
Proteoma de vesículas de P. brasiliensis
As informações de que o P. brasiliensis aparentemente libera vesículas, as quais
contêm moléculas antigênicas e potencialmente imunorregulatórias, direcionou nosso
interesse imediato em realizar o proteoma desse material. Paralelamente, foi realizada a
análise do proteoma da parede do fungo (manuscrito anexo), não somente pela
importância de conhecer as proteínas associadas a esse compartimento, com também para
estabelecer uma base de comparação com as proteínas de vesículas que necessariamente
devem transitar por esse compartimento antes de alcançarem o meio externo.
Os isolados a serem estudados foram selecionados segundo sua capacidade
secretora e pelo seu grau de virulência. A Pb339 tem sido usada há décadas como fonte
de antígenos extracelulares e purificação de gp43. Geralmente apresenta crescimento
abundante e secreção de inúmeros componentes moleculares. O Pb18 tem sido
igualmente utilizado há décadas em experimentos de infecção de camundongos devido à
sua alta virulência e representa a maior espécie filogenética (S1), segundo Matute et al.
(2006). O Pb12 foi o isolado mais agressivo em experimentos de PCM murina em
camundongos B10.A, para os quais IFNγ estava ausente durante a infecção (Carvalho et
al., 2005). O Pb3 representa uma espécie filogenética distinta (PS2) e tem um padrão
regressivo de PCM em camundongos B10.A (Carvalho et al., 2005; Matute et al., 2006).
O perfil em SDS-PAGE das proteínas contidas no precipitado 100,000 x g de
vesículas pode ser observado na figura 6. As preparações aparecem com vários e
abundantes componentes. Somente as preparações provenientes de Pb3 apresentaram
95
baixa quantidade de proteínas. Isso já era esperado, já que os precipitados de 100.000 x g
eram consideravelmente menores para esse isolado. Apesar das preparações conterem
uma quantidade razoável de proteínas, as digestões com tripsina mostraram poucos “hits”
significativos com o banco de dados. Para validar o proteoma de vesículas serão
necessárias preparações contendo maior quantidade de proteínas. O conhecimento do
genoma completo do P. brasiliensis também facilitará enormemente sua identificação.
Figura 6. Padrão de banda das proteínas do precipitado 100.000 x g secretadas pelo P. brasiliensis Pb18,
Pb12, Pb3 e Pb339 em gel de SDS-PAGE 10% corado pelo método da prata.
•
Marcação da preparação de vesículas com anticorpos anti-alfa galactosil
Um dos nossos objetivos foi detectar um marcador para as vesículas
extracelulares de P. brasiliensis de forma a otimizar sua purificação. Para tal, já foram
testados anticorpos anti-PbLon, anti-gp43 e anti-PbMdj1, porém todos produziram
reações negativas.
96
Em T. cruzi, parte da população de vesículas extracelulares é reconhecida por
anticorpos anti-epitopo alfa-galactosil (anti-Gal), os quais podem ser isolados em grandes
quantidades a partir de soros de pacientes chagásicos (Almeida et. al., 1994). Esses
epitopos estão presentes em cadeias longas de oligossacarídeos O-ligados, principalmente
em resíduos de treonina de mucinas-like do complexo antigênico F2/3 de T. cruzi. O
complexo F2 é composto por glicoproteínas de 74 e 95 kDa e F3 por glicoproteínas de
120 a 200 kDa.
Apesar de epitopos contendo resíduos de α-galactosil não terem sido descritos em
fungos até o momento, foi feita uma reação de “immunoblot” de precipitados 100.000 x g
de sobrenadantes de P. brasiliensis Pb339 com anticorpos anti-Gal. Observou-se uma
reação intensa com um componente de cerca de 70 kDa (Fig. 7) de migração difusa no
gel de SDS-PAGE.
Figura 7. “Immunoblot” com revelação por quimioluminescência mostrando a reatividade de anticorpos
anti-alfa galactosil isolados de soro chagásico com precipitados de sobrenadantes de cultura (100.000 x g)
de P. brasiliensis Pb339: 1, crescido em YPD ou 3, crescido em meio F12/glu. Em 2, componentes do
sobrenadante de cultura < 300.000 Da (não retidos na concentração em Amicon).
97
Ensaios de ELISA utilizando concentrações crescentes do anticorpo (10, 20 e 40
µg/mL) mostraram que a reação foi dose-dependente, como ocorre com o controle
positivo, a fração F2/3 (Fig. 8).
