| Helicobacter pylori, is a Gram-negative bacterium that colonizes the gastric mucosa causing chronic gastritis which can progress to peptic ulcer and gastric cancer. H. pylori is a neutralophile and depends on bacterial urease activity to generate sufficient ammonia to combat acidity in vivo and in vitro. The H. pylori urease has a neutral pH optimum that functions effectively at the neutral pH of the bacterial cytoplasm. The activity of intrabacterial urease increases at least 20 fold with medium acidification, half maximally at pH 6.0, and maintains the proton-motive force across the inner membrane by buffering the bacterial periplasm. Intrabacterial urease activity is controlled by an acid-gated urea channel, UreI, that enhances urea permeability 300-fold at acidic pH. UreI is shown to have six transmembrane spanning segments, two periplasmic loops and periplasmic C- and N-termini. Expression of UreI in Xenopus oocytes results in urea transport that is increased ∼10-fold by decreasing external pH and is also half-maximal at pH 6.0. S. salivarius expresses a UreI homolog, 84% similar in the membrane domain, that enhances urea transport equally at neutral and acidic pH. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI shows that these residues in the first periplasmic loop, PL1, are not involved in acid activation whereas His123, His131, Asp 129, Asp 140, Glu138, Lys132 in PL2, and His 193 in the C-terminus (Ct) are important for transport. These amino acids are absent in SsUreI. Chimeras, where PL1 or PL1 and Ct of HpUreI are replaced by the corresponding regions of SsUreI, retain activity at acidic pH and are 60% active at neutral pH. Substitution of PL2 inactivates HpUreI. Chimeras, where either PL1 or PL2 of HpUreI substitutes for of the corresponding loop of SsUreI, retain wild-type transport, but replacement of Ct or both loops abolishes transport. PL1, in HpUreI, appears important for suppression of transport at neutral pH, whereas protonation of three histidines in PL2 and Ct, possibly charge-pairing with three carboxylic amino acids in PL2, seem necessary for acid activation. The periplasmic domain of HpUreI is, therefore, the region important for regulation of urea transport. |
