Permeation through the bacterial urea transporter UreI from Heliobactor pylori

More than half of the world`s population is infected with Helicobactor pylori, a bacterium that colonizes the gastric mucus or the gastric mucosa. In many patients the infection leads to gastritis or a duodenal ulcer. Since standard therapies have to cope with increasing antibiotic resistance, other strategies are necessary in the long run. The pH-dependent urea channel, HpUreI of H. pylori, provides a possible starting point. It spans the inner membrane of the bacterium surrounded by two cell membranes and ensures that H. pylori can survive in the acidic environment of the stomach. HpUreI transports urea from the compartment between the two membranes, the periplasmic space, into the cell interior. There, urea is cleaved into ammonia and carbon dioxide by means of an enzyme, the urease. These two substances diffuse back into the periplasm and bind protons there. Thus, the periplasmic space can only serve as a buffer zone between the acidic stomach environment and the neutral bacterial interior as long as HpUreI functions. In order to be able to selectively eliminate the channel with drugs, it must first be clarified how it can selectively transport urea and water without passing protons and hydrogen ions. It has hitherto been known that HpUreI has an hourglass-shaped design with a central selectivity filter. However, in contrast to purely water-conducting channels, it has no electrostatic barrier and is exclusively equipped with uncharged and hydrophobic amino acids. In the course of this project we will test whether the hydrophobic nature of the constriction is sufficient for proton exclusion or whether the strongly charged entrance and exit of the channel are decisive. For this purpose we will purify HpUreI from genetically modified yeast cells and incorporate the channel into lipid membranes. Different mutants that have modified charges at the channel mouth or a less hydrophobic central bottleneck will reveal the mechanism of substrate selectivity and the nature of the proton barrier. The use of single-molecule techniques will allow the number of protons, urea and water molecules to be measured. Our preliminary experiments have shown that we can not only incorporate HpUreI into membranes in its natural six-unit ring assembly, but also in smaller alloys. Functional studies on differently sized networks will give us valuable information on the importance of the interactions between the individual subunits for its function. The advancement of light scatter-based measurement methods will pave the way for future high-throughput analytical methods in the search for inhibitors of HpUreI.