Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca2+-sensitive fluorescent d yes acid confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-typ e bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial peripla smic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also indu ced a slower increase in the pH of the medium. Pretreatment of the mutant b acteria with the detergent C12E8 before adding urea resulted in rapid eleva tion of bacterial cytoplasmic pH and medium pH, UreI-dependent NH3 generati on by intrabacterial ut ease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusio n of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca2+](in), unless the conditioned medium was first neut ralized to elevate the NH3/NH4+ ratio, Therefore, cellular effects of intra bacterial ammonia generation under acidic conditions are indirect and not t hrough a type IV secretory complex, The pH(in) and [Ca2+](in) elevation tha t causes the NH3/NH4+ ratio to increase after neutralization of infected ga stric juice may contribute to the gastritis seen with H. pylori infection.