Figura 8. Reatividade em Elisa revelada por quimioluminescência de diferentes concentrações de
anticorpos anti-alfa-galactosil isolados de soro chagásico com precipitados de sobrenadantes de cultura
(100.000 x g) de P. brasiliensis Pb339 (A) ou fração F2/3 (B) de T. cruzi.
Para verificar se os anticorpos reconhecem epitopos de caboidratos, as vesículas
foram tratadas com metaperiodato de sódio em 3 concentrações diferentes (5, 10 e 20
98
mM). Pode-se observar na Fig. 9 que 5 mM do reagente praticamente aboliram a ligação
do anticorpo com o precipitados a 100.000 x g, sugerindo que a reatividade era devida a
epitopos de galactose.
Figura 9. Reatividade em Elisa, revelada por quimioluminescência, de anticorpos anti-alfa galactosil (20
mg/mL) isolados de soro chagásico com precipitado de sobrenadantes de cultura (100.000 x g) de P.
brasiliensis tratado com diferentes concentrações de metaperiodato de sódio (NaIO4).
Um ensaio de inibição com diversos açúcares em concentração de 0,3 M
incubados com o anticorpo primário mostrou inibição principalmente com d(+)-galactose
(45%), seguida de methyl-β-D-xilopiranosídeo (38%) e methyl-α-D-galactopiranosídeo
(35%) (figura 10).
99
Figura 10. Reatividade em Elisa, revelada por quimioluminescência, de anticorpos anti-alfa-galactosil (20
mg/mL) isolados de soro chagásico com precipitado de sobrenadantes de cultura (100.000 x g) de P.
brasiliensis Pb339 na presença de 0,3 M dos açúcares indicados. O controle foi incubado na ausência de
competidor.
Ensaios para confirmar a presença do epitopo anti-alfa galactosil foram realizados
pela Dra. Luciane Ganiko, no laboratório do Dr. Igor C. Almeida da University of Texas
at El Paso (UTEP). Nesses ensaios, o substrato foi tratado com a enzima alfagalactosidase, a qual hidroliza resíduos de alfa-galactosidase de cadeias lineares. Na Fig.
11 observamos que o tratamento das vesículas de T. cruzi com alfa-galactosidase impediu
substancialmente o reconhecimento de uma lectina específica para resíduos alfagalactosil, conhecida como MOA (Marasmium oreades agglutinin). O reconhecimento da
lectina foi drasticamente reduzido, indicando que esse epitopo também está presente em
preparações de vesículas de P. brasiliensis. Tanto o anticorpo quanto a lectina poderão
100
ser utlizados na purificação das vesículas de P. brasiliensis em coluna de afinidade ou
como marcador.
Figura 11. Reatividade em Elisa, revelada por quimioluminescência, de MOA com precipitado de
sobrenadantes de cultura (100.000 x g) de P. brasiliensis (PbVs) ou vesículas de T. cruzi (TcaGalVes)
tratados (barras negras) ou não (barras brancas) com alfa-galactosidase. Controle: na ausência de antígeno.
101
Capítulo 2: conclusões
Em nosso laboratório foi especulada a possibilidade da serino-tiol proteinase
(PbST) ou algum modulador positivo da enzima ser veiculado ao meio extracelular por
vesículas secretadas pelo fungo. Os resultados preliminares de caracterização de
vesículas extracelulares de P. brasiliensis apontam que:
1. Foi observada por microscopia eletrônica a presença de estruturas semelhantes a
vesículas delimitadas por membrana em precipitados a 100.000 x g de
sobrenadantes de P. brasiliensis. Frações do precipitado correspondentes à
densidade de vesículas em gradiente de sacarose foram capazes de estimular a
produção de NO por macrófagos, porém não de IL-10 ou Il-12 nas condições
testadas. Preparações de vesículas foram reconhecidas especificamente por
anticorpos
de
pacientes
portadores
de
PCM,
anticorpos
policlonais
monoespecíficos anti-alfa galactosil e também pela lectina MOA, que reconhece
epitopos contendo α galactose. A atividade da PbST não foi detectada nas
preparações de vesículas até o momento, possivelmente porque sua secreção não é
estimulada no meio F12/glucose.
Esses resultados preliminares abriram a
perspectiva de um amplo projeto do laboratório para o estudo de vesículas
extracelulares de P. brasiliensis.
