Genetic and biochemical study ov V. cholerase RTX toxin

V.霍乱酶 RTX 毒素的遗传和生化研究

• 批准号: 6577364
• 负责人: Karla J F Satchell
• 金额: $ 29.78万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6577364
• 关键词: Vibrio cholerae; actins; active sites; bacteria infection mechanism; bacterial cytopathogenic effect; binding sites; chemical structure; cholera; cholera toxin; computer simulation; crosslink; cytotoxicity; high performance liquid chromatography; host organism interaction; immunofluorescence technique; matrix assisted laser desorption ionization; microorganism culture; nucleic acid sequence; polymerization; posttranslational modifications; protein binding; protein localization; protein protein interaction; protein purification; protein structure function; proteolysis

DESCRIPTION (provided by applicant): Vibrio cholerae strains that do not produce cholera toxin induce a more inflammatory diarrhea than normal cholera disease, implicating other potent toxins in the pathogenesis of cholera. Several accessory toxins of V cholerae are purported to account for this reactogenic response. One of these factors is the newly discovered VcRtxA toxin of V cholerae. VcRtxA is a large protein toxin that is a unique member of the RTX family Production of this toxin has been evolutionarily conserved by Vibrio sp indicating that maintenance of this large toxin is essential for virulence or survival in the environment. Its cytotoxicity has been further shown to function by a novel mechanism This toxin causes depolymerization of actin stress fibers in both polarized and non-polarized cell lines by a unique pathway Concurrent with depolymerization, the actin molecules become covalently linked together into dimers, trimers, and higher order multimers This observation suggests that covalent crosslinking of actin by the toxin drives actin depolymerization This unusual reaction distinguishes VcRtxA from all other bacterial toxins that cause actin depolymerization. Research performed under this grant proposal will investigate further the novel biochemical properties of this important virulence factor The VcRtxA toxin is the largest single polypeptide protein toxin ever described, however, it is unclear whether the full 4545 amino acid protein is the toxic moiety The size of the toxic moiety and potential post-translational modifications will by identified by examining biochemical properties of the active form of purified toxin The catalytic activity will then be investigated and the role of a putative catalytic domain in this reaction will be assessed. It will then be established whether the toxin is active at the membrane or within the cytoplasm of the target cells, and the role of a putative actin-binding domain will be characterized.

描述（由申请人提供）：不产生霍乱毒素的弧菌霍乱菌株比正常霍乱疾病诱导炎症性腹泻更大，这暗示了其他有效的毒素在霍乱的发病机理中。 V霍乱的几种辅助毒素据称是为了解释这种反应。这些因素之一是V霍乱的新发现的VCRTXA毒素。 VCRTXA是一种大蛋白毒素，是该毒素RTX家族产生的独特成员，这在进化上是由颤音中的SP在进化中保守的，表明这种大毒素的维持对于环境中的毒力或生存至关重要。 Its cytotoxicity has been further shown to function by a novel mechanism This toxin causes depolymerization of actin stress fibers in both polarized and non-polarized cell lines by a unique pathway Concurrent with depolymerization, the actin molecules become covalently linked together into dimers, trimers, and higher order multimers This observation suggests that covalent crosslinking of actin by the toxin drives actin这种异常反应的解聚将VCRTXA与引起肌动蛋白解聚化的所有其他细菌毒素区分开。根据该赠款提案进行的研究将进一步研究该重要毒力因子的新型生物化学特性，VCRTXA毒素是有史以来描述的最大的单多肽蛋白毒素毒素，然而，尚不清楚整个4545氨基酸蛋白是否毒性的毒性部分是有毒的毒物大小和毒性的构成，该毒物的大小和毒品的构成能力是毒品的毒品，并且是通过毒性的毒品构成，而毒品的构成构成了毒物的构成，而不是毒性的构成，而不是毒性蛋白质的毒性构成，而不是毒性蛋白质的毒性构成，则可以通过毒性构建体系来进行毒性构成。毒素将研究催化活性，并评估推定催化结构域在该反应中的作用。然后将确定毒素是在膜上还是在靶细胞的细胞质内活性，并且将表征假定的肌动蛋白结合结构域的作用。