Acid pH Sensing by EnvZ–OmpR Alters H-bonding Networks to Allosterically Activate Virulence Gene Expression

The molecular basis for how acidic pH environments trigger noncanonical phosphorylation-independent conformational changes in the EnvZ–OmpR two-component signal transduction system for turning on virulence gene expression is not understood. Using amide hydrogen–deuterium exchange mass spectrometry (HDXMS), we compared kinetics of deuterium exchange of the cytosolic domain of EnvZ (EnvZc) with its cognate response regulator (RR) OmpR under conditions mimicking the intracellular environments encountered during host phagocytosis (pH range – 6.5–7.5). At pH 6.5 compared to pH 7.5, EnvZc showed lower deuterium exchange in its four-helical bundle subdomain, particularly in the region surrounding the conserved His 243, indicating acid stabilization. Similarly, pH-dependent changes were observed in OmpR, notably in peptides associated with its aromatic switch in the receiver and DNA binding domains. Interestingly, acidic pH elicited changes in the same allosteric loci associated with phosphorylation-dependent regulation in response to osmosensing. These findings suggest that the EnvZ–OmpR system adapts to acidic environments via a mechanism distinct from canonical phosphorylation but with different gene expression outcomes to favor expression of virulence factor genes. The study provides new insights into bacterial adaptation to host-induced stress and highlights potential targets for antimicrobial development.