102
A parede celular de fungos é uma estrutura coerente e dinâmica formada
principalmente por polissacarídeos de quitina e glucana, mas contem também proteínas,
glicoproteínas e lipídeos. A parede celular tem fundamental importância na biologia do
fungo e na interação parasita-hospedeiro, além de ser um ótimo alvo farmacológico.
Nossos estudos sobre o proteoma diferencial de frações da parede celular de dois isolados
do P. brasiliensis, que diferem geneticamente e quanto à virulência em camundongos,
podem ser assim resumidos:
1. Foram identificadas proteínas associadas à parede cellular (CWAPs)
diferencialmente expressas no fungo dimórfico P. brasiliensis. Os isolados Pb3 e Pb18
foram cultivados em F12 glucose na presença ou não de 5% de soro fetal bovino (SFB).
Esses isolados pertencem a grupos filogenéticos distintos e provocam tipos diferentes de
resposta immune e progressão da PCM em modelo murino. Preparações de parede
celular, lavadas extensivamente com sal e tratadas com ditiotreitol, foram extraídas com
NaOH e HF, e os peptídeos tripticos foram identificados por espectrometria de massas
(LC-MS/MS) utilizando os softwares Mascot e Phenyx. 60 proteínas foram identificadas
com 4 ou mais peptídeos e as mais abundantes foram o fator de elongação 1, Hsp 70 e
histona H2B. Proteínas ribosomais, hsp70, hsp90, catalase, calmodulina e várias
proteínas metabólicas foram mais abundantes nas culturas em FSB. Entre as CWAPs
prevalentes em Pb18, oxidante tiol específica e gliceraldeído-3-fosfato desidrogenase
poderiam estar relacionadas com sua maior virulência. A prevalência de gp43 na parede
de Pb3, por outro lado, poderia contribuir com a sua eliminação mais eficaz pelo sistema
imune.
103
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Anexo 1: Resultados complementares sobre a PbST
1. Purificação da PbST
A purificação da serino-tiol ativa tem sido impedida por ocorrer em baixas
concentrações proteicas, ter tendência à autólise e agregação com componentes
polissacarídeos de alto peso molecular. Anteriormente ao desenvolvimento desta tese,
várias estratégias aplicadas com esse fim tiveram sucesso parcial. Desta forma, um dos
objetivos deste trabalho foi testar outras técnicas no sentido de conseguir preparações
suficientemente enriquecidas para a proteinase que pudessem ser analisadas quanto à
composição de aminoácidos. Nos trabalhos apresentados anteriormente, utilizamos dois
tipos de preparações enzimáticas, a saber, fração 3 eluída de uma coluna de interação
hidrofóbica de Phenyl Superose (Matsuo et al., 2006) e a fração 4 resultante de
cromatografia de afinidade em uma coluna de p-aminometilbenzamidina (pABA),
utilizada apenas recentemente no laboratório (Matsuo et al., 2007). Outras estratégias
usadas na purificação da PbST estão comentadas abaixo e foram extensivamente
detalhadas em relatórios científicos apresentados à FAPESP.
a) Cromatografia de fase reversa em coluna de Sephasil Protein C4: Frações
eluídas da coluna de Phenyl-Superose correspondentes ao pico de atividade proteolítica
(Matsuo et al., 2006) eram fracionados por f.p.l.c, em ÄKTA, em uma coluna de fase
reversa Sephasil Protein C4 (Amersham Biociences) que continha 1,66 ml de resina
equilibrada com 0,1% de TFA (ácido triclorofluoroacético). As frações eram eluídas
através de um gradiente de 0 a 90% acetonitrila em 0,1% de TFA, rapidamente
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congeladas em gelo-seco, secas em speed-vac, e estocadas a -20ºC. Antes de testadas, as
frações eram ressuspendidas em 100µL de Tris 50 mM, pH 8,0, e então analisadas quanto
ao padrão de bandas e atividade frente ao substrato peptídico específico AbzMKALTLQ-EDDnp. Tínhamos consciência de que uma cromatografia em condições
drásticas com solventes orgânicos poderia resultar na morte da atividade enzimática. No
entanto, foi encontrada uma intensa atividade nas frações 5 (pico fino e homogêneo) e 12.
