The role of cyclic nucleotide-binding efflux pumps in Mycobacterium tuberculosis and antimicrobial resistance
File(s)
Author(s)
Tounta, Vivian
Type
Thesis
Abstract
Tuberculosis (TB) is an ancient disease caused by the intracellular bacterial pathogen
Mycobacterium tuberculosis (Mtb), and even to this day it poses a global health challenge.
The rise of antimicrobial resistance (AMR) threatens to bring TB to the forefront of bacterial
pathogens as the current treatments are increasingly becoming ineffective. Understanding the
development of AMR and the virulence processes of Mtb is crucial for the identification of new
drug targets and the rational design of anti-TB treatments. One of the established mechanisms
of resistance is through the function of efflux proteins, transmembrane transporters that bind
and remove antibiotic molecules out from the cell. The Major Facilitator Superfamily (MFS) of
efflux pumps is a large and diverse family, and almost 65 % of putative MFS proteins in Mtb
have been annotated as multidrug efflux transporters. Two of these putative multidrug
proteins, Rv3239c and Rv3728, were also predicted to bind 3',5'-cyclic adenosine
monophosphate (cAMP). The second messenger cAMP is significant for Mtb virulence,
adaptation, and survival during infection. cAMP translates external signals and starts an
intracellular downstream cascade of events by binding effector proteins. During my PhD, I
focused on characterising the structure and function of Rv3239c and Rv3728 in Mtb using a
combination of bioinformatic models and biochemical experiments. Conserved profile
recognition demonstrated that the proteins were comprised of 3 domains: a transmembrane
MFS, and intracellular cyclic nucleotide binding (CNB) and patatin-like phospholipase domains
(PLP). Based on the predictive model, Rv3239c and Rv3728 are antiporters that couple efflux
of substrates to the import of protons and belong to the drug/proton antiporter 2 (DHA2)
subfamily. Using a combination of an M. smegmatis model and Mtb, I found that transporter
expression has a correlation to higher MIC to isoniazid, one of the front-line antibiotics used
to treat TB. I also present the first experimental evidence to suggest Rv3728 and Rv3239c
have efflux function and that Rv3728 can bind cAMP in vitro which can be decreased
significantly by mutating key residues in the CNB. This work offers some of the first insights
into the structure and function of Rv3239c and Rv3728 and provides the necessary constructs
to complete their characterisation in the future.
Mycobacterium tuberculosis (Mtb), and even to this day it poses a global health challenge.
The rise of antimicrobial resistance (AMR) threatens to bring TB to the forefront of bacterial
pathogens as the current treatments are increasingly becoming ineffective. Understanding the
development of AMR and the virulence processes of Mtb is crucial for the identification of new
drug targets and the rational design of anti-TB treatments. One of the established mechanisms
of resistance is through the function of efflux proteins, transmembrane transporters that bind
and remove antibiotic molecules out from the cell. The Major Facilitator Superfamily (MFS) of
efflux pumps is a large and diverse family, and almost 65 % of putative MFS proteins in Mtb
have been annotated as multidrug efflux transporters. Two of these putative multidrug
proteins, Rv3239c and Rv3728, were also predicted to bind 3',5'-cyclic adenosine
monophosphate (cAMP). The second messenger cAMP is significant for Mtb virulence,
adaptation, and survival during infection. cAMP translates external signals and starts an
intracellular downstream cascade of events by binding effector proteins. During my PhD, I
focused on characterising the structure and function of Rv3239c and Rv3728 in Mtb using a
combination of bioinformatic models and biochemical experiments. Conserved profile
recognition demonstrated that the proteins were comprised of 3 domains: a transmembrane
MFS, and intracellular cyclic nucleotide binding (CNB) and patatin-like phospholipase domains
(PLP). Based on the predictive model, Rv3239c and Rv3728 are antiporters that couple efflux
of substrates to the import of protons and belong to the drug/proton antiporter 2 (DHA2)
subfamily. Using a combination of an M. smegmatis model and Mtb, I found that transporter
expression has a correlation to higher MIC to isoniazid, one of the front-line antibiotics used
to treat TB. I also present the first experimental evidence to suggest Rv3728 and Rv3239c
have efflux function and that Rv3728 can bind cAMP in vitro which can be decreased
significantly by mutating key residues in the CNB. This work offers some of the first insights
into the structure and function of Rv3239c and Rv3728 and provides the necessary constructs
to complete their characterisation in the future.
Version
Open Access
Date Issued
2023-04-27
Date Awarded
01/09/2023
License URL
Advisor
Larrouy-Maumus, Gerald
Meier, Thomas
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Grant Number
BB/M011178/1
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)