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Transcriptional regulation of the mtrCDE efflux pump operon: importance for Neisseria gonorrhoeae antimicrobial resistance.

ABSTRACT: This review focuses on the mechanisms of transcriptional control of an important multidrug efflux pump system (MtrCDE) possessed by Neisseria gonorrhoeae, the aetiological agent of the sexually transmitted infection termed gonorrhoea. The mtrCDE operon that encodes this tripartite protein efflux pump is subject to both cis- and trans-acting transcriptional factors that negatively or positively influence expression. Critically, levels of MtrCDE can influence levels of gonococcal susceptibility to classical antibiotics, host-derived antimicrobials and various biocides. The regulatory systems that control mtrCDE can have profound influences on the capacity of gonococci to resist current and past antibiotic therapy regimens as well as virulence. The emergence, mechanisms of action and clinical significance of the transcriptional regulatory systems that impact mtrCDE expression in gonococci are reviewed here with the aim of linking bacterial antimicrobial resistance with multidrug efflux capability.

SUBMITTER: Ayala JC

PROVIDER: S-EPMC10235802 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Transcriptional regulation of the mtrCDE efflux pump operon: importance for Neisseria gonorrhoeae antimicrobial resistance.

Ayala Julio C JC Balthazar Jacqueline T JT Shafer William M WM

Microbiology (Reading, England) 20220801 8

This review focuses on the mechanisms of transcriptional control of an important multidrug efflux pump system (MtrCDE) possessed by Neisseria gonorrhoeae, the aetiological agent of the sexually transmitted infection termed gonorrhoea. The mtrCDE operon that encodes this tripartite protein efflux pump is subject to both cis- and trans-acting transcriptional factors that negatively or positively influence expression. Critically, levels of MtrCDE can influence levels of ...[more]

PMID: 35916832

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Project description:Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hot spot of interspecific recombination introducing alleles from Neisseria meningitidis and Neisseria lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic mtrD allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel mtrD alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents.IMPORTANCENeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through mtr alleles from other Neisseria species, demonstrating that commensal Neisseria bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.

Project description:UNLABELLED:MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20-30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci. IMPORTANCE:Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a "superbug." One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.

Project description:Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

Dataset Information

Transcriptional regulation of the mtrCDE efflux pump operon: importance for Neisseria gonorrhoeae antimicrobial resistance.

Publications

Transcriptional regulation of the <i>mtrCDE</i> efflux pump operon: importance for <i>Neisseria gonorrhoeae</i> antimicrobial resistance.

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