Requirement of Histidine Kinases HP0165 and HP1364 for Acid Resistance in Helicobacter pylori

Abstract

In this study, we investigated a potential requirement of two-component signal transduction systems for acid resistance in Helicobacter pylori. In comparison to a wild-type strain, isogenic strains with null mutations in either HP0165 or HP1364 histidine kinases were impaired in their ability to grow at pH 5.0. The growth of complemented mutant strains was similar to that of the wild-type strain. H. pylori DNA array analyses and transcriptional reporter assays indicated that acid-responsive gene transcription was altered in the HP0165 and HP1364 null mutant strains compared to the parental wild-type strain. These results indicate that intact HP0165 and HP1364 histidine kinases are required for acid resistance in H. pylori.Within the human stomach, Helicobacter pylori encounters a range of acidic pH conditions. The ability of H. pylori to live in this environment is likely to be dependent on regulation of bacterial gene expression in response to pH. Consistent with this view, changes in the pH of the culture medium result in alterations in H. pylori gene expression during growth in vitro (2,4,8,13,23,24,40). Two-component signal transduction systems (TCSTSs), comprised of a sensor histidine kinase and a cognate response regulator, are commonly used by bacteria to detect and respond to environmental signals (reviewed in references 18 and 31). The H. pylori genome is predicted to encode three histidine kinases (3, 33), which have been designated ArsS, AtoS (FlgS, FleS), and CrdS (26,28,38). Other designations include HP0164/HP0165, HP0244, and HP1364 (the respective gene numbers in H. pylori strain 26695) and JHP0151, JHP0229, and JHP1282 (gene numbers in H. pylori strain J99). A genome sequence analysis of H. pylori strain J99 indicated that arsS (JHP0151) is a 1,326-bp open reading frame (ORF) in this strain (3). In contrast, genome sequence analysis of H. pylori strain 26695 indicated the presence of two smaller arsS ORFs, designated HP0165 (519 bp) and HP0164 (762 bp) (33), due to a single nucleotide deletion (at position ϩ497 from the HP0165 translation initiation site). We have sequenced HP0165/HP0164 in our laboratory's copy of H. pylori strain 26695, and it contains a single arsS ORF, similar to that found in strain J99. Consistent with previous studies (8,12,15,28), we use the H. pylori 26695 nomenclature in the current study, and we use the designation HP0165 when describing arsS.H. pylori mutant strains containing deletions of individual histidine kinase genes (HP0165, HP0244, and HP1364) are defective in the capacity to colonize the mouse stomach (27). A recent study (28) reported that the transcription of three genes (HP0119, HP1432, and ureA) was upregulated by acidic pH in a wild-type H. pylori strain but not in an isogenic HP0165 mutant strain. Therefore, it was suggested that the HP0165 histidine kinase may function as an acid sensor (28). Binding of the cognate response regulator (HP0166) to sequences upstream from HP0119 and ureA has been demonstrated experimentally (12,29).Although there is evid...

Regulation of Helicobacter pylori cagA Expression in Response to Salt

“…Extraction of RNA was done using the Trizol method (Invitrogen), and the extracted RNA was further purified with RNeasy spin columns (Qiagen). Labeling of RNA with [ 33 P]dATP was carried out with SuperScript II Reverse Transcriptase (Invitrogen) as described previously (20). Each labeled RNA preparation was then hybridized to H. pylori Panorama arrays (Sigma Genosys) according to the manufacturer's instructions.…”

“…Plasmids containing the cat cassette in the desired orientation were identified by PCR. The plasmid used for construction of the ureA-cat transcriptional fusion has been described previously (20). H. pylori reporter strains were generated by transformation of H. pylori strain 26695 SC#7 with the plasmids described above, and transformants were selected on BB-FBS-0.5% agar plates containing 5 Ag/mL chloramphenicol.…”

Structural Analysis of the DNA-binding Domain of the Helicobacter pylori Response Regulator ArsR

“…Evidence supporting a cognate relationship between ArsS and ArsR includes immediate proximity of the two genes in the H. pylori chromosome and the demonstration that the two purified proteins can participate in a phosphotransfer reaction in vitro (12). H. pylori ArsS null mutants are viable, but such mutants are impaired in the ability to grow in low pH in vitro, and these mutant strains are unable to colonize mice (14,15). Attempts to generate ArsR null mutants have been unsuccessful, which suggests that ArsR is essential for H. pylori viability (12).…”

“…Several recent studies reported that an H. pylori TCS, ArsSArsR (acid-responsive signaling sensor/response regulator), responds to acidic conditions (12)(13)(14)(15)(16)(17)(18)(19)(20). This TCS consists of the histidine kinase protein, ArsS, and the RR protein, ArsR.…”

“…Therefore, H. pylori has evolved mechanisms to survive severe acid shock and grow under moderately acidic conditions. One of the important mechanisms by which H. pylori responds to acidic pH involves regulation of gene expression through a two-component signal transduction system (12)(13)(14)(15)(16)(17)(18)(19)(20).…”