A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes.
A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes.
复制标题
DOI：
10.1371/journal.ppat.1002803
复制
发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Iida T
中科院分区：
文献类型：
--
作者：
Matsuda S;Okada N;Kodama T;Honda T;Iida T
副溶血性弧菌是人类致病性弧菌之一。在哺乳动物细胞感染期间，这种病原体表现出依赖于其III型分泌系统（T3SS1）的细胞毒性。VepA是一种通过T3SS1分泌的效应蛋白，在副溶血性弧菌依赖T3SS1的细胞毒性中起主要作用。然而，VepA参与t3ss1依赖性细胞毒性的机制尚不清楚。在这里，我们发现VepA蛋白转染HeLa细胞导致细胞死亡，表明VepA单独具有细胞毒性。酵母中VepA的异位表达干扰了酵母的生长，表明VepA对酵母也有毒性。酵母全基因组筛选发现酵母基因VMA3是VepA抑制酵母生长的必要基因。虽然VMA3编码液泡H+-ATPase （V-ATPase）的c亚基，但VepA的毒性与V-ATPase的功能无关。在HeLa细胞中，敲低V-ATPase亚基c可降低vepa介导的细胞毒性。我们还证明了VepA与V-ATPase亚基c相互作用，而VepA的羧基末端截断突变体（VepAΔC）没有表现出毒性。在感染过程中，溶酶体内容物渗漏到细胞质中，表明溶酶体膜渗透发生在细胞溶解之前。在无细胞系统中，VepA足以诱导分离溶酶体释放组织蛋白酶D。因此，我们的数据表明，细菌效应物VepA靶向v - atp酶的c亚基，诱导宿主细胞溶酶体破裂和随后的细胞死亡。副溶血性弧菌是一种细菌性病原体，可引起食源性胃肠炎、伤口感染和败血症。在哺乳动物细胞感染期间，它表现出依赖于其III型分泌系统（T3SS1）的细胞毒性。虽然效应体VepA在细胞毒性中起主要作用，但其机制尚不清楚。在这里，我们发现VepA靶向液泡H+- atp酶（v - atp酶）的c亚基并诱导宿主细胞溶酶体的破裂。我们发现VepA单独对HeLa细胞有细胞毒性，对酵母也有毒性。通过酵母全基因组筛选，我们确定了酵母v - atp酶亚基c对VepA对酵母的毒性至关重要。我们还证明了V-ATPase亚基c的敲除降低了VepA介导的对HeLa细胞的细胞毒性，并且VepA与V-ATPase亚基c相互作用。在感染过程中，溶酶体内容物在细胞裂解之前渗漏到细胞质中，而VepA对于这种渗漏是必要和充分的。我们的数据表明，细菌效应物VepA通过靶向进化上保守的v - atp酶，破坏宿主细胞的“自杀袋”溶酶体，并引发随后的细胞死亡。
Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. Vibrio parahaemolyticus is a bacterial pathogen that causes food-borne gastroenteritis and also wound infection and septicemia. It exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1) during the infection of mammalian cells. Although an effector VepA plays a major role in the cytotoxicity, the mechanism was unknown. Here we show that VepA targets subunit c of the vacuolar H+-ATPase (V-ATPase) and induces the rupture of host cell lysosomes. We found that VepA alone is cytotoxic in HeLa cells and also toxic in yeast Saccharomyces cerevisiae. Using a yeast genome-wide screening, we identified yeast V-ATPase subunit c as essential for the toxicity of VepA to yeast. We also demonstrated that knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity toward HeLa cells and that VepA interacted with subunit c of V-ATPase. During infection, lysosomal contents leaked into the cytosol prior to cell lysis, and VepA was necessary and sufficient for this leakage. Our data suggest that a bacterial effector VepA ruptures lysosomes, the “suicide bags” of host cells, by targeting the evolutionarily conserved V-ATPase, and elicits subsequent cell death.
登录
查看更多内容
影响因子：
3.6
作者：
Burdette DL;Seemann J;Orth K
通讯作者：
Orth K
影响因子：
6.4
作者：
Daniels, NA;MacKinnon, L;Slutsker, L
通讯作者：
Slutsker, L
影响因子：
6
作者：
Appelqvist, Hanna;Nilsson, Cathrine;Ollinger, Karin
通讯作者：
Ollinger, Karin
影响因子：
3.4
作者：
Kodama, Toshio;Rokuda, Mitsuhiro;Honda, Takeshi
通讯作者：
Honda, Takeshi
影响因子：
4.8
作者：
Jehn, BM;Dittert, I;Bielke, W
通讯作者：
Bielke, W