presentation

A Novel “Shock and Kill' Strategy
for Targeting Mtb Persisters
Petros C. Karakousis, M.D.
Associate Professor of Medicine
and International Health
Center for Tuberculosis Research
Johns Hopkins University
June 16, 2015
The Stymphalian Birds:
The Sixth Labor of Hercules
•
Man-eating birds with beaks of bronze and sharp metallic
feathers they could launch at their victims.
•
They migrated to a lake in Arcadia to escape a pack of
wolves, and bred quickly, taking over the countryside,
destroying local crops, fruit trees, and townspeople.
The Stymphalian Birds:
The Sixth Labor of Hercules
•
After cleaning the Augean Stables, Hercules was sent
by Eurystheus to defeat the Stymphalian birds.
•
However, Hercules could not go too far into the swamp,
for it would not support his weight.
•
Athena, taking pity on the hero's plight, gave Hercules a
rattle which Hephaestus had made especially for the
occasion.
•
Hercules shook the rattle and frightened the birds into
the air. He then was able to shoot them down with his
poisoned arrows.
Microbial persistence
“Capacity of drug-susceptible organisms to survive drug
attack when subsisting in an animal body”1
Results from:
1. Host immune containment
2. Antibiotic stress
3. Stochastic phenomenon
1 McDermott
W. 1958. Yale J Biol Med 30:257-91
Antibiotic tolerance and relationship to
bacterial growth/metabolism
• “Antibiotic tolerance”1
• The rate of bacterial killing by cell wall synthesis
inhibitors is directly proportional to the rate of
bacterial replication and metabolic activity2
• In vitro and in vivo phenomenon
• Not unique to mycobacteria
• S. pneumoniae, S. pyogenes, E. coli, T.
pallidum3
1 Tomasz
A, et al. 1970. Nature 227:138-40
2 Tuomanen
3
E. 1986. Rev Infect Dis 8:S279-91
Eagle H. 1952. Am J Med 13:389-99
The Stringent Response
Osmotic stress
Nutrient starvation
Stringent response
Heat shock
(p)ppGpp
Inorganic
polyphosphate (poly P)
RNA polymerase
3’
DNA
σ
5’
RelA
Expression of components for
stress resistance, growth restriction
Avarbock A, et al. 2005. Biochem; Kornberg A, et al. 1999. Ann. Rev. Biochem
Mtb stringent response: Positive feedback loop
Rv0496 (PPX1)
Rv1026 (PPX2)
Poly P
Hypoxia
Phosphate starvation
A
Amino acid starvation
Carbon starvation
Respiration inhibition
(p)ppGpp
A
Persistence
Stress response
Growth restriction
Sureka, et al. 2007. Mol Microbiol; Rifat D, et al. 2009 J Infect Dis; Thayil et al. 2011 PLoS One; Chuang Y-M et al. 2015 Mbio
Karakousis PC et al. 2004 JEM
The Mtb Stringent Response: Growth
restriction and antibiotic tolerance
• Induction of the stringent response through
accumulation of poly(P) causes Mtb growth restriction
and tolerance to isoniazid.1,2,3
• RNA-seq and metabolomics analysis of poly(P)accumulating strains show metabolic changes
associated with persistence.3
• Basal “noise” in expression of stringent response genes
and positive feedback may account for spontaneous
“persisters”.4
1Thayil
SM, et al. 2011. PLoS One.
Y-M, et al. 2013. Mbio.
3Chuang Y-M, et al. 2015. Mbio.
4Sureka K, et al. 2008 PLoS One.
2Chuang
Immune pressure
Hypoxia
Acidic pH
Starvation
Cell death
Relief of stress
(p)ppGpp ↑
Poly P ↑
Antibiotic tolerance
Mtb lifestyle
Antibiotic susceptibility
“Persister”
Growing
Stochastic elevation of
poly P and (p)ppGpp
Dutta and Karakousis. MMBR. 2014
(p)ppGpp deficiency is associated with
poly(P) deficiency
(p)ppGpp is required for Mtb metabolic
downshift during nutrient starvation
•
•
•
We hypothesized that (p)ppGpp deficiency (ΔrelA) would lead to
continued Mtb metabolism during nutrient starvation.
