The Helicobacter pylori UreI protein: role in adaptation to acidity and identification of residues essential for its activity and for acid activation

Abstract

Helicobacter pylori is a human gastric pathogen that survives the strong acidity of the stomach by virtue of its urease activity. This activity produces ammonia, which neutralizes the bacterial microenvironment. UreI, an inner membrane protein, is essential for resistance to low pH and for the gastric colonization of mice by H. pylori. In the heterologous Xenopus oocytes expression system, UreI behaves like an H+‐gated urea channel, and His‐123 was found to be important for low pH activation. We investigated the role of UreI directly in H. pylori and showed that, in the presence of urea, strains expressing wild‐type UreI displayed very rapid stimulation of extracellular ammonia production upon exposure to pH ≤ 5. This response was not observed when acetamide was used as a source of ammonia; therefore, it is specific for urea hydrolysis. To identify residues critical for UreI activity or activation, we constructed H. pylori strains carrying individual chromosomal mutations of UreI (i) in the four conserved histidine residues (H71, H123, H131, H193) and (ii) in a conserved region of the third intracellular loop (L165, G166, K167, F168). The distal H193 (and not H123) was found to be crucial for stimulating the production of ammonia at low pH; a single mutation in this residue uncoupled the UreI activity from its acid activation. The third intracellular loop of UreI was shown to be important for UreI activity. Thus, in H. pylori, UreI is necessary for the adaptation of urease activity to the extracellular pH. UreI behaves like a novel type of urea transporter, and the identification of residues essential for its function in H. pylori provides new insight into the unusual molecular mechanism of low pH activation.

Production of anti-Helicobacter pylori urease-specific immunoglobulin in egg yolk using an antigenic epitope of H. pylori urease

“…To address this problem, we identified the following H. pylori proteins as those that reacted immunodominantly to IgY-Hp: UreA, UreB, heatshock proteins (HSP60), probable peroxiredoxin and probable thiol peroxidase (Shin et al, 2003). Of the five immunodominant proteins, urease plays a pivotal role in the pathogenesis of H. pylori infection by protecting the bacteria from the acid environment of the stomach, promoting colonization and inducing the production of ammonia (Bury-Mone et al, 2001;Graham et al, 1992). Urease also appears to be necessary for Helicobacter organisms to establish an infection.…”

“…In a previous study, we elucidated the five immunodominant H. pylori proteins that react strongly with IgY-Hp to address the limitations of IgY-Hp (Shin et al, 2003). Among the immunodominant proteins identified was urease, a wellknown critical virulence factor (Bury-Mone et al, 2001) and the antigen most often studied in vaccine trials.…”

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).…”

Mechanism of Proton Gating of a Urea Channel

“…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).…”

“…Being composed of only 170 amino acids, HhUreI represents the simplest known acid-gated channel. In contrast to mutations of HpUreI (9,10,14), active mutants of all six protonatable periplasmic amino acids of HhUreI could be generated with almost full retention of urea transport at acidic pH, allowing analysis of each of their roles in pH gating. While it was possible to observe acid activation in histidine-less mutants, both histidines and carboxylic acid residues likely coordinate to create a distributed pH sensor with a pH 0.5 of 6.8, nearly one pH unit higher than in HpUreI.…”