Valérie POLONAIS, G. PRENSI ER et F. DELBAC

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Valérie POLONAIS, G. PRENSI ER et F. DELBAC
Valérie POLONAIS, G. PRENSI ER et F. DELBAC
Laborat oire Biologie des Prot ist es UMR CNRS 6023
Parasitologie Moléculaire et Cellulaire
Université Blaise Pascal, Clermont- Ferrand
Unicellular eukaryot es
Obligat e int racellular parasit es
Opport unist ic inf ect ions in human
Environment al- resist ant spores wit h a unique
invasive apparat us (polar t ube)
Encephalit ozoon hellem
Human: t he unique known mammalian host
Zoonot ic opport unist ic inf ect ion
Anchorage disk
Polaroplast
Exospore
Endospore
Cell Wall
Plasma membrane
Polar tube
Ribosomes
Nucleus
200 nm
Posterior
vacuole
Ex
En
I n E. cuniculi
I n E. int est inalis
(Bohne et al., 2000)
(Hayman et al., 2001)
2 genes encoding
1 gene encoding
EiSWP1 (388 aa, 41kDa)
EcSWP1 (450 aa,46 kDa)
EiSWP2 (1002 aa, 107 kDa)
Labelling restricted to the electron- dense exospore
Signal peptide: 18 aa
Conserved N - terminal regions
Specific C- terminal parts
EcSWP1
5 repeats Gly and
Ser- rich of 16 aa
EiSWP1
EiSWP2
34 aa
50 repeats Glu and Asprich of 12- 15 aa
Southern blot experiments suggested E. hellem contains a swp1
homologous gene (Bohne et al., 2000)
Cross reactivity with antibodies raised against Encephalit ozoon
int est inalis exospore (Mab1E4)
1 µm
66 kDa
1. non- infested
HFF cells
45 kDa
2. HFF- Ec
E. hellem spores
3. HFF- Ei
1 2 3
4
4. HFF- Eh
5
3
SWD1
SWD2
2
1
SWR1
EhR1
EhD1
5
3
OligodT
3 UTR
Coding region : 1530 bp
146 bp
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
MMKLSLLLGLVSFSAVLASENRRGN-CQMCPEG-TRYFEKNNLLGRRFKNDVRKLCGDGM
MIKLSLLLSLASFTAVLANQRPR---CQRCPVSSSKYFQQNNLLGSRFQNEVQRLCARRV
MLKLSLLLGLANFTAILAGGERRRSGLLQCSANGSRYFREQNLLGIKYKDDVKRLCGERP
* * **************
*
** **
* ** ***** ****** **** *
SSDVHDESSSHSSSSSDCKPRRRRHHRRYEDSCSFGSSDCDDSSTYSSCVSSECSPPCRP
REESSSESSSSSSSE -DCSRRRRRPHREWEDSCSSSYSSCS-----STDSCSSSAPCPPP
EGPHPSESSSCSSSCSEECGRRRFPGIRDDCEYSSWDACCSS----SWDECTDSTPCTTP
***** *** *** **** *** ****** * **
* * ** * ** *
VPLNCDYELKTPIINMGERIFEFLKNYEDQYKKAVVLFLTRILSQIDGFAPSYPSADYEP
VAQRCDIELKTPIILMGERIYEFLKNYEDQYKKAVLLFLTNILSQISGFNPVFPGGDYDA
TPLRCDAELRVPIINMGERIYEFLKNYEDQYESAVTLALNNILSQVSGFNPIFAGADYAA
* **** ******************************************** ****** *
LIEQLETLGVTVPSNMAADLAALDAAEATSLAGTIRANAQKVIGDLLARVNTMCYLDLMS
LIEQLKTLGVTVPANTAAELAAIDAAESSALTRAIQANAQKVISDLLTRVSAMCYLDIMS
LVEQLETLGVNVPANTAAELASIDSSESAALSRIIQGNAQKIISDLFARVGSMCYSDITS
