Association of Vibrio parahaemolyticus Thermostable Direct Hemolysin with Lipid Rafts Is Essential for Cytotoxicity but Not Hemolytic Activity
Association of Vibrio parahaemolyticus Thermostable Direct Hemolysin with Lipid Rafts Is Essential for Cytotoxicity but Not Hemolytic Activity
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DOI：
10.1128/iai.00946-09
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发表时间：
2010-02-01
影响因子：
3.1
通讯作者：
Iida, Tetsuya
中科院分区：
文献类型：
--
作者：
Matsuda, Shigeaki;Kodama, Toshio;Iida, Tetsuya
耐热直接溶血素（TDH）是副溶血性弧菌的主要毒力因子，对培养细胞具有毒性作用。然而，TDH的细胞毒作用的机制，包括其靶分子对真核细胞的质膜仍不清楚。在这项研究中，我们确定了脂筏的作用，胆固醇和鞘脂丰富的微结构域，在TDH细胞毒性。用甲基-β-环糊精（M β CD）（一种筏破坏剂）处理细胞可抑制TDH的细胞毒性。TDH与耐洗涤剂膜（DRM）相关，M β CD消除了这种关联。相反，无毒TDH突变体和DRM之间没有这种关联。脂筏的破坏既不影响溶血作用，也不抑制TDH诱导的HeLa细胞内钙内流。这些结果表明，TDH的细胞毒性，而不是溶血活性是依赖于脂筏。细胞鞘磷脂的外源性和内源性消耗也防止TDH的细胞毒性，但TDH和鞘磷脂之间的直接相互作用没有检测到与脂质覆盖试验或脂质体吸收试验。用100 mU/ml的鞘磷脂酶（SMase）处理破坏了TDH与DRM的关联，但不影响脂筏标记蛋白（小窝蛋白-1和flotillin-1）与DRM的定位。这些结果表明，鞘磷脂是重要的协会TDH与脂筏，但不是一个分子靶TDH。我们假设TDH可能靶向对一定浓度的SMase敏感的某组筏，这不会影响其他类型的筏。
Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, induces cytotoxicity in cultured cells. However, the mechanism of TDH's cytotoxic effect including its target molecules on the plasma membrane of eukaryotic cells remains unclear. In this study, we identified the role of lipid rafts, cholesterol-and sphingolipid-enriched microdomains, in TDH cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (M beta CD), a raft-disrupting agent, inhibited TDH cytotoxicity. TDH was associated with detergent-resistant membranes (DRMs), and M beta CD eliminated this association. In contrast, there was no such association between a nontoxic TDH mutant and DRMs. The disruption of lipid rafts neither affected hemolysis nor inhibited Ca2+ influx into HeLa cells induced by TDH. These findings indicate that the cytotoxicity but not the hemolytic activity of TDH is dependent on lipid rafts. The exogenous and endogenous depletion of cellular sphingomyelin also prevented TDH cytotoxicity, but a direct interaction between TDH and sphingomyelin was not detected with either a lipid overlay assay or a liposome absorption test. Treatment with sphingomyelinase (SMase) at 100 mU/ml disrupted the association of TDH with DRMs but did not affect the localization of lipid raft marker proteins (caveolin-1 and flotillin-1) with DRMs. These results suggest that sphingomyelin is important for the association of TDH with lipid rafts but is not a molecular target of TDH. We hypothesize that TDH may target a certain group of rafts that are sensitive to SMase at a certain concentration, which does not affect other types of rafts.