AlamarBlue (resazurin) may be used to assess cellular metabolism
(NADH/NAD+ ratio).
The luciferase assay may be used to assess intracellular ATP content.
(p)ppGpp is required for Mtb growth
restriction during nutrient starvation
A “replication clock” plasmid may be used to calculate
the rate of bacterial division, since the unstable
plasmid is lost at a steady, quantifiable rate from
dividing cells in the absence of antibiotic selection.1
1Gill
WP et al. Nat Med. 2009
(p)ppGpp is required for Mtb antibiotic
tolerance to isoniazid
MIC: Minimum Inhibitory Concentration required to inhibit visible growth
MBC: Minimum Bactericidal Concentration kills 2 log10 CFU (99%) in 14-day cultures
7H9: OADC-supplemented Middlebrook 7H9 broth
NS: Nutrient starvation
(p)ppGpp is required for development of
INH tolerance in mouse lungs
7
wild type
Log10 CFU/Lung
6
Δrel
5
wild type +INH
4
Δrel +INH
3
2
1
0
0
7
14
21
28
Days after infection
35
42
Survival of C3Heb/FeJ mice is markedly improved
after aerosol infection with relA-deficient Mtb
(p)ppGpp is required for Mtb survival under
in vitro stress conditions and in vivo
relA-deficient Mtb exhibits reduced survival during nutrient
starvation and high temperature1, in mouse lungs2, and in a murine
hypoxic granuloma model of LTBI3.
Log10 CFU/Lung
6
5
4.97
4
3.91
4.36
3.57
3
2
2.09
1
0
ΔrelA
H37Rv
0
7
14
21
28
35
0.62
Comp
42
49
56
63
70
Time After Infection (days)
1Primm
TP et al. J Bacteriol 2000 2Dahl JL et al. PNAS 2003 3Karakousis PC et al. J Exp Med 2004
Klinkenberg LG et al. J Infect Dis 2010
GSK: High-throughput screening strategy for Mtb
RelA inhibitors
GSK-X9 shows RelA-specific anti-TB activity
GSK-X9 shows synergy with INH
against nutrient-starved Mtb
Conclusions: Can we wake up “persisters”
to more rapidly clear TB infection?
• The alarmone (p)ppGpp is required for Mtb growth restriction,
metabolic downshift, and INH tolerance during nutrient starvation,
as well as long-term survival in animal lungs.
• RelA represents an attractive drug target, as specific enzyme
inhibitors may synergize with tuberculocidal drugs to shorten the
duration of TB treatment.
• RelA and (p)ppGpp are absent in the host.
• Inhibition of RelA is theoretically safe, as complete deficiency of
the enzyme leads to a profound defect in Mtb survival in the host
and should not predispose to opportunistic infections, unlike
immune-modulating strategies.
Acknowledgments
Johns Hopkins
Lee Klinkenberg, Ph.D.
Yu-Min Chuang, M.D., Ph.D.
Seema Thayil, Ph.D.
Dalin Rifat, Ph.D.
Noton Dutta, Ph.D.
Michael Pinn
Vicky Campodónico, M.D., Ph.D.
Will Matern
JHU-High-Throughput Biology
Center and Biomedical Engineering
Nirmalya Bandyopadhay, Ph.D.
Will Matern
Joel S. Bader, Ph.D.
SBRI
GSK-DDW
David Sherman, Ph.D.
Alfonso Mendoza-Losana, Ph.D.
Esther Pérez-Herrán, Ph.D.
U Penn
Harvey Rubin, M.D., Ph.D.
UCT/HHMI
Valerie Mizrahi, Ph.D.
R01AI083125
R01HL106786
R01AI106613