Bacterial Anti -Virulence Strategy as an Alternative …...Anti-Virulence approach: Disarming...
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Bacterial Anti-Virulence Strategy as an Alternative To Antibiotics Center for Surgery, Innovation & Bioengineering Department of Surgery, Microbiology and Immunobiology Massachusetts General Hospital & Harvard Medical School, Boston, USA Damien Maura, PhD 2 nd International Symposium on Alternatives to Antibiotics Dec. 15 th 2016
Transcript of Bacterial Anti -Virulence Strategy as an Alternative …...Anti-Virulence approach: Disarming...
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Bacterial Anti-Virulence Strategy as an Alternative To Antibiotics
Center for Surgery, Innovation & BioengineeringDepartment of Surgery, Microbiology and ImmunobiologyMassachusetts General Hospital & Harvard Medical School, Boston, USA
Damien Maura, PhD2nd International Symposium on Alternatives to AntibioticsDec. 15th 2016
Source: WHO
70 % of nosocomial infections in the US are now resistant to
one or more antibiotics
Antibiotic Resistance is One of Our Most Serious Health Treats
Excessive Antibiotic Use for Food Producing Animals
Increase in antibiotic use from 2009 to 2014in food producing animals
Medically important
Non- medically important
Dantas & Sommer. Antibiotic Resistomes Review. American Scientist 2014
Just one gram of soil is estimated to contain about one billion bacterial cells
Antibiotic Resistant Bacteria are Transmitted to Humans via Food, Water and Environment
Adapted from Outterson et al, JLME 2013
Drop in New Antibiotics Approved by the FDA since 2000
Anti-Virulence approach: Disarming bacterial pathogens from their ability to cause infection by targeting key mechanisms required for acute and persistent infections without affecting bacterial growth or viability.
Anti-Virulence as an Alternative Approach to Antibiotics
Antibiotics Anti-Virulence
growth, viability virulence
Selective pressure for survival
Resistance & Tolerance to Treatment
“Superbug”
Selective pressure for survival
Resistance & Tolerance to Treatment
Advantages over antibiotics:
Less resistance to treatment
Preservation of beneficial flora
Active even on MDR bacteria
anti-virulence
Biofilms
Quorum sensing
Protein secretionToxins
Two component systems
Capsule
c-di-GMPJM Ghigo, Institut Pasteur
Buttner et al. 2010 FEMS Micro. Rev.
Pearson Education Inc. 2011
Bretl et al. 2011 MMBR
Main Conserved Targets for Anti-Virulence Strategies
anti-virulence
Biofilms
Quorum sensing
Protein secretionToxins
Two component systems
Capsule
c-di-GMPJM Ghigo, Institut Pasteur
Buttner et al. 2010 FEMS Micro. Rev.
Pearson Education Inc. 2011
Bretl et al. 2011 MMBR
Main Conserved Targets for Anti-Virulence Strategies
Quorum Sensing is an Cell-to-Cell Density-Dependent Communication System that Synchronizes
the Production of Multiple Virulence Factors
Phenotypes shown to be Regulated by Quorum SensingIn Gram Negative and Gram Positive Bacteria
Pseudomonas aeruginosa infects multiple organisms
PyocyaninCyanideLectinsProteasesElastaseLasR QS RhlR QSRhamnolipidsPeptidesChitinases
MvfR = Multiple virulence factor regulator
3 QS systems in P. aeruginosa:- MvfR- LasR- RhlR
PyochelinPyoverdinType VI secretionType II secretionHAQs, 2-AAEfflux pumpsFimbriae/piliFlagellaProtein translationmore…
Model: Quorum Sensing in Pseudomonas aeruginosa
66%
34%
Down-regulateUpregulatedRest of the genomeMvfR Regulated Cao et. .al. PNAS 2001; Deziel et. al. PNAS, 2004; Deziel et. al. Mol Micr. 2005; Xiao
et. al. Mol Micr. 2006; Kesarwani et al. 2011 PLoS Pathogens
MvfR regulated genes MvfR regulated virulence factors
PA14 mvfR-0%
20%
40%
60%
80%
100%
% le
thal
ity
PA14 mvfR-105
106
107
108
Gro
wth
on
Arab
idop
sis
leav
es
lethality in flies
PA14 mvfR-0%
20%
40%
60%
80%
100%
% le
thal
ity
lethality in mice multiplication in plants
MvfR QS System is Critical for Infection in Multiple Hosts
Adapted from Rahme et.al. 1997 PNASAdapted from Lau et al. 2003 Infect. & Immun.
MvfR = LTTR family
Ilangovan et al. 2013 PLoS Pathogens
LysR-type transcriptional regulators (LTTR)
Maddocks et al. 2008 Microbiology
MvfR is Part of a Family of Transcriptional Regulators Widely Conserved in Bacterial Pathogens
LasR/lasI RhlR/rhlIMvfR
PqsE
pqsABCDE
PqsHPqsABCD
PQS
pqsH
HHQHHQ
PQS
2-AA
Acute infection
Chronic infectio
nHQNO
phnAB
Adapted from Maura et.al. 2016 Scientific Reports, 2016
MvfR QS Systemand its Interconnection with the LasR & RhlR QS systems
virulence factors virulence factors virulence factors
Hazan et al. 2016 Current Biology
HQNO and 2-AA Promote Chronic Infection Phenotypes
2-AA
bacterialtranslation
“dormancy”stage
host immuneresponse
reduced bacterial clearance
HQNO
antibiotictolerance
chronic infection
antibiotic
antibiotic tolerant cell
antibiotic sensitive cell
Que et al. 2013 PLoS One, Bandyopadhaya et al. 2012 PLoS Pathogens, Bandyopadhaya et al. 2016 Nature Microbiology
Que Y-A et. al. Journal of Pathogens, 2011
High Levels of MvfR Controlled Small Molecules are Produced in Human Tissues
MvfR
Acute infections
Chronic infections
inhibitor???
