Histidine Residue 94 Is Involved in pH Sensing by Histidine Kinase ArsS of Helicobacter pylori

Citation: M uller S, Go tz M, Beier D ( Histidine Residue 94 Is Involved in pH Sensing by Histidine Kinase ArsS of Helicobacter pylori Stefanie Mu ller 0 Monika Go tz 0 Dagmar Beier 0 Niyaz Ahmed, University of Hyderabad, India 0 Theodor-Boveri-Institut fu r Biowissenschaften, Lehrstuhl f u r Mikrobiologie, Universita t W u rzburg , Am Hubland, Wu rzburg , Germany Background: The ArsRS two-component system is the master regulator of acid adaptation in the human gastric pathogen Helicobacter pylori. Low pH is supposed to trigger the autophosphorylation of the histidine kinase ArsS and the subsequent transfer of the phosphoryl group to its cognate response regulator ArsR which then acts as an activator or repressor of pHresponsive genes. Orthologs of the ArsRS two-component system are also present in H. pylori's close relatives H. hepaticus, Campylobacter jejuni and Wolinella succinogenes which are non-gastric colonizers. Methodology/Principal Findings: In order to investigate the mechanism of acid perception by ArsS, derivatives of H. pylori 26695 expressing ArsS proteins with substitutions of the histidine residues present in its periplasmic input domain were constructed. Analysis of pH-responsive transcription of selected ArsRS target genes in these mutants revealed that H94 is relevant for pH sensing, however, our data indicate that protonatable amino acids other than histidine contribute substantially to acid perception by ArsS. By the construction and analysis of H. pylori mutants carrying arsS allels from the related e-proteobacteria we demonstrate that WS1818 of W. succinogenes efficiently responds to acidic pH. Conclusions/Significance: We show that H94 in the input domain of ArsS is crucial for acid perception in H. pylori 26695. In addition our data suggest that ArsS is able to adopt different conformations depending on the degree of protonation of acidic amino acids in the input domain. This might result in different activation states of the histidine kinase allowing a gradual transcriptional response to low pH conditions. Although retaining considerable similarity to ArsS the orthologous proteins of H. hepaticus and C. jejuni may have evolved to sensors of a different environmental stimulus in accordance with the non gastric habitat of these bacteria. - Funding: This work was funded by a grant from the Deutsche Forschungsgemeinschaft (BE1543/6-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The human pathogen Helicobacter pylori thrives in the mucous layer covering the gastric epithelium. The neutralophilic bacterium has developed efficient mechanisms to cope with both the high acidity encountered during the passage of the stomach lumen in the initial phase of colonization and moderately acidic conditions expected to prevail in the mucous layer. Central to the acid adaptation of H. pylori is the urease system which is essential for maintaining both the cytoplasmic and periplasmic pH near neutrality when the bacteria are exposed to low pH [1]. The cytoplasmic urease enzyme is a nickel-containing dodecameric heterodimer consisting of the subunits UreA and UreB [2] which cleaves urea present in the gastric juice in millimolar concentrations to ammonia and carbon dioxide. Ammonia then acts as a buffering compound in both the cytoplasm and the periplasm. Moreover, carbon dioxide which rapidly diffuses to the periplasmic space is converted to HCO3- by the periplasmic a-carbonic anhydrase providing an additional buffering compound [3]. The enzymatic activity of the cytoplasmic urease is controlled by the inner membrane pH-gated channel UreI, which regulates the access of the substrate urea to the bacterial cell in response to acidic pH [4,5]. Both urease and the channel protein UreI are essential for colonization in several animal infection models [68]. Furthermore, urease-independent mechanisms of pH-homeostasis are likely to exist [9,10]. Accordingly, global transcriptional profiling performed by several research groups revealed the differential expression of 100 to about 280 genes in response to the exposure of H. pylori to low pH [1114]. The ArsRS two-component system is the master regulator of H. pyloris intricate acid response. Acidic pH triggers the autophosphorylation of the histidine kinase ArsS and the subsequent phosphorylation of its cognate response regulator ArsR. Phosphorylated ArsR (ArsR,P) then acts both as an activator and repressor of pH-responsive genes [14]. The ArsR,P regulon comprises the urease genes, the amidase genes amiE and amiF, hp1186 encoding a periplasmic a-carbonic anhydrase, as well as genes encoding antioxidant systems, Ni2+-storage proteins, proteins affecting the composition of the cell envelope and H. pylorispecific proteins of unknown function [1417]. Consistent with a prominent role of the ArsRS two-component system in the transcriptional control of the acid response, arsS null mutants of H. pylori were unable to colonize in a mouse infection model [18]. The metal dependent regulators Fur and NikR also contribute to pH-responsive gene regulation, since Fur- and NikR-deficient mutants showed an aberrant transcription profile upon exposure of H. pylori to low pH [13,19]. Furthermore, it was reported that in the H. pylori strain J99 the two-component system CrdRS (HP1365-HP1364) which positively regulates the expression of the copper resistance determinant CrdAB-CzcAB in response to increasing concentrations of copper ions [20] is also involved in the pH-responsive regulation of major acid-resistance determinants including the urease gene cluster [21]. This regulatory effect was not observed when CrdR-deficient mutants of the H. pylori strains 26695 and G27 were analysed [22]. Recently, the histidine kinase HP0244 which governs the expression of flagellar class II genes was also implicated in pH-responsive transcriptional control [23,24]. However, the ratios of differential expression were modest in an hp0244 negative mutant and differential expression of most target genes including several members of the ArsR,P regulon was detected only at extremely low pH [24]. In this study we investigated the mechanisms by which the sensor protein ArsS perceives acidic pH. It was assumed that protonation of specific amino acid residues in the periplasmic input domain of ArsS eliciting a conformational change of the histidine kinase is involved in pH sensing. Furthermore, we analysed the ability of ArsS orthologs from other members of the e-proteobacteria to respond to acidic pH. Materials and Methods Bacterial strains and growth conditions H. pylori 26695 and G27 are clinical isolates which have been described previously [25,26]. H. pylori strains were grown at 37uC under microaerophilic conditions (Oxoid) on Columbia agar plates containing 5% horse blood, 0.01% c (...truncated)