Phosphorylation-dependent and Phosphorylation-independent Regulation of Helicobacter pylori Acid Acclimation by the ArsRS Two-component System

Abstract

Background: The pH-sensitive Helicobacter pylori ArsRS two-component system (TCS) aids survival of this neutralophile in the gastric environment by directly sensing and responding to environmental acidity. ArsS is required for acid-induced trafficking of urease and its accessory proteins to the inner membrane, allowing rapid, urea-dependent cytoplasmic and periplasmic buffering. Expression of ArsR, but not its phosphorylation, is essential for bacterial viability. The aim of this study was to characterize the roles of ArsS and ArsR in the response of H. pylori to acid.

Materials and methods: Wild-type H. pylori and an arsR(D52N) phosphorylation-deficient strain were incubated at acidic or neutral pH. Gene and protein expression, survival, membrane trafficking of urease proteins, urease activity, and internal pH were studied.

Results: Phosphorylation of ArsR is not required for acid survival. ArsS-driven trafficking of urease proteins to the membrane in acid, required for recovery of internal pH, is independent of ArsR phosphorylation. ArsR phosphorylation increases expression of the urease gene cluster, and the loss of negative feedback in a phosphorylation-deficient mutant leads to an increase in total urease activity.

Conclusions: ArsRS has a dual function in acid acclimation: regulation of urease trafficking to UreI at the cytoplasmic membrane, driven by ArsS, and regulation of urease gene cluster expression, driven by phosphorylation of ArsR. ArsS and ArsR work through phosphorylation-dependent and phosphorylation-independent regulatory mechanisms to impact acid acclimation and allow gastric colonization. Furthering understanding of the intricacies of acid acclimation will impact the future development of targeted, nonantibiotic treatment regimens.

Keywords: ArsRS; Helicobacter pylori; acid acclimation; two-component system; urease.

Figure 1

Phosphotransfer from His₆-tagged ArsS to His₆-tagged ArsR(D52N) protein is abolished. ArsS-His₆ protein was incubated with [γ-³²P] ATP before addition of ArsR-His₆ wildtype (WT) or ArsR(D52N) mutant (D52N). Phosphotransfer from ArsS to ArsR was only seen with WT ArsR. No phosphotransfer to ArsR(D52N) was detected. Representative gels analyzed by autoradiography from a phosphotransfer assay with incubation times of 15 (A) and 30 (B) minutes are shown. The presence (+) or absence (−) of ArsS and ArsR protein used in each reaction are indicated above the lanes.

Figure 2

Phosphorylation of ArsR is not required for survival of H. pylori in acid. Wildtype and arsR(D52N) bacteria were incubated in dialysis chambers suspended in 1500 mL media for 4 hours at pH 3.0 or 7.4 with 5 mM urea. Medium pH did not change after 4 hours. Bacteria were then serially diluted and plated in duplicate. Colony counts are expressed as percent of pH 7.4 control for each strain. There was no significant difference in acid survival. N=3 complete experiments, error bars represent SEM, p=0.66.

Figure 3

Total urease activity is increased in the absence of ArsR phosphorylation. Wildtype and arsR(D52N) bacteria were harvested from plates (neutral pH) and lysed. Urease activity was measured. Total urease activity was increased in the arsR(D52N) strain, likely reflecting the absence of negative feedback on the urease gene cluster provided by phosphorylation of ArsR at neutral pH. N=6, error bars represent SEM, p=0.02.

Figure 4

ArsR phosphorylation affects expression of the urease gene cluster and α-carbonic anhydrase. Wildtype and arsR(D52N) strains were incubated at pH 3.0 and 7.4 for 4 hours, followed by RNA isolation and subsequent qPCR. The level of ureA transcript of the arsR(D52N) strain was identical to the wildtype at both pHs and there was a 2 fold increase at pH 3.0 vs pH 7.4. The level of ureB, ureI, ureE, ureF, ureG, and ureH transcripts of the arsR(D52N) strain were decreased at both pHs. In contrast, the relative difference between the level of transcript at pH 3.0 and pH 7.4 were the same for both the wildtype and arsR(D52N) strains. The level of α-carbonic anhydrase transcript of the arsR(D52N) strain was twice that of the wildtype strain at pH 3.0 and pH 7.4 and there was an increase in transcription of both strains when challenged with acid. n=3, error bars represent standard deviation. SQ refers to the starting quantity of RNA.

Figure 5

Trafficking of urease proteins to the membrane in acid is not affected by absence of phosphorylation of ArsR, but overall protein levels are decreased. Wildtype, ΔarsS and arsR(D52N) bacteria were incubated at pH 4.5 or 7.4 for 30 minutes without urea. Membranes were then purified and fractionated by SDS-PAGE. UreA and UreB were detected by Western blot. Representative blots for each antibody are shown in (A). Densitometry at pH 4.5 expressed as percent of pH 7.4 for each strain is shown in (B). N=3 for each condition, error bars represent the s.e.m.

Figure 6

Urea addition increases cytoplasmic pH in acid. Cytoplasmic pH was monitored using the pH sensitive fluorophore, BCECF-AM. Cytoplasmic pH decreased in the wildtype (black), arsR(D52N) (grey) and ΔarsS (light grey) strains when presented with an acid challenge (pH 4.5). The addition of 5 mM urea resulted in a rapid increase of cytoplasmic pH in the wildtype and arsR(D52N) strains, but not the ΔarsS strain. These results demonstrate that ArsS is required for UreI/urease complex function independent of ArsR phosphorylation

Figure 7

UreA, UreE, and UreI proteins are decreased in the arsR(D52N) strain in a pH independent manner. Wildtype and arsR(D52N) bacteria were incubated in dialysis cassettes suspended in media for 4 hours at pH 3.0 or 7.4 with 5 mM urea. Medium pH did not change after 4 hours. Bacteria were then lysed, fractionated by SDS-PAGE, and proteins detected by Western blot. Representative blots for each antibody are shown in the top portion of the figure, and averaged densitometry results are shown in the graph on the bottom. N=3 for each condition, pH_o = medium pH, error bars represent standard deviation.