Mechanism of Cell Membrane Targeting by Vibrio Cholerae Cytolysin

霍乱弧菌溶细胞素靶向细胞膜的机制

• 批准号: 8366847
• 负责人: RICHARD A OLSON
• 金额: $ 46.02万
• 依托单位: WESLEYAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366847
• 关键词: Affinity; Antineoplastic Agents; Bacteria; Binding; Binding Sites; Biological Process; Carbohydrates; Cell Death; Cell membrane; Cells; Cellular Membrane; Cholesterol; Communicable Diseases; Complex; Cytolysins; Cytolysis; Data; Development; Drug Delivery Systems; Engineering; Eukaryotic Cell; Food Poisoning; Goals; Health; Homologous Gene; Human; Intestines; Knowledge; Lectin; Leukocytes; Ligands; Link; Lytic; Malignant Neoplasms; Mediating; Membrane; Mission; Mole the mammal; Nature; Oligosaccharides; Outcome Study; Pathogenesis; Pharmaceutical Preparations; Polysaccharides; Process; Proteins; Public Health; Research; Research Support; Resolution; Role; Screening procedure; Sepsis; Site; Structure; Surface; Targeted Toxins; Technology; Tertiary Protein Structure; Toxin; Vibrio cholerae; Vibrio vulnificus; Viral Tumor Antigens; Virulence Factors; Work; X-Ray Crystallography; base; combat; design; drug development; human disease; improved; molecular rearrangement; pathogen; pathogenic bacteria; perforin; prevent; receptor; sugar; therapy development

DESCRIPTION (provided by applicant): Vibrio cholerae Cytolysin (VCC) is a potent pore-forming toxin (PFT) that attacks human cells, although the specific targets of the toxin and role in pathogenesis are unknown. Understanding how pathogenic virulence factors recognize host membranes is an important first step towards developing therapies to block their action. This knowledge may also benefit the development of drugs that target diseased cells, such as cancer or virally infected cells. The long-term goal is to elucidate how pore-forming toxins recognize, assemble on, and disrupt cellular membranes from a structural and mechanistic perspective. The overall objective of this application, which is the next step towards obtaining this goal, is t understand how VCC targets motifs on cell membranes, including carbohydrate and cholesterol molecules. The VCC toxin contains two structural domains with folds similar to sugar-binding lectin proteins. Similar carbohydrate-binding domains are found on a variety of VCC toxin homologs with important human health implications, including toxins produced by Vibrio vulnificus, a cause of deadly food poisoning and sepsis. The central hypothesis of this project is that VCC recognizes membranes in a specific and reversible manner. This involves binding sites on the toxin molecule that increase the local concentration of toxin on the surface of the membrane. The rationale for the proposed work is that structural and functional characterization of VCC binding motifs will provide a clear understanding of the contribution that recognition sites make towards the picomolar cytolytic activity of the toxin. In order to accomplish this goal, three specific aims will be pursued: 1) Identify the carbohydrate ligands that VCC accessory domains target on cell membranes; 2) Structurally characterize interactions between VCC accessory domains and their respective ligands to understand the mechanism of ligand selectivity; and 3) Investigate the mechanism by which VCC recognizes cholesterol in eukaryotic membranes. Under the first aim, a combination of glycan screening and biophysical characterization will be used to identify and quantify carbohydrate ligands recog nized by VCC. Under the second aim, high-resolution structural analysis by X-ray crystallography will be used to understand the nature of ligand selectivity. Under the third aim, putative cholesterol binding-sites will be ana lyzed fo their contribution towards the recognition of susceptible cell membranes by VCC. The approach is in novative because it combines a comprehensive structural analysis with new screening technologies to answer fundamental questions about biological function. The proposed research is significant, because it will illuminate how this potent channel-forming toxin utilizes receptorsto assemble on cell membranes with high-affinity. This information will inform efforts to target toxins and drugs to specific cell membranes in order to treat infectious diseases and cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because PFTs, such as VCC, are involved in the process of pathogen colonization and host damage. Understanding how toxins attack cells is essential for developing strategies to combat infectious disease. Therefore, the proposed research is relevant to NIH's mission to improve the health of the Nation by supporting research in the causes and cure of human diseases.

描述（由申请人提供）：霍乱弧菌溶细胞素（VCC）是一种有效的成孔毒素（PFT），可攻击人体细胞，但该毒素的具体靶标和在发病机制中的作用尚不清楚。了解致病毒力因子如何识别宿主膜是开发阻断其作用的疗法的重要的第一步。这些知识也可能有利于针对患病细胞（例如癌症或病毒感染细胞）的药物的开发。长期目标是从结构和机制的角度阐明成孔毒素如何识别、聚集和破坏细胞膜。该应用程序的总体目标是实现这一目标的下一步，即了解 VCC 如何靶向细胞膜上的基序，包括碳水化合物和胆固醇分子。 VCC 毒素包含两个结构域，其折叠类似于糖结合凝集素蛋白。在多种对人类健康具有重要影响的 VCC 毒素同源物中发现了类似的碳水化合物结合结构域，包括创伤弧菌产生的毒素，这是致命的食物中毒和败血症的原因。该项目的中心假设是 VCC 以特定且可逆的方式识别膜。这涉及毒素分子上的结合位点，增加膜表面毒素的局部浓度。拟议工作的基本原理是 VCC 结合基序的结构和功能表征将提供对识别位点的贡献的清晰理解 使毒素具有皮摩尔细胞溶解活性。为了实现这一目标，三 将追求的具体目标： 1) 识别 VCC 辅助结构域针对细胞膜的碳水化合物配体； 2) 结构表征VCC辅助结构域与其各自配体之间的相互作用，以了解配体选择性的机制； 3) 研究VCC识别真核细胞膜中胆固醇的机制。第一个目标是结合聚糖筛选和生物物理表征来鉴定和量化 VCC 识别的碳水化合物配体。第二个目标是通过 X 射线晶体学进行高分辨率结构分析，以了解配体选择性的本质。在第三个目标下，将分析假定的胆固醇结合位点对 VCC 识别易感细胞膜的贡献。该方法具有创新性，因为它将全面的结构分析与新的筛选技术相结合，回答了有关生物功能的基本问题。拟议的研究意义重大，因为它将阐明这种有效的通道形成毒素如何利用受体以高亲和力在细胞膜上组装。这些信息将为将毒素和药物靶向特定细胞膜以治疗传染病和癌症提供信息。 公共健康相关性：拟议的研究与公共健康相关，因为 VCC 等 PFT 参与病原体定植和宿主损害的过程。了解毒素如何攻击细胞对于制定对抗传染病的策略至关重要。因此，拟议的研究与 NIH 通过支持人类疾病的原因和治疗研究来改善国家健康的使命相关。