CpxR and HicB exert independent regulatory action on the gonococcal hicAB -encoded toxin-antitoxin system

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to front-line antibiotics has focused efforts on understanding how alternative therapies, such as the expanded use of gentamicin (Gen), might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for the treatment of gonorrheal infections, we previously used RNA-seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level and showed that expression of the putative HicA-HicB toxin-antitoxin (TA) system was increased in response to sub-lethal Gen. Importantly, loss of this TA system resulted in reduction of Ng biofilm formation in a strain specific manner. Focusing on this strain specificity, we found that the CpxR/CpxA two-component system (TCS) influences expression of the hicAB operon independently of HicB autoregulation. We now report that CpxR selectively binds to the hicAB operon to enhance expression of hicAB but does not interfere with binding of HicB to the promoter region. Furthermore, we show that single base pair differences in the intergenic region between hicA and hicB impact regulation by CpxR. Hence, the regulation of the HicAB TA in gonococcal strains is a highly coordinated response that can involve autoregulation by HicB and the CpxRA TCS. We propose that this dual regulatory scheme maximizes the ability of Ng to respond to Gen and hostile environmental conditions.

Antibiotic concentrations within the body change over time as the drug is absorbed and spread through tissues and later eliminated. Therefore, during antibiotic treatment of disease, bacteria that survive the initial high dose exposure could be exposed to sub-lethal concentrations until the antibiotic clears. Currently, little is understood about the response of Ng to sub-lethal antibiotic concentrations; we believe closing this gap is crucial to combat antibiotic resistance and disease spread. We hypothesize that during antibiotic treatment, these sub-lethal concentrations could serve as a stress signal, allowing for internal changes that increase bacterial survival. In our previous work, we showed that sub-lethal levels of the aminoglycoside gentamicin can influence levels of the Ng HicAB toxin-antitoxin system. Here, we show that a two-component system CpxRA, a major stress response system in bacteria, directly influences expression of hicAB . Furthermore, we find that CpxR and HicB work synergistically, but independently, to regulate expression of the hicAB locus. Our study highlights the importance of understanding the complex interplay between regulatory systems and provides new insight into how Ng can use multiple stress responses simultaneously to survive the threat posed by antibiotics.