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

幽门螺杆菌是一种重要的致病菌，与包括慢性胃炎在内的多种疾病有关。消化性溃疡和胃癌。幽门螺杆菌通过IV型分泌系统将CagA蛋白转运到胃上皮细胞中，并诱导宿主细胞的形态发生反应。为了了解CagA在真核细胞中的功能，我们在野生型二倍体酿酒酵母或一组4792个非必需基因的纯合缺失突变体中表达了cagA基因。CagA的过表达导致野生型酵母的生长抑制和更严重的生长抑制在18个突变株，其中7个菌株属于液泡生物合成和内吞途径的功能类别，是高度保守的所有真核生物。实际上，对应于缺失菌株的所有7个基因都具有哺乳动物同源物。我们表达了与酵母优化的GFP（yEGFP）融合的CagA，发现CagA类似地定位于哺乳动物细胞中的酵母细胞外周。我们使用膜脂质探针FM 4 -64观察了在存在或不存在yEGFP-CagA的情况下酵母中的内吞作用的动力学。表达的yEGFP-CagA，但不是单独的yEGFP引起显着延迟内体从质膜到空泡的过渡。接下来，我们使用转铁蛋白（网格蛋白介导的内吞作用的探针）和霍乱毒素B（CT-B）（脂筏介导的内吞作用的探针）观察了CagA表达对哺乳动物细胞内吞作用动力学的影响。CagA特异性地抑制CT-B的内吞作用，而不干扰胃上皮AGS细胞中转铁蛋白的内吞作用。从这些结果。我们认为H pylon CagA抑制哺乳动物细胞中脂筏介导的内吞作用，这可能在与H pylon感染相关的各种疾病的发病机制中起重要作用。

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.