Infelizmente, no entanto, esse resultado não se repetiu em qualquer das 8 vezes
subsequentes e a estratégia foi abandonada.
b) Cromatografia em colunas de troca-iônica: Frações eluídas da coluna de
Phenyl-Superose correspondentes ao pico de atividade hidrolítica sobre AbzMKALTLQ-EDDnp (Matsuo et al., 2006) foram fracionadas em colunas trocadoras
catiônicas de Sepharose SP (forte) e Sepharose CM (fraca), e em resinas trocadoras
aniônicas Sepharose Q (forte) e Sepharose DEAE (fraca), da Amersham Pharmacia
Biotech, de acordo com os procedimentos sugeridos pelo fabricante. Os ensaios de
hidrólise de substrato peptídico de fluorescência apagada Abz-MKALTLQ-EDDnp
mostraram que a recuperação da atividade foi melhor com as resinas catiônicas Sepharose
DEAE (28,4%) e Sepharose Q (26,5%), porém o rendimento foi baixo e tornou este tipo
de cromatografia inviável.
c) Precipitação de sobrenadante total com etanol: para enriquecer a atividade
da PbST, sobrenadantes de cultura ativos de P. brasiliensis foram precipitados em série
com etanol nas proporções de 20%, 40%, 60%, 80% ou 95%. As proteínas do
sobrenadante concentraram-se nas frações 20% a 40% e 40% a 60% de etanol. Quando
testadas quanto à clivagem de fibronectina (Fn) por 30 min a 37oC, verificou-se que a
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fração 20% a 40%, não apresentou atividade proteolítica, enquanto as frações 40% a 60%
e 60% a 80% clivaram a Fn totalmente. Coincidentemente, estas mesmas frações
continham um componente de alta massa molecular, aparentemente a galactomanana do
fungo com efeito modulatório sobre a PbST (Matsuo et al., 2006). A atividade do
precipitado com 40% a 60% de etanol foi inibida por PMSF e pHMB. Não foi dada
sequência aos experimentos de precipitação com etanol porque a coluna de pABA foi
adquirida nesse período e mostrou boa resolução (Matsuo et al., 2007). No entanto, uma
preparação ativa e com reduzido conteúdo proteico resultante da precipitação com 50 a
80% de etanol foi analisada quanto à composição peptídica, com resultados
desanimadores.
2. Resultados de experimentos sugeridos pela banca de
qualificação
Por ocasião do exame de qualificação, dois experimentos foram sugeridos pela
banca, os quais foram realizados como descrito a seguir:
1. Obter anticorpos policlonais reativos com alguma subtilina-tiol e verificar reação
cruzada com a PbST. Para tal, inoculamos uma coelha com 10 µg de proteinase K
(Sigma) purificada do fungo Tritirachium album via intradérmica com adjuvante de
Freund (50 µL). Após duas imunizações, o soro foi recolhido e testado em ELISA,
“Western blot” ou “Dot blot” contra proteinase K, sobrenadante total de P. brasiliensis
ativo para PbST, fração eluída ou não-ligada em coluna de pABA. Porém os resultados
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foram positivos apenas para a proteinase K, mostrando que o anticorpo não foi capaz de
reagir cruzadamente com a serino-tiol nas condições testadas.
2. Verificar se há anticorpos no soro de paciente com PCM capazes de inibir a atividade
proteolítica, uma vez que a proteinase é secretada. Para tal, imunoglobulinas de um
paciente com PCM foram purificadas em coluna de afinidade de proteína A e testadas em
diversas concentrações (0,5, 0.25 ou 0.1 µg/ µL) quanto à capacidade de inibir a clivagem
de fibronectina pela PbST eluída de coluna pABA. Em nenhuma concentração testada as
imunoglobulinas foram capazes de inibir a ação da proteinase, sugerindo que o soro de
paciente não continha anticorpos anti-PbST ou nas condições testadas os anticorpos não
foram capazes de inibir ou interagir com os epitopos da molécula ao ponto de interferir
com o sítio ativo.
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Anexo 2: Resultados complementares sobre a PbLon
1. Expressão da PbLon em sistema heterólogo
Um dos objetivos do projeto de doutorado foi a expressão da enzima PbLon
recombinante ativa para estudos de especificidade utilizando peptídeos de fluorescência
apagada do tipo O-aminobenzoil-peptidil-etilenodiaminobenzidina (Abz-EDDnp), os
quais poderiam levar à detecção de inibidores específicos da enzima. Para tal, a
construção deveria conter os fragmentos de cDNA correspondentes aos sítios ativos da
enzima. No trabalho apresentado anteriormente (Batista et al., 2006) obteve-se a
expressão em bactéria de uma porção N-terminal da PbLon, na forma de corpos de
inclusão, a qual foi essencial para obtenção de anticorpos policlonais de coelho usados
em experimentos de localização celular. Foram realizadas, entretanto, inúmeras tentativas
frustadas de expressão da molécula solúvel e ativa, cujas construções e sistemas estão
esquematizamos na Figura 1.
