A H ⁺ -Gated Urea Channel: The Link Between Helicobacter pylori Urease and Gastric Colonization

Abstract

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.

Mechanism of Proton Gating of a Urea Channel

“…Mutagenic PCR products were digested with XbaI and KpnI (New England Biolabs) and ligated into pcDNA3.1Ϫ (Invitrogen) downstream of a T7 promoter and upstream of a 170-bp poly(A) tract. The poly(A) tract, not present in the parental vector used previously, increased protein expression ϳ5-fold over levels reported in previous studies (7,9). To achieve comparable steady state levels of uptake and to avoid premature equilibration, the time of incubation was shortened to 6 min from the previous time of 30 min.…”

“…This family has homology to the sixtransmembrane segment putative amide bacterial transporters but has none with the 10-transmembrane segment-containing family of urea channels present in multiple species of bacteria and mammals (4 -6). HpUreI has been shown previously to be acid-activated, alternating between open and closed confirmations with only extracellular protons as activating ligands (7)(8)(9)(10).…”

Analysis of geospecific markers for Helicobacter pylori variants in patients from Japan and Nigeria by triple-locus nucleotide sequence typing

“…Diluted DNA (100 ng) was used to amplify an internal fragment from three genes (ureI, atpA and ahpC) which were selected because of their potential roles in core cytoplasmic metabolic housekeeping activities and not in encoding outer-membrane proteins or secreted proteins that might be under greater selection pressure in a stressful environment such as the gut mucosal surface. The ureI locus, which was included previously in a panel of seven housekeeping genes used for a population genetic study (Achtman et al, 1999), is part of the H. pylori urease gene cluster and encodes a urea accessory (transporter) protein thought to be an integral cytoplasmic membrane protein forming a proton-gated urea-channel-regulating cytoplasmic urease (Weeks et al, 2000;Mobley, 2001). The following primers were designed for amplification and sequencing of a 585 bp fragment within the ureI sequence (HP0071) of H. pylori strain 26695: forward primer (ureIS4) 59-GGAAGGAAAAGGCAATGC-39 and reverse primer (ureIAS2) 59-CTAAACGCTCTATGATCA-39.…”

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

“…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). The UreI protein is required for survival of the bacterium in the mouse or the gerbil gastric mucosa (Skouloubris et al 1998) (Mollenhauer-Rektorschek et al 2002).…”