**************************** ** * **************** ********
LVTSGLFASQVTSAFSNTQPIITIAGNDLFTKQMAVFQRLPGTLPSAAITAITNALQANK
LVNSGLLASQVSSVFNNIQPIITITGNDLFAKQMAVFQKIPGTLPSAAITAITNALQGNR
LINSGLFASQISSAFSNTQPVITIASNDLYAKQMAVFQRIPGTLPAAAVTAITNALQTNR
********************************************************* **
NNFVTFFTTQTTNLQTDVQNALTALITALTTLTSTTSTEFTQFANSEIGALTGRIFGSSG
TNFVTFFTTQTSNLQTSVQNSLTTLISELEKLATDTETAFTAFANAEISALISRIFPTST
NNFATFFTTQATTLQTNVQNILTALTTALTTLTNSTSTEFTTFANSEIAALAARIFPATT
**************** *** ***********
****** ****** ** **** **
SGSGGSSGGSSGSGSDGSGSGGSSGGSSGSGSDGSGSGGSSGGSSGSGSDGSGSGGSSGG
TSSGSGD-------STGSGSTGDAGDAGDTGEDGGDDEGTEGTG---------------ASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDPVTALLNTIKEEGEKLGKAAM
*** **
* * * **** *
* *
** *
*
SSGSGSDGSGSGGSSGGSSGSGSGGESGGSSS--------------------------------------------------------------------------------------EEVMTAIKEQGTTQGAEAVQEMMDAVKNEADTKKTETVDEILSKVKDEADTKKTETVTEI
EcSWP1
EiSWP1
EhSWP1
----------------------------------------------------------------MDKVKTEADTKKTDTVTEIMEKAKETWEAGKSS
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
58
57
60
118
111
116
178
171
176
238
231
236
298
291
296
358
351
356
418
388
416
450
388
476
450
388
509
Length
( amino acids)
Protein
%
pI
S
G
A
T
E
Nb of
Cys
Nb of
potential Oglycosylation
sites
5.2
18.9
14.1
6.5
7.6
3.9
11
41
370
5
14
6.2
8.6
8.9
5.6
10
19
1002
984
4
5
17.2
3
7.8
25
10
3
509
491
4.6
8.9
6.3
10.1
11
10.6
12
2
Precursor
Mature
protein
EcSWP1
450
432
EiSWP1
388
EiSWP2
EhSWP1
I mmunolabelling wit h ant isera raised
against recombinant prot eins
Mab1E4
Anti- EhSWP1N
66 kDa
Anti- EhSWP1C
60 kDa
55 kDa
1 µm
45 kDa
NI Ec Ei Eh
NI Ec Ei
Eh
NI
Ec
Ei
Eh
Identification of 2 major bands (60 and 55
kDa) in E. hellem
1 µm
Anti- EhSWP1N cross react with EcSWP1
and EiSWP1
EhSWP1 is localized in the spore wall
I mmunolocalizat ion in Transmission
Elect ron Microscopy
Spore
Sporonte
Sporoblaste
En
Ex
200
200 nm
nm
200 nm
EhSWP1 is localized in t he exospore
Genot yping E. hellem isolat es by analysis of t he
region encoding t he C- t erminal end of SWP1
600 bp
500 bp
EhD EhV213 EhV242 EhV257
PCR amplification from genomic DNA : 2 bands for each isolate
By RT- PCR, 2 transcrits are obtained for each isolate
1. Intra- isolate variability
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
TTASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDP-----------------TTASGDNNSGSTGGDDTGDEGDGGEEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPVYP
