Characterization of Bacterial Homologs of Helicobacter pyloris UreI Urea Channel with Yeast Complementation Assays

Description

Chronic gastric infections with Helicobacter pylori, a bacterium that is linked to diseases like peptic ulcer and stomach cancer, are present in over 50% of the world population.
Conventional antibiotic therapies are becoming increasingly ineffective due to the rise in antibiotic resistance. An alternative therapeutic target is the pH-gated inner membrane
urea channel UreI, which is crucial for the survival of the bacteria in the acidic environment of the human stomach. Despite extensive research, including in vivo studies and highresolution
structures in both open and closed states, the precise gating mechanism of HpUreI remains elusive. A comprehensive sequence- and structure-based analysis of
homologous bacterial urea channels revealed intriguing subgroup variants that differ in terms of length, gram status, and charge distribution within the loops. To further elucidate
the molecular basis of UreI gating, we examined bacterial urea channel representatives from clinically relevant organisms within these subgroups using yeast complementation
assays. To assess their urea permeability, we employed a urea uptake-deficient Saccharomyces cerevisiae strain (YNVW1 Δdur3) that expresses our constructs. This strain was
grown in liquid media at various pH levels with urea as the exclusive nitrogen source. The relative urea permeabilities were then compared to those of our role model, the HpUreI
wild-type protein, within the physiologically relevant pH range of 4.0 to 7.0. The results of this study call into question the hypothesis that periplasmic loops PL1 and PL2 constitute
the main pH-sensor of HpUreI, as for instance the urea channel from S. salivarius, which lacks such extensive periplasmic loops, exhibits a similar pH-dependent activity as HpUreI.
This suggests that alternative structural elements may contribute to pH gating. Yeast complementation assays offer a cost-effective and versatile approach to qualitatively assess
the functional differences among protein variants for a wide range of solutes. Our results provide new insights into the HpUreI gating mechanism and highlight the value of
heterologous expression systems for investigating membrane channel function.

Period	21 Jul 2025
Event title	YEAST 25
Event type	Conference
Location	Paris, FranceShow on map
Degree of Recognition	International