Helicobacter pylori is a gram-negative bacterium that colonizes the mucosal layer of the human stomach. Today, nearly half of the world population is infected with H. pylori. This infection leads to chronic inflammation, and potentially peptic ulcer disease, or gastric cancer. Developing therapeutics based on the colonization mechanism of this bacterium holds great promise as a therapeutic paradigm to promote human gastric health. To adapt to the hostile acidic environment in human stomach, H. pylori utilizes a Two-Component Signal Transduction system (TCS), ArsRS, to mediate the expression of acid response genes, such as the adhesin gene sabA and the urease component gene ureA. Here, with Electrophoretic Mobility Shift Assays (EMSA), we further demonstrated the importance of ArsR in adaptation and adherence by showing its binding activities in the promoter regions of other adhesin genes such as labA, hopZ, and sabB. Our lab has previously shown that the substitution with a phosphomimetic amino acid, such as glutamic acid (ArsR D52E) and asparagine (ArsR D52N), in the highly conserved 52nd position in ArsR preserves its acid response function, despite its inability to serve as a phosphoacceptor. Here, through site-directed mutagenesis and real-time qPCR, we discovered that the alanine substitution for adjacent aspartic acids at the 47th and 59th positions of mutant D52E retained normal sabA repression under pH5, but showed an increase in ureA induction under pH5. This study also demonstrates that ArsRS TCS failed to regulate sabA and ureA in response to pH 5 exposure when the phophoacepting histidine (H214) in ArsS was substituted with alanine (ArsS H214A), which disabled its enzymatic function. Furthermore, a kinetics experiment under constant acidity revealed that in ArsR D52E mutant, the repression of sabA occurred immediately upon exposure to acidity (time 0 min), peaked at time 150 min, and disappeared at time 210 min. The induction of ureA started at time 60 min, maximized at time 120 min, and stopped at 210 min. Together, our results demonstrate the complex nature of this TCS system. We speculate that this complex acclimation to the acid regimen of the stomach helps facilitate the decades long infection of arguably the most extreme environment associated with the human condition
