ELUCIDATING pH SENSITIVITY OF THE SALMONELLA TYPHIMURIUM SPI-2 TRANSCRIPTIONAL REGULATOR SsrB

Abstract

SsrB is a transcriptional regulator that acts as a switch between the virulent and biofilm lifestyles of the non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes of the Salmonella Pathogenicity Island-2 (SPI-2), which are essential for survival and replication within the macrophage. The low pH inside the macrophage is a key inducing factor for the expression of SsrB. Previous studies from the lab demonstrated an increase in the DNA-binding affinity of SsrB at low pH. The molecular basis of this acid pH- sensitivity was unknown, and our work aimed to elucidate its underlying mechanism and functional significance. We employed site-directed mutagenesis, single-molecule techniques and transcriptional assays to reveal the SsrB domain conferring pH-sensing. We report that the SsrB DNA binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, a well-conserved residue in the N-terminal receiver domain, His12, confers pH sensitivity to SsrB, hinting at an allosteric mechanism. Interestingly, the aromatic nature of the histidine residue appears to be important in contributing to this phenomenon. We also report that His12 plays a role in SsrB phosphorylation, as substituting His12 reduced SsrB phosphorylation at neutral pH and abolished pH-dependent differences observed in phosphorylation.