Sites of pH regulation of the urea channel of Helicobacter pylori.

David L. Weeks and George Sachs*University of California, Los Angeles, and Building 113,Room 324, VA Greater Los Angeles Health Care System,Los Angeles, CA 90073, USA.SummaryHelicobacter pylori (Hp) and Streptococcus salivarius(Ss) require intrabacterial urease for acid resistanceandexpressaureachannel,UreI.ThepresenceofUreIwas shown to increase urea permeability <300-foldover that of a non-polar ureI deletion mutant.Expression of SsUreI in Xenopus oocytes increasedurea uptake pH independently, whereas HpUreIshows an acidic pH dependence, half-maximal atpH6.0. Mutagenesis of all histidines, aspartates,glutamates and the lysine in the periplasmic domainof HpUreI showed that His-123, His-131, Asp-129,Asp-140, Glu-138 and Lys-132 in the second periplas-mic loop (PL2) and His-193 in the C-terminus (Ct) wereimportant for activation of transport. With theexception of a lysine that was shown to substitutefor His-193 in HpUreI, these charged amino acids areabsent in SsUreI. A chimera in which PL1 of HpUreIwas replaced by PL1 of SsUreI retained activity atacidic pH and gained partial activity at neutralpH. Exchange of PL2 inactivated transport, whereasexchange of Ct had no effect. Chimeras, in whicheither PL1 or PL2 of HpUreI replaced those of SsUreI,retained wild-type transport, but replacement ofthe Ctor both loops inactivated transport. PL1 appears to beimportant for restricting transport through HpUreI atneutral pH, whereas protonation of three histidines inPL2 and Ct and the presence of three dicarboxylicamino acids in PL2 appears to be necessary toactivate HpUreI at acidic pH.IntroductionThe urease of neutralophilic microorganisms can servedifferent functions. The ammonia produced can provide anitrogen source, but it can also buffer the cytoplasm orperiplasm to allow acid resistance. The former functionhas been demonstrated for both Helicobacter pylori (Hp)and Streptococcus salivarius (Ss) (Mobley et al. 1995;Chen etal., 2000). Thesecond function, acid resistance, isdiscussed here.Various Helicobacter spp. are acid-tolerant neutralo-philes that colonize mammalian stomachs. H. pylori isthe human gastric pathogen responsible for peptic ulcerdisease and increased risk of gastric cancer (Covacciet al., 1999). UreI, a six-transmembrane segment ureachannel encoded in the urease gene cluster of H. pylori,isa unique adaptation to the varying acidity of the gastricenvironment. Urea transport by UreI allows this pathogento use a constitutively expressed, neutral pH optimumintrabacterial urease for acid resistance (Weeks et al.,2000) and gastric colonization (Skouloubris et al., 1998).Channel closure at neutral pH prevents excessiveammonia generation by this urease. There is no homologybetween UreI and other non-ionic channels such asaquaporins (Scheuring et al., 1999; Zeuthen and Klaerke,1999), glycerol facilitators (Maurel et al., 1994) ormammalian urea transporters (Hediger et al., 1996).Streptococcus salivarius, a neutralophile inhabiting themouth, also produces urease and UreI (Sissons et al.,1990). In contrast to the urease genes of H. pylori, all eightstructural and accessory genes are expressed mainlywhen cells are grown at an acidic pH similar to thoseencountered in the mouth as a result of bacterial glycolysis(Chen et al., 1998). Urease activity is required for acidresistance of S. salivarius; however, the function of UreI,the ﬁrst gene in the operon, 64% homologous to UreI ofH. pylori, has not been determined (Chen et al., 2000)Here, SsUreI is shown to facilitate urea transport at bothneutral and acidic pH.The urease of H. pylori is largely intracellular, althoughas much as 10% can be found on the surface in oldercultures as a result of bacterial autolysis (Phadnis et al.,1996). Activation of urea transport by UreI increasesurease activity of intact bacteria 10- to 40-fold as mediumpH falls between pH6.5 and 5.5 (Rektorschek et al., 1998;Scott et al., 1998; Weeks et al., 2000). Urease activityremains elevated down to pH2.0, a pH far below that atwhich surface urease becomes inactivated. Non-polardeletion of ureI abolishes the pH-induced increase inintrabacterial urease activity, while allowing normal ureasebiosynthesis (Weeks et al., 2000). Also, the effects of ureI