Neisseria gonorrhoeae - etouches · 8/10/2015 1 Efflux pumps in Neisseria gonorrhoeae: cause of...
Transcript of Neisseria gonorrhoeae - etouches · 8/10/2015 1 Efflux pumps in Neisseria gonorrhoeae: cause of...
8/10/2015
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Efflux pumps in Neisseria gonorrhoeae: cause of resistance and targets for therapeutics/vaccines?
William M. Shafer, Ph.D.Emory University School of
Medicine Emory Antibiotic Resistance Center
Atlanta VA Medical Center
No financial disclosures or conflicts
“It is time to sound the alarm. During the past 3 years, the wily gonococcus has become less susceptible to our last line of antimicrobial defense, threatening our ability to cure
gonorrhea and prevent severe sequelae.”
1930 1940 1950 1960 1970 1980 1990 2000 2010 2020
History of Resistance & Shrinking Pipeline
Sulfo
nam
ide
s
SUL resistance
common
PEN resistance,
dose repeatedly
increased
SPT resistance
High-level PEN
resistance(β-lactamase plasmids)
Plasmid-mediated TET
resistance.
Chromosomal TET and PEN resistance
spreading. SPT
resistance frequent in Korea
CIP/OFX
resistance in Asia
CFM resistance in Japan
AZM resistance in
Latin America
First high-level AZM
resistance in Europe
First high-
level CRO
resistance in Japan
2007: Super Bug Status!
CFM and CRO clinical failures in
Japan, Europe, Canada, Australia
and South Africa
Unemo and Shafer, 2014
To Effectively Counteract Resistance With New Drug
Development, It Is Important To Understand Mechanisms of
Resistance
Mechanisms of Antibiotic Resistance N. gonorrhoeae Efflux Pumps
OM
IM
PS
Transporter
family
FQs
EtBr
MATE
NorM
Ery
Azi
ABC
MacAB-
MtrE
FAs
MF
FarAB-
MtrE
Host
Agents
β-Lactams
Ery/Azi
N-9
RND
MtrCDE
Sulfonamides
AbgT
MtrFGC
Transporters
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For ceftriaxone resistance: mosaic PBP2 and overproduction of the MtrCDE efflux pump
Benzyl Penicillin
Ceftriaxone Cefixime
Important β-Lactams Previously or Currently Used to Treat Gonorrhea: Clinically Resistant Strains of Historical Importance
NC epidemicIsolate-1983
FA6140
Japan isolate2009(HO41)
CAT....ATTATAAAAAAGACTTTTTATCCGTGCAATCGTGTATGTATATT....TATAAC....ATG
mtrR mtrC
Inverted
Repeat-35mtrC -10mtrC
-35mtrR-10mtrR
The mtrCDE locus
Multiple Transferable Resistance
Maness & Sparling, 1973. J. Infect. Dis.Pan & Spratt. 1994. Mol. Microbiol. Hagman et al. 1995, 1997. Microbiol.Delahay et al. 1997. MicrobiologyVeal & Shafer. 2003. JAC.
WT
mtrD::kan
0
1
2
3
4
5
6
7
LL-37 Ery CV
Loss of the MtrCDE Efflux Pump Increases Gonococcal Susceptibility to Antimicrobials
WT mtrD::kan
Mtr Efflux Pump-Deficient Gonococci Have a Survival Defect in a Murine Model of Lower Genital Tract In Females (Jerse
et al. 2003)
EL1 (farB)
FA19 (wt)
RD1 (mtrE)*KH14 (mtrD)*
1
2
3
4
5
6
log10
CFU
day post-inoculation2 4 6 8 10 12
* p < 0.05
Mean durationof recovery
days (range)
FA19 9.0 (3-12)
EL1 12.0 (12)
KH14 5.0 (0-12)
RD1 3.4 (0-12)
mtrR mtrCDE
Cis- and Trans-Acting Mutations That Increase Resistance
HO41
MS11
HTHA39T,G45D
X
Promoter mutations: High RMtrR mutations: Intermediate R
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CAT....ATTATAAAAAAGACTTTTTATCCGTGCAATCGTGTATGTATATT....TATAAC....ATG
mtrR mtrC
Inverted
Repeat-35mtrC -10mtrC
-35mtrR-10mtrR
A Single Nucleotide Change Can Account for the MtrPhenotype in High Level Resistant Strains
Western Blot Showing Over-Production of MtrCDE
0
1
2
3
4
5
6
7
8
Pen Cef Ctx Az
MICµg/ml
Loss of the MtrCDE Efflux Pump Enhances Gonococcal (resistant strain HO41) Susceptibility to
Antibiotics
HO41HO41 mtrD::kan
Golparian et al. 2014 Antimicrob. Agents Chemother.
Can We Exploit the MtrCDE Efflux Pump In Efforts to Counteract Antibiotic Resistance
Expressed by Gonococci?
Efforts By Two Groups
•Ann Jerse Ph.D. (USUHS, Bethesda, MD. USA): vaccine development targeting surface exposed domains of MtrE, the OMP channel of the pump
• Edward Yu Ph.D. (Iowa State University, Ames, IA, USA): developing peptides that target MtrC, the periplasmic adapter protein of the pump, and block assembly of a functional pump
MtrCDE
MtrE
MtrC
MtrD
MtrE
Cholera Toxin Adjuvant
N=17-22 mice/ group N=18-22 mice/ group
P= 0.94
Log-Rank Test
MtrE + CTCT alone
PBS alone
MtrE+ CpGCpG alone
PBS alone
P= 0.04
Log-Rank Test
Comparison of MtrE3-467 Immunization/Challenge Results Using CT versus CpG as the Adjuvant
CpG Adjuvant
Data from Ann Jerse Ph.D.
Conclusions
•The MtrCDE efflux pump is an important part of how gonococci develop resistance to antibiotics
•This pump is also important for bacterial survival during an experimental infection
•Expression of the mtrCDE operon is regulated by cis- and trans-acting factors
•Propose that the MtrCDE efflux is a target for new drug discovery or an antigen for a future vaccine
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Special Thanks To Collaborators
•Ann Jerse Ph.D. and her lab group (Bethesda, USA): all mouse work and antibiotic resistance collaborations for nearly 15 years•Magnus Unemo Ph.D. and Daniel Golparian (Orebro, SE): clinical significance of mtr mutations.•Edward Yu Ph.D. and his lab group (Ames, USA): structural biology of Mtr efflux pump proteins MtrD and MtrE and EPIs.•Fred Sparling M.D. (Chapel Hill, USA)
Shafer Lab 2015
Many thanks to the NIH and VA for funding support