A Novel Mechanism of Enhanced Efflux Pump Expression in Neisseria gonorrhoeae: Implications for Antimicrobial Resistance and Cell Physiology.

摘要

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, a significant global public health concern as it is estimated that over 106 million cases occur each year worldwide. Infection control is complicated by the lack of a vaccine against N. gonorrhoeae and widespread antibiotic resistance mediated by multiple mechanisms. The N. gonorrhoeae MtrCDE multidrug efflux pump confers resistance to a diverse array of antimicrobial agents. Expression of this pump is tightly regulated by a transcriptional repressor, MtrR, and an inducible transcriptional activator, MtrA. The mtrR gene lies 250 base pairs upstream and is transcribed divergently from the mtrCDE operon. Isolates of N. gonorrhoeae expressing clinically significant levels of resistance to MtrCDE substrate antimicrobials often contain mutations in mtrR or in the mtrR-mtrCDE intergenic region. Recently, a C-to-T transition mutation was identified 120 base pairs upstream of the mtrC start codon and shown to be sufficient to confer high-level resistance to MtrCDE substrates. This work was designed to characterize the mechanism by which this mutation, termed mtr120, affects MtrCDE-mediated resistance. It was determined that the mtr120 mutation generates a second, highly active promoter for mtrCDE transcription, resulting in increased MtrCDE production and correspondingly high-level resistance. This novel promoter was shown to act outside of MtrR or MtrA regulation, thereby offering a unique opportunity to study the physiological effects of efflux pump overexpression on gonococcal cells. This work also demonstrates that global transcriptional changes occur in gonococci when mtrCDE is overexpressed by the mtr120 promoter, including the downregulation of a previously uncharacterized transcriptional regulatory protein, GepR, that appears to be involved in regulation of genes important for antimicrobial resistance, including the mtrCDE operon. Disruption of GepR was shown to cause hypersusceptibility to MtrCDE substrates and clinically relevant antibiotics. In summary, this research characterizes a novel mechanism of high-level efflux-mediated antimicrobial resistance with global physiological implications in the gonococcus, and identifies a new transcriptional regulator important for clinically significant levels of antibiotic resistance, which may offer a unique target in the development of novel drugs to combat N. gonorrhoeae..