Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

Abstract

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3′,4′,5‐tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB‐kanR‐EFGH operon all showed wild‐type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild‐type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid‐derived H. pylori ureI restored wild‐type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy‐ethylene‐8‐laurylether; C12E8), suggested a membrane‐limited access of urea to internal urease at neutral pH. Measurement of 14C‐urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI‐independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.

Mechanism of Proton Gating of a Urea Channel

“…However, with regards to characterization of one key residue, His-123, the results of Bury-Mone et al (10) were found to contradict the findings obtained using Xenopus oocytes. With the exception of this report by Bury-Mone and colleagues (10), functional descriptions of HpUreI expressed in oocytes and H. pylori have been indistinguishable (17). Further, the urea channel of Yersinia pseudotuberculosis, Yut, has been shown to function similarly both in oocytes and in H. pylori (4).…”

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells

“…Mutants were made according to a published procedure that is only presented in outline here (30). Standard procedures (31) were used for isolation and treatment of plasmid DNA.…”

“…The electroporated bacteria were plated onto LB plates (LB agar base; GIBCO BRL) supplemented with 50 mg/mL ampicillin. The DNA was generated by PCR of selected primers as detailed previously (30).…”

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

“…UreI (21.7 kDa) is a proton-gated inner membrane protein involved in urea transport as well as acid resistance (Rektorschek et al 2000;Skouloubris et al 1998). UreI possesses six membrane-spanning segments and conserved histidine residues, including His123, are essential for acid activation of the channel (Bury-Mone et al 2001;Weeks et al 2000;Weeks and Sachs 2001).…”