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Figura 1. Construções contendo o gene da PbLON para expressão em sistema procarioto (E. coli) ou
sistema eucarioto (Pichia pastoris e S. cerevisiae).
Expressão de PbLon recombinante truncada no N-terminal
A primeira tentativa de expressão da PbLon foi realizada pela Dra. Tânia Fraga
Barros utilizando o cDNA completo da proteinase inserido no vetor pHIS1, porém
nenhuma banda correspondente à proteína foi observada. A segunda construção privou a
molécula dos primeiros 178 aminoácidos do N-terminal, o que não comprometeu os sítios
catalíticos. Para tal, usou-se um plasmídeo pHIS1Lon do laboratório (Barros, 2001), onde
o cDNA do gene PbLON truncado no nt 151 foi inserido no sítio EcoRI de pHIS1. Esse
plasmídeo foi presentemente clivado com BamHI (nt 537) e re-ligado no sítio BamHI do
vetor. Essa construção foi usada na transformação das bactérias de expressão
BL21(DE3), BL21(DE3) pLysS resistente ao clorofenicol, BL21(DE3) trxB resistente à
kanamicina e BL21(DE3) Rosetta.
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As bactérias induzidas com IPTG por 3 horas foram lisadas e as proteínas
submetidas à cromatografia em coluna de Ni-NTA (Qiagen). As proteínas eluídas foram
analisadas em “Western blot” com anti-rPbLon onde foram vizualizadas duas bandas
específicas, uma de 32 kDa e outra de 80 kDa (figura 2), quando o peso molecular
esperado era de 95 a 100 kDa.
Figura 2. Imunoblot das proteínas eluídas de uma coluna de níquel a partir do sobrenadante das bactérias
recombinantes induzidas por 3 horas com IPTG. As massas moleculares estão indicadas em kDa.
Para verificar se essa proteína truncada teria atividade, foram realizados ensaios
de proteólise de azocaseína com extrato total de bactéria, porém não foi observada
nenhuma clivagem ATP-dependente ou diferente do controle. O produto de eluição de
colunas de níquel foi igualmente negativo quanto à atividade proteolítica de azocaseína
dependente de ATP.
123
Expressão de PbLon recombinante usando o vetor pET44a+: Este vetor tem como
características principais a expressão da proteína recombinante com uma cauda da
proteína Nus (495 aminoácidos), que confere solubilidade às proteínas expressas, uma
cauda de histidina na região N-terminal para facilitar da purificação e o gene de
resistência à ampicilina. O fragmento de cDNA de 3,1 kb do gene PbLON foi subclonado
no sítio EcoRI do vetor pET44a+ e transformado em E. coli BL21 pLysS.
Análises em “Western blot” utilizando anti-PbLon não revelaram nenhuma banda
na altura esperada de 160 kDa (~45 kDa da cauda de Nus mais ~117 kDa da proteinase),
porém detectou uma banda com cerca de 120 kDa marcada especificamente (figura 3).
Quando sobrenadantes de bactérias recombinantes foram testados quanto à clivagem de
azocaseína, não foi observada nenhuma clivagem ATP-dependente ou diferente do
controle (bactéria sem plasmídeo de expressão).
124
Figura 3. A) Padrão de bandas do extrato total de bactérias recombinantes pET44a+PbLon após 3 horas de
indução com 1 mM de IPTG. Análise em gel de SDS-PAGE 10% corado por Comassie Blue. Controle,
extrato de bactéria transformada com o vetor expressando uma proteína não relacionada. B) Imunoblot
revelado por quimioluminescência e anti-PbLon (1:500). Foram testados extratos de 3 clones
recombinantes e o mesmo controle de A.
Expressão de PbLon em P. pastoris: ainda com objetivo de expressar PbLon completa e
ativa testamos o sistema de expressão na levedura P. pastoris, o qual foi utilizado com
sucesso em nosso laboratório na secreção de gp43 recombinante solúvel (Carvalho,
2004). Foram usados os vetores pPIC9 e pHIL-S1, que possuem os marcadores de
resistência à ampicilina para seleção em bactéria, e o gene HIS4 (da histidinol
desidrogenase) para seleção em leveduras. Nesse sistema, o fragmento de cDNA
heterólogo é sub-clonado entre as sequências promotora e terminadora do gene da
oxidase alternativa (AOX1), para indução da proteína recombinante em grandes
125
quantidades com metanol. Além disso, o fragmento é clonado em fase com uma uma
sequência sinal na região 5’ para promover a secreção da proteína expressa.
