Screening of host facters affected by CagA, a virulence factor of Helicobacter pylori

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Helicobacter pylon is an important pathogen that is associated with various diseases including chronic gastritis. peptic ulcer and gastric cancer. H pylori translocates CagA protein into gastric epithelial cells by the type IV secretion system and induces morphogenetic response of the host cells. To understand CagA function in eukaryotic cells, we expressed cagA gene in wild type diploid Saccharomyces cerevisiae or a set of homozygous deletion mutants for 4792 nonessential genes. Overexpression of CagA caused growth inhibition in wild type yeast and more severe growth inhibition in 18 mutant strains, 7 strains of which fell in the functional category of vacuole biogenesis and endocytosis pathway that is highly conserved in all eukaryotes. Indeed, all of 7 genes corresponding to the deletion strains have mammalian homologues. We expressed CagA that is fused to the yeast-optimized GFP (yEGFP) to find that CagA was localized at yeast cell periphery similarly in mammalian cells. We observed the dynamics of endocytosis in yeast using a membrane lipid probe FM4-64 in the presence or absence of yEGFP-CagA. Expression of yEGFP-CagA but not yEGFP alone caused marked delay in the transition of endosomes from the plasma membrane to vacuoles. We next observed the impact of CagA expression on the endocytosis dynamics in mammalian cells using transferrin, a probe for the clathrin-mediated endocytosis and cholera toxin B (CT-B), a probe for the lipid-raft mediated endocytosis. CagA specifically inhibited CT-B endocytosis without interfering with the endocytosis of transferrin in the gastric epithelial AGS cells. From these results. We propose that H pylon CagA inhibits the lipid raft-mediated endocytosis in mammalian cells, which may be important in pathogenesis of various diseases associated with H pylon infection.