Adapted from O’Connell 2007 Nat. Rev. Micro
Objective of the project
4 6 8 10
50
100
150 vehiclecpd#1cpd#2cpd#3cpd#4cpd#5
time (h)
pqsA
-GFP
exp
ress
ion
(AU
)
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
High Throughput Screening for MvfR Inhibitors
Benzamide-Benzimidazole (BB) core structure
High Throughput Screening for MvfR Inhibitors
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
HTS
Commercially available
Synthesized
M64
IC50 = 200-300nM
HNCH2C
OSN
NH O
N+–O
O
H = HHQ
P = PQS
N = Pyocyanin
BB Compounds Optimization
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
MvfR PqsBC
R = resistantI = intermediateS = sensitive
P. aeruginosa MDR Strains are Sensitive to M64
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
JM Ghigo, Institut Pasteur
M64 Blocks Biofilm Formation
survival bacterial load in underlying muscle
Abdominal burn + PA14 infection assess survival
M64 IV 4mg/kg
1 2 3 4 5 6days
CFUs CFUs CFUs CFUs
11 14
CFUs CFUs
//
8
CFUs
// //
M64 Rescues Mice from P. aeruginosa Burn Infection
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
bacterial load (lung)
HHQ production
PA14 lung infection
assess survival
M64 IV 4mg/kg
1 2 3 4 5 6days
14h
survival
CFUsHHQ
CFUs
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
M64 Rescues Mice from P. aeruginosa Lung Infection
Antibiotic tolerant cells2-AA
PA14 +
DMSO
PA14 +
M64
PA14 +
M50
PA14 +
M59
PA14 +
M510%
20%
40%
60%
80%
100%
*** ******
***
% o
f Mer
open
em to
lera
nt c
ells
PA14 +
DMSO
PA14 +
M64
PA14 +
M59
PA14 +
M50
PA14 +
M510%
20%
40%
60%
80%
100%
% o
f 2-A
A p
rodu
ctio
n
Maura et al. submitted
BB Compounds Block the Formation of Antibiotic Tolerance Cells in vitro
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
ciprofloxacin
Burn + PA14 infection
M64 IV 4mg/kg
1 2 3 4 5 6days
CFUs CFUs CFUs CFUs
11 14
CFUs CFUs
//
8
CFUs
// //
Ciprofloxacin IV 10mg/kg
M64
M64 Prevents Relapsing P. aeruginosa Infection in Mice
Starkey*, Lepine*, Maura* et al. 2014 PLoS Pathogens
MvfR = LTTR family
Ilangovan et al. 2013 PLoS Pathogens
LysR-type transcriptional regulators (LTTR)
Maddocks et al. 2008 Microbiology
Can these inhibitors have a broad applicability?
Invasion of Caco2 cells
DMSO Mµ
M64 10
Mµ
M64 15
Mµ
M64 25
0%
20%
40%
60%
80%
100%
****** ***
% in
vasi
on o
f Cac
o2 c
ells
DMSO Mµ
M64 25
103
104
105
106
107
108
109
CFU
/mL
DMSO Mµ
M64 25
0%
20%
40%
60%
80%
100%
% s
urvi
val o
f Cac
o2 c
ells
0%
20%
40%
60%
80%
100%
120%
fimD fimH flhB flhDtype 1 pilus flagella
rela
tive
gene
exp
ress
ion
(nor
mal
ized
to 1
6S)
M64 Blocks Escherichia coli Invasion of Intestinal Epithelial Cells
Bacterial viability Caco2 cells viability
Bacterial gene expression
Bacteria
Quantify gut inflammatory cells(immuno-histo-chemistry)
Fly food 24h
M64
M64 also Blocks the Virulence of Many Bacterial Pathogens
HTS
M64
Chronic virulence- antibiotic tolerance- biofilms- relapsing infection
Acute virulence- pyocyanin - acute infection
HNCH2C
OSN
NH O
N+–O
OSAR
MvfR
PhnAB & PqsABCDE
HAQs
PqsE
virulence functions
virulence functions
virulence functions
HHQ & PQS
2-AA, PQS & HQNO
M64HNCH2C
OSN
NH O
N+–O
O
BB compounds = potential to be the next generation anti-bacterial drugs for Pseudomonas infections
Summary & Perspectives
BB compounds have an anti-virulence efficacy on a broad range of bacterial pathogens
Potential applications?
Summary & Perspectives
Melissa StarkeyArunava BandyopadhayaBiliana LesicJianxin HeHadi GharedaghiTomoe KitaoYiorgos ApidianakisAlicia BallokRonen HazanJeremy YanAmy TsurumiLaurence Rahme
Valeria RighiAria Tzika
Sylvain MilotFrancois Lepine
Funding:
Steffen DreesSusanne Fetzner
Michele NegriAntonio Felici
Acknowledgements
