Since LPS species migrating in this region likely include only core oligosaccharide and lipid A moieties, we directed our attention to these components in trying selleck kinase inhibitor to identify specific cholesterol-dependent
structural modifications. We selectively disrupted two lipid A modification genes, either lpxE or eptA, encoding the lipid A 1-phosphatase and lipid A phosphoethanolaminetransferase, respectively . Then, LPS profiles were compared in pairwise cultures of these mutated G27 strains grown in the presence or RGFP966 absence of cholesterol (Figure 9C). We found that the eptA::cat strain retained an LPS response to cholesterol that was even more distinct than in the wild type. In contrast, cholesterol-responsive bands were abolished in the lpxE::cat selleck chemicals strain. These results implied that the aberrant bands which accumulated under conditions of cholesterol depletion in the wild type, but not in lpxE::cat, may represent forms of LPS in which the lipid A moiety has been dephosphorylated at the 1-position. It is also possible that, in these bands, the
core may have undergone further modification subsequent to lipid A dephosphorylation (see Discussion). The LPS gel results described above (Figure 9C) contrasted with the outcome of whole cell ELISA analysis of the lpxE::cat strain. This mutant strain retained its capacity to respond to cholesterol availability with enhanced surface Lewis X and Lewis Y expression (Figure 10, Table 2), as did the eptA::cat strain (data not shown) and the cgt::cat strain (Fig. 10). These contrasting results show that www.selleck.co.jp/products/Cisplatin.html the enhanced surface display of Lewis antigen in response to growth in cholesterol occurred independently of the structural modifications to the core/lipid A moiety seen on silver-stained gels. Figure 10 H. pylori G27 retain Lewis antigen response to cholesterol after disruption of cgt or lpxE. Whole cell ELISA assays were performed in duplicate on samples of H. pylori G27 cgt::cat (panel A) or lpxE::cat (panel B), which were cultured in parallel in the
absence (open symbols) or presence of cholesterol (filled symbols). Absorbance readings for individual wells are plotted. Discussion In eukaryotic membranes, cholesterol modulates curvature and fluidity, and cholesterol-rich lipid subdomains influence numerous membrane functions, including signal transduction and transport activity , yet very little is known about the physiological roles of cholesterol among the prokaryotes that utilize it. In this study, we used chemically defined medium to begin to characterize these roles of cholesterol in H. pylori. Growth of H. pylori in the presence of cholesterol proved to be essential for gastric colonization in the gerbil, even though it is not necessary for growth in vitro. This colonization experiment was conducted under standard dietary conditions, where cholesterol should be abundant in gastric mucus [2, 3, 60]. Taking into account that H.