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.

Keywords: Neisseria gonorrhoea; antimicrobial resistance; efflux; regulation; transcription.

Fig. 1.

Genomic locus and structural organization of the MtrCDE efflux pump. (a) Genomic localization of the mtr locus in the gonococcal reference strain FA1090. (b) Structure and stoichiometry of the MtrCDE efflux pump localized across the bacterial membranes. The structure of the pump components was drawn using the 3D viewer of the Protein Data Bank [76] and the PDB entries 4MT0 for MtrE [77], 4MT1 for MtrD [74] and 2K32 (AcrA of Campylobacter jejuni ) for MtrC [78]. (c) Bottom view of pump structure from the cytoplasm. OM, outer membrane; PGN, peptidoglycan; IM, inner membrane.

Fig. 2.

Cis-acting elements controlling transcription of the mtr locus. Alignment of the mtrC/mtrR intergenic sequences of wild-type strain FA19 and a clinical isolate bearing a mosaic-like mtr locus (NCBI accession: SRR15204408) was done with Clustal-O. The mtrC and mtrR start codons are indicated in bold font. Promoter −10 and −35 elements, transcriptional start sites (+1) and ribosome-binding site (SD) are underlined and indicated on top of the sequence for mtrC (red font, determined in Ref. [12]) and mtrR (blue font, determined in Ref. [26]). The positions for the mtr120 [42], the A-to-C transition in the −35 region of mtrC that occurs in mosaic mtr sequences [29] and the Adenine deletion [26, 63] mutations are highlighted in yellow. The black double arrow line represents the inverted repeat sequence (IRS) present in the spacer of the mtrR promoter elements.

Fig. 3.

Trans-acting factors controlling transcription of the mtrCDE operon. (a) The transcriptional regulators MtrR and MtrA act directly on the mtrC promoter to differentially regulate transcription. MtrR is the repressor of the operon while binding to the promoter region in the form of an apo dimer (i.e. in the absence of known inducers such as bile salts, which induce structural changes that are incompatible with binding to DNA) [45, 47]. MtrA induces mtrCDE transcription when gonococci are exposed to sub-lethal levels of antimicrobials [60]. MtrA increases its binding affinity for the mtrC promoter in the presence of the known pump substrate Triton X-100, most likely acting as an MtrR anti-repressor [46]. The DNase I-protected regions for MtrR (determined in Ref. [44]) and MtrA (determined in Ref. [46]) are indicated by striped boxes relative to the mtrC (red font) and mtrR (blue font) −10 and −35 promoter elements. 5′UTR, 5′ untranslated region; IRS, 13 bp inverted repeat sequence. (b) DNA sequence required for MtrR (left, determined in Ref. [45]) and MtrA (right, determined in Ref. [46]) binding. For the MtrR-recognized sequence the two grey boxes indicate the sequence recognized by each monomer with nucleotide bases making direct contact with the HTH domain highlighted in yellow. For MtrA, nucleotide bases highlighted in yellow indicate bases required for MtrA binding determined by mutagenesis [46]. (c) Factors controlling the gene expression or DNA-binding activity of MtrR that influence mtrCDE gene expression, such as the AraC-like regulator MpeR and the iron sensor regulator Fur [58], bile salts [47] and autorepression of its own gene [29, 45]. →, transcriptional activation; ─┤, transcriptional repression/DNA-binding inactivation.

Fig. 4.

The gonococcal MtrR regulon. (a) The genome-wide MtrR regulon was determined by microarray from gonococcal cells grown to mid-logarithmic phase [28]. Repressed genes are shown in red and genes activated are in green and these designations are labelled as such. Deviation from the average GC content of the genome is depicted by the black plot. (b) MtrR is a direct repressor of multiple genes controlling antimicrobial resistance [44], the stress response sigma factor RpoH that activates expression of multiple genes (grpE, clpB, dnaJ and dnaK) [28] and virulence gene expression [48, 79]. →, transcriptional activation; ─┤, transcriptional repression.