Molecular Characterization of H. Pylori Ure I

The Gram-negative, spiral shaped bacterium Helicobacter (H.) pylori colonizes the human gastric mucosa and is the causative agent of gastritis. Subsequently, an ulcus duodenii or carcinoma of the stomach may develop. The prevalence of the infection is about 30 in developed countries and may rise up to 90% in developing countries. This implies that gastritis is one of the most common infectious diseases woldwide. Currently a triple therapy (proton pump inhibitor + 2 different antibiotics) is used to eradicate the infection. Because of therapy side-effects and the emergence of antibiotic resistance, a H. pylori specific monotherapy is desirable. One of the most important virulence factors of H. pylori is a massive urease production. This enzyme, which cleaves urea to yield ammonia and carbondioxide, is responsible for the remarkable acid tolerance of the bacterium as well as for a successful colonization of the gastric mucosa and in part for the observed pathological changes. Moreover, urease is an important diagnostic marker. The production of a functional urease is dependent on the structural subunits UreA and UreB as well as on several accessory proteins (UreE, UreF, UreG, UreH). The destruction of another protein, UreI, leads to the loss of acid tolerance although the overall urease production is not affected. According to a priliminary model of UreI, which was developed by Dr. Sachs` group, the integral membrane protein is an acid-activated urea transporter, which is able to saturate the intra-bacterial urease under physiologic conditions. The aim of the research plan is to identify functionally important amino acid residues in UreI. Towards this end, several mutations will be introduced in the ureI gene and their impact on the acid tolerance of H. pylori as well as the transport of urea in an eucaryotic model system (Xenopus oocytes) will be tested. In addition, we will attempt to isolated and crystallize the protein in order to subject the material to X-ray diffraction. Furthermore, we will investigate wether phosphorylation of UreI or interaction with other proteins is a prerequisite for urea transport, since a sequence comparison analysis has already identified interesting similarities. This information combined with already existing facts will be introduced into computer-assisted UreI modeling and should facilitate the identification of potential inhibitors of the urea transport or the molecular mechanism of the acid-activation of the protein. Such compounds could be potentially useful for the development of therapeutic drugs designed to eradicate the organism with monotherapy.