A P. pastoris é uma levedura metilotrópica capaz de metabolizar metanol como
única fonte de carbono, a partir de sua oxidação em formaldeído pela álcool oxidase. Em
nosso experimento foi utilizada a cepa GS115 de P. pastoris, que apresenta o genotipo
HIS4. Essa linhagem sofre inserção do plasmídio linearizado no sítio AOX1 e a levedura
passa a apresentar o fenótipo His+ Mut+ / His+ Muts (com alta ou baixa utilização de
metanol, respectivamente), dependendo da enzima utilizada para linearizar o plasmídeo.
O vetor foi clivado com a enzima SacI, que resulta no fenótipo His+ Mut+. Esse evento
pode ocorrer com plasmídeos fechados ou religados, porém em freqüência baixa.
Foi subclonado no sítio EcoRI dos vetores pPIC9 e pHIL-S1um fragmento de 3,1
kb correspondente ao cDNA da PbLON partir do nucleotídeo 45. As construções foram
selecionadas em bactérias através de resistência à ampicilina e as leveduras selecionadas
em meio mínimo desprovido de histidina, após crescimento por 2 dias a 30ºC. Foram
selecionados 10 clones para análise da expressão de PbLon. Para tal, as leveduras foram
induzidas com metanol por 2, 3, 4, 5 e 6 dias e uma alíquota de 5 µL do sobrenadante das
culturas foi testado. Nenhum deles demonstrou atividade ATP-dependente em ensaios de
azocaseína. Não detectamos a proteína recombinante em gel de SDS-PAGE, “Western
blot” ou “Dot blot”.
Expressão de PbLon em S. cerevisiae: testamos o sistema de expressão na levedura S.
cerevisiae, o qual foi utilizado com sucesso na expressão de genes LON de outros
126
organismos (van Dijl et al., 1998). Presentemente, foram usados os vetores pYES2/NT B
e pYES2/NT C (Invitrogen), que possuem os marcadores de resistência à ampicilina para
seleção em bactéria, e o gene URA3 para seleção das leveduras em meio seletivo URA-.
Nesse sistema, o fragmento de cDNA heterólogo é subclonado em fase com a sequência
promotora GAL1, a qual induz fortemente a expressão de proteínas recombinantes na
presença de galactose e é reprimido na presença de glucose.
No vetor pYES2/NT C, foi subclonado um fragmento de 3,1 kb no sítio EcoRI,
correspondente ao cDNA da PbLON partir do nucleotídeo ~100. No vetor pYES2/NT B,
foi subclonado um fragmento de 2,6 kb nos sítios BamHI e EcoRI, com a região Nterminal truncada em 178 aminoácidos.
As bactérias foram transformadas com as construções e selecionadas através de
resistência à ampicilina. Os produtos de ligação foram utilizados para transformar as
leveduras, de acordo com os protocolos sugeridos pelo fabricante. As leveduras foram
induzidas, coletadas e analisadas em gel de SDS-PAGE, “Western blot” ou “Dot blot”
porém não foi detectado nenhum indício da proteína recombinante. Ressaltamos que este
kit da Invitrogen possui um vetor controle contendo o gene da β-galactosidade que
funcinou perfeitamente.
2. Localização da PbLon por microscopia eletrônica
Em Batista et al. (2006) foram registrados os resultados de localização
mitocondrial da PbLon usando microscopia confocal. Presentemente mostramos que em
microscopia eletrônica as células marcadas com o anticorpo policlonal monoespecífico de
coelho anti-rPbLon mostraram uma discreta marcação na mitocôndria (setas), como visto
127
na figura 4A representativa das demais. A marcação discreta, que contrasta com aquela
vista em microscopia confocal (Batista et al., 2006), pode estar relacionada ao plano do
corte ultrafino analisado. Com o anticorpo pré-imune, houve uma ligeira marcação
inespecífica na parede celular (figura 4B).
Figura 4. A) Microscopia eletrônica de P brasiliensis marcado com anticorpos policlonais anti-rPbLon
monoespecíficos e B) soro pré-imune. As setas indicam marcações intramitocondriais.
128

Paracoccidioides brasiliensis

Transcription

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