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; 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）：沃利奥霍乱细胞溶素（VCC）是一种有效的孔形成毒素（PFT），它会攻击人类细胞，尽管毒素的特定靶标和发病机理的作用尚不清楚。了解致病性毒力因素如何识别宿主膜是开发疗法以阻止其作用的重要第一步。这些知识还可能有益于靶向患病细胞（例如癌症或病毒感染细胞）的药物的发展。长期目标是阐明孔形成毒素如何从结构和机械的角度识别，组装和破坏细胞膜。该应用的总体目标是获得该目标的下一步，是t了解VCC如何靶向细胞膜（包括碳水化合物和胆固醇分子）上的基序。 VCC毒素包含两个结构结构域，其折叠与结合凝集素蛋白相似。在各种具有重要人类健康影响的VCC毒素同源物上发现了类似的碳水化合物结合域，包括颤动的食物中毒和败血症的原因。该项目的中心假设是，VCC以特定和可逆的方式识别膜。这涉及在毒素分子上的结合位点，从而增加毒素在膜表面上的局部浓度。拟议工作的理由是，VCC结合基序的结构和功能表征将对识别位点的贡献有清晰的理解 朝着毒素的皮摩尔细胞溶解活性。为了实现这一目标，三个 将追求具体目的：1）确定VCC辅助域在细胞膜上靶向的碳水化合物配体； 2）结构表征VCC辅助结构域与其各自的配体之间的相互作用，以了解配体选择性的机理； 3）研究VCC在真核膜中识别胆固醇的机制。在第一个目标下，将使用聚糖筛选和生物物理表征的结合来识别和量化VCC识别的碳水化合物配体。在第二个目标下，将使用X射线晶体学的高分辨率结构分析来理解配体选择性的性质。在第三个目标下，推定的胆固醇结合位置将是对VCC对易感细胞膜识别的贡献。这种方法之所以成为新手，是因为它将全面的结构分析与新的筛查技术结合在一起，以回答有关生物学功能的基本问题。拟议的研究很重要，因为它将阐明这种有效的通道形成的毒素如何利用受体聚集在具有高亲和力的细胞膜上。这些信息将为靶向毒素和药物靶向特定的细胞膜，以治疗传染病和癌症。 公共卫生相关性：拟议的研究与公共卫生有关，因为PFT（例如VCC）参与了病原体定植和宿主损害的过程。了解毒素攻击细胞的方式对于制定打击传染病的策略至关重要。因此，拟议的研究与NIH的使命是通过支持人类疾病的原因和治愈的研究来改善国家健康。