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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