Regulatory mechanisms that impact Neisseria gonorrhoeae survival of host innate immunity and antibiotics: The roles of LptA, TbpBA, and MisR

Neisseria gonorrhoeae is a Gram-negative bacterium and causes the sexually transmitted infection, gonorrhea. Gonococci can be cultured from purulent exudate in symptomatic individuals, which also contains numerous polymorphonuclear cells (neutrophils). This potent proinflammatory response is effective largely due to the killing action of cationic antimicrobial peptides (CAMPs) and proteins, but not all gonococci are killed. To further explore how gonococci survive the innate immune response, we investigated the importance of a two component regulatory system response regulator, MisR, for gonococcal resistance to CAMPs. Though loss of MisR did not affect the function of the LptA lipid A phosphoethanolamine transferase required for gonococcal resistance to CAMPs, we observed that lptA can be transcribed from two distinct promoters, and is post-transcriptionally regulated by a phase variable poly-T8 tract present within the lptA open reading frame. Importantly, lptA-deficient gonococci are much less fit than WT gonococci in competitive infections of mice and men. RNA-Seq analysis demonstrated that MisR significantly regulates the transcription of nearly 100 genes (including the transferrin-binding protein genes tbpB and tbpA, which are essential for in vivo survival). Interestingly, we found a previously unknown RNA species that impacts tbpBA transcript and TbpBA protein levels by an undefined mechanism. Phagocytosis of gonococci by human macrophage-like monocytic cells greatly upregulated transcription of tbpBA (and other iron-responsive genes), and did not kill 100% of the internalized bacteria, suggesting that the iron-limiting environment of the macrophage interior can be sensed by gonococci to increase survival during the ironlimiting innate immune response. Loss of MisR increased susceptibility to CAMPs and aminoglycosides by approximately the same factor (4-8 fold). Furthermore, function (but not expression) of the MtrCDE antimicrobial efflux pump was impaired by loss of MisR in both WT and mtrCDE-overexpressor genetic backgrounds, which are common among gonococcal clinical isolates. We propose that loss of MisR increases membrane permeability due to misfolded protein accumulation. The characterization herein of a novel multi-antimicrobial resistance mechanism in gonococci is of special interest in light of the dwindling number of curative antibiotics for gonorrhea, and the approaching implementation of an aminoglycoside (gentamicin) as a first-line therapy in the United States.