****************************:***** ..::..
--------VTALLNTIKEEGEKLGKAAMEEVMTAIKEQGTTQGAEAVQEMMDAVKNEADT
DRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEVDT
:*******************:*** *:*.:. *: :*:*::::: **:*.**
KKTETVDEILSKVKDEADTKKTETVTEIMDKVKTEADTKKTDTVTEIMEKAKETWEAGKS
KKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE****** **:.*** *** **::******::****.:*: . *.*::*:** ::* :
S
54.7% of identity
-
The 60 kDa band is recognized by Ab
raised against t he 26 AA insert ion
Anti-EhSWP1C (26 AA)
Anti-EhSWP1C
60 kDa
60 kDa
55 kDa
Ec Ei
Eh
Ec
Ei
Eh
2. I nt er - isolat e variabilit y
EhV242-S
EhV257-S
Eh-Didier-S
EhV213-S
TTASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDPVTALLNTIKEEGEKLGKA
TTASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDPVTALLNTIKEEGEKLGKA
TTASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDPVTALLNTIKEEGEKLGKA
TTASGDNNSGSTGGDDTGGE---GEEEDGGEEESPEEEEEDLLTALLNTIKEEGEKLGKA
EhV242-S
EhV257-S
Eh-Didier-S
EhV213-S
AMEEVMTAIKEQGTTQGAEAVQEMMDAVKNEADTKKTETVDEILSKVKDEADTKKTETVT
AMEEVMTAIKEQGTTQGAEAVQEMMDAVKNEADTKKTETVDEILSKVKDEADTKKTETVT
AMEEVMTAIKEQGTTQGAEAVQEMMDAVKNEADTKKTETVDEILSKVKDEADTKKTETVT
AMEEVMTAIKEQGTAQGTEAVEEVMKAVKNEADTKKTTTAQEIMDAVKNEVDTKKTTTAQ
EhV242-S
EhV257-S
Eh-Didier-S
EhV213-S
EIMDKVKTEADTKKTDTVTEIMEKAKETWEAGKSS
EIMDKVKTEADTKKTDTVTEIMEKAKETWEAGKSS
EIMDKVKTEADTKKTDTVTEIMEKAKETWEAGKSS
EIMDAVKTEAEGKKTETVTEIMNKAKETWGAGKSG
EhV242-L
EhV257-L
Eh-Didier-L
EhV213-L
TTASGDNNSGSTGGDDTGDEGD--GGEEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPV
TTASGDNNSGSTGGDDTGDEGD--GGEEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPV
TTASGDNNSGSTGGDDTGDEGD--GGEEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPV
TTASGDNNSGSTGGDDTGDEGEEEDGGEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPV
