Acidic media trigger cytoplasmic urease activity of the unique human gastric pathogen Helicobacter pylori. Deletion of ureI prevents this activation of cytoplasmic urease that is essential for bacterial acid resistance. UreI is an inner membrane protein with six transmembrane segments as shown by in vitro transcription/translation and membrane separation. Expression of UreI in Xenopus oocytes results in acid-stimulated urea uptake, with a pH profile similar to activation of cytoplasmic urease. Mutation of periplasmic histidine 123 abolishes stimulation. UreI-mediated transport is urea specific, passive, nonsaturable, nonelectrogenic, and temperature independent. UreI functions as a H+-gated urea channel regulating cytoplasmic urease that is essential for gastric survival and colonization.

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ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced H. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

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Helicobacter pylori is a Gram-negative neutralophile associated with peptic ulcers and gastric cancer. It has a unique ability to colonize the human stomach by acid acclimation. It uses the pH-gated urea channel, UreI, to enhance urea access to intrabacterial urease and a membrane-anchored periplasmic carbonic anhydrase to regulate periplasmic pH to approximately 6.1 in acidic media, whereas other neutralophiles cannot regulate periplasmic pH and thus only transit the stomach.

Survival of Helicobacter pylori in acid depends on intrabacterial urease. This urease is a Ni(2+)-containing oligomeric heterodimer. Regulation of its activity and assembly is important for gastric habitation by this neutralophile. The gene complex encodes catalytic subunits (ureA/B), an acid-gated urea channel (ureI), and accessory assembly proteins (ureE-H). With the use of yeast two-hybrid analysis for determining protein-protein interactions, UreF as bait identified four interacting sequences encoding UreH, whereas UreG as bait detected five UreE sequences. These results were confirmed by coimmunoprecipitation and beta-galactosidase assays. Native PAGE immunoblotting of H. pylori inner membranes showed interaction of UreA/B with UreI, whereas UreI deletion mutants lacked this protein interaction. Deletion of ureE-H did not affect this interaction with UreI. Hence, the accessory proteins UreE/G and UreF/H form dimeric complexes and UreA/B form a membrane complex with UreI, perhaps enabling assembly of the urease apoenzyme at the membrane surface and immediate urea access to intrabacterial urease to allow rapid periplasmic neutralization.

Helicobacter pylori is a neutralophilic, gram-negative, ureolytic organism that is able to colonize the human stomach but does not survive in a defined medium with a pH <4.0 unless urea is present. In order to live in the gastric environment, it has developed a repertoire of acid resistance mechanisms that can be classified into time-independent, acute, and chronic responses. Time-independent acid resistance depends on the structure of the organism's inner and outer membrane proteins that have a high isoelectric point, thereby reducing their proton permeability. Acute acid resistance depends on the constitutive synthesis of a neutral pH optimum urease that is an oligomeric Ni(2+)-containing heterodimer of UreA and UreB subunits. Gastric juice urea is able to rapidly access intrabacterial urease when the periplasmic pH falls below approximately 6.2 owing to pH-gating of a urea channel, UreI. This results in the formation of NH3, which then neutralizes the bacterial periplasm to provide a pH of approximately 6.2 and an inner membrane potential of -101 mV, giving a proton motive force of approximately -200 mV. UreI is a six-transmembrane segment protein, with homology to the amiS genes of the amidase gene cluster and to UreI of Helicobacter hepaticus and Streptococcus salivarius. Expression of these UreI proteins in Xenopus oocytes has shown that UreI of H. pylori and H. hepaticus can transport urea only at acidic pH, whereas that of S. salivarius is open at both neutral and acidic pH. Site-directed mutagenesis and chimeric analysis have identified amino acids implicated in maintaining the closed state of the channel at neutral pH and other amino acids that play a structural role in channel function. Deletion of ureI abolishes the ability of the organism to survive in acid and also to colonize the mouse or gerbil stomach. However, if acid secretion is inhibited in gerbils, the deletion mutants do colonize but are eradicated when acid secretion is allowed to return, showing that UreI is essential for gastric survival and that the habitat of H. pylori at the gastric surface must fall to pH 3.5 or below. The chronic response is from increased Ni(2+) insertion into the apo-enzyme, which results in a threefold increase in urease, which is also dependent on expression of UreI. This allows the organism to live in either gastric fundus or gastric antrum depending on the level of acidity at the gastric surface. There are other effects of acid on transcript stability that may alter levels of protein synthesis in acid. Incubation of the organism at acidic pH also results in regulation of expression of a variety of genes, such as some outer membrane proteins, that constitutes an acid tolerance response. Understanding of these acid resistance and tolerance responses should provide novel eradication therapies for this carcinogenic gastric pathogen.