EhV242-L
EhV257-L
Eh-Didier-L
EhV213-L
YPDRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEV
YPDRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEV
YPDRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEV
YPDRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEV
EhV242-L
EhV257-L
Eh-Didier-L
EhV213-L
DTKKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE
DTKKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE
DTKKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE
DTKKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE
500 bp band
600 bp band
EhSWP1 is a good t arget f or genot ype
analysis
E. hellem isolate
Marker
EhDidier
EhV242
EhV257
EhV213
ITS
1A
1A
1A
1A
SSU rRNA
1A
1A
1A
1A
PTP1
1A
1A
1A
1B
SWP1
1A
1A
1A
1B
SWP1 as PTP1 is more discriminating than ITS or SSU rDNA
1. Sout hern blot analysis (2 genes?)
2. Use of EhC- t erminal specif ic recombinant
prot eins in serodiagnost ic st udies
3. I nvolvement of SWP1 in cell invasion ?
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
MMKLSLLLGLVSFSAVLASENRRGN-CQMCPEG-TRYFEKNNLLGRRFKNDVRKLCGDGM
MIKLSLLLSLASFTAVLANQRPR---CQRCPVSSSKYFQQNNLLGSRFQNEVQRLCARRV
MLKLSLLLGLANFTAILAGGERRRSGLLQCSANGSRYFREQNLLGIKYKDDVKRLCGERP
* * **************
*
** **
* ** ***** ****** **** *
SSDVHDESSSHSSSSSDCKPRRRRHHRRYEDSCSFGSSDCDDSSTYSSCVSSECSPPCRP
REESSSESSSSSSSE -DCSRRRRRPHREWEDSCSSSYSSCS-----STDSCSSSAPCPPP
EGPHPSESSSCSSSCSEECGRRRFPGIRDDCEYSSWDACCSS----SWDECTDSTPCTTP
***** *** *** **** *** ****** * **
* * ** * ** *
VPLNCDYELKTPIINMGERIFEFLKNYEDQYKKAVVLFLTRILSQIDGFAPSYPSADYEP
VAQRCDIELKTPIILMGERIYEFLKNYEDQYKKAVLLFLTNILSQISGFNPVFPGGDYDA
TPLRCDAELRVPIINMGERIYEFLKNYEDQYESAVTLALNNILSQVSGFNPIFAGADYAA
* **** ******************************************** ****** *
LIEQLETLGVTVPSNMAADLAALDAAEATSLAGTIRANAQKVIGDLLARVNTMCYLDLMS
LIEQLKTLGVTVPANTAAELAAIDAAESSALTRAIQANAQKVISDLLTRVSAMCYLDIMS
LVEQLETLGVNVPANTAAELASIDSSESAALSRIIQGNAQKIISDLFARVGSMCYSDITS
**************************** ** * **************** ********
LVTSGLFASQVTSAFSNTQPIITIAGNDLFTKQMAVFQRLPGTLPSAAITAITNALQANK
LVNSGLLASQVSSVFNNIQPIITITGNDLFAKQMAVFQKIPGTLPSAAITAITNALQGNR
LINSGLFASQISSAFSNTQPVITIASNDLYAKQMAVFQRIPGTLPAAAVTAITNALQTNR
********************************************************* **
NNFVTFFTTQTTNLQTDVQNALTALITALTTLTSTTSTEFTQFANSEIGALTGRIFGSSG
TNFVTFFTTQTSNLQTSVQNSLTTLISELEKLATDTETAFTAFANAEISALISRIFPTST
NNFATFFTTQATTLQTNVQNILTALTTALTTLTNSTSTEFTTFANSEIAALAARIFPATT
**************** *** ***********
****** ****** ** **** **
SGSGGSSGGSSGSGSDGSGSGGSSGGSSGSGSDGSGSGGSSGGSSGSGSDGSGSGGSSGG
TSSGSGD-------STGSGSTGDAGDAGDTGEDGGDDEGTEGTG---------------ASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDPVTALLNTIKEEGEKLGKAAM
*** **
* * * **** *
* *
** *
*
SSGSGSDGSGSGGSSGGSSGSGSGGESGGSSS--------------------------------------------------------------------------------------EEVMTAIKEQGTTQGAEAVQEMMDAVKNEADTKKTETVDEILSKVKDEADTKKTETVTEI
EcSWP1
EiSWP1
EhSWP1
----------------------------------------------------------------MDKVKTEADTKKTDTVTEIMEKAKETWEAGKSS
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
EcSWP1
EiSWP1
EhSWP1
58
57
60
118
111
116
178
171
176
SWD1
EhR1
238
231
236
298
291
296
358
351
356
418
388
416
450
388
476
450
388
509
SWR1
EhD1
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
Eh-Didier-s
Eh-Didier-l
TTASGDNNSGSTGGDDTGDEGDGGEEEETPEEEEGDEEEEDP-----------------TTASGDNNSGSTGGDDTGDEGDGGEEEESPEEEEESRKQRSIQKPKKGLRNKRQTLPVYP
****************************:***** ..::..
--------VTALLNTIKEEGEKLGKAAMEEVMTAIKEQGTTQGAEAVQEMMDAVKNEADT
DRESPKAILTALLNTIKEEGEKLGKAAMKEVMDAVKSEADTKKTETVEDILNKVKDEVDT
:*******************:*** *:*.:. *: :*:*::::: **:*.**
KKTETVDEILSKVKDEADTKKTETVTEIMDKVKTEADTKKTDTVTEIMEKAKETWEAGKS
KKTETVTEIMDKVKTEADGKKADTVTEIMNEVKTEGETEVDKIVKEFIEEAKTAYEEKE****** **:.*** *** **::******::****.:*: . *.*::*:** ::* :
S
-

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