Determine host surface interactions of MARTX toxin of foodborne Vibrio vulnificus

确定食源性创伤弧菌 MARTX 毒素宿主表面相互作用

• 批准号: 10537885
• 负责人: Jiexi Chen
• 金额: $ 4.2万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537885
• 关键词: Address; Animals; Antibiotic Resistance; Antibodies; Bacteria; Bacterial Proteins; Bacterial Toxins; Basic Science; Binding; Biological Assay; Biology; C-terminal; CRISPR screen; CRISPR/Cas technology; Calcium; Calcium ion; Caspase; Cell Death; Cell Line; Cell Membrane Proteins; Cell membrane; Cell surface; Cells; Cellular Assay; Cessation of life; Communicable Diseases; Communication; Consensus Sequence; Consumption; Core Facility; Cytolysis; Cytosol; Cytotoxin; Data; Defect; Disease; Environment; Exhibits; Family; Fatality rate; Flow Cytometry; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Foundations; Gastroenteritis; Generations; Genes; Genetic Screening; Goals; Gram-Negative Bacteria; Grant; Hour; Human; Immunofluorescence Immunologic; In Vitro; Incidence; Individual; Infection; Institutes; Intoxication; Journals; Knock-out; Knowledge; Lead; Life; Lysine; Membrane; Membrane Proteins; Microscopic; Mission; Modeling; Monoclonal Antibodies; National Institute of Allergy and Infectious Disease; Pathogenesis; Pathogenicity; Pharmaceutical Preparations; Phosphotransferases; Phytic Acid; Play; Principal Investigator; Property; Protease Domain; Protein Biochemistry; Protein Fragment; Protein Kinase; Proteins; Public Health; Research; Research Personnel; Research Support; Resistance; Role; Seafood; Sepsis; Septicemia; Signal Transduction; Surface; System; TGFBR2 gene; Targeted Toxins; Techniques; Terminal Repeat Sequences; Testing; Toxin; Training; Universities; Vibrio; Vibrio vulnificus; Virulence Factors; Water; Work; Wound Infection; Writing; base; career; climate change; coastal water; combat; design; economic cost; enteric infection; extracellular; foodborne; foodborne illness; genetic manipulation; genome-wide; gulf coast; instrument; intestinal epithelium; medical schools; meetings; novel therapeutics; open wound; pathogen; prevent; programs; protein purification; protein structure; receptor; receptor binding; skills; therapeutic development; toxin V; training opportunity; wound

Project Summary/Abstract The marine pathogen Vibrio vulnificus is a highly lethal Gram-negative bacteria inhabiting coastal waters. It causes intestinal infections via consumption of raw seafood and wound infections via open wounds. The infection progresses rapidly to septicemia with high fatality rates. This bacterium exerts the highest economic cost of any seafood-related disease. As multi-antibiotic resistant isolates have been detected and infection incidence is climbing due to climate change, studies elucidating the pathogenic mechanisms of V. vulnificus are increasingly important to support novel therapeutic development. The Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) toxin has been shown in animal studies to be the primary virulence factor of V. vulnificus associated with death. The goal of the proposed project is to determine pathogenic mechanisms of MARTX at host cell surfaces that are necessary for intoxication. Addressing this question could provide critical information toward design of antibodies that would function to block MARTX from binding to its targeted cells. This goal is particularly pertinent to the NIAID given the institute's mission to support research to better understand, treat, and prevent infectious diseases. Specifically, the first aim will determine host receptors for the V. vulnificus MARTXVv toxin. Our preliminary data using an unbiased Genome-wide CRISPR-Cas9 Screening approach identified a host cell membrane protein kinase as the most promising receptor candidate. In this aim, we will use a combination of genetic manipulation, in vitro cellular assays and FRET based flow cytometry to validate the role of this kinase as a MARTXVv receptor. The second aim will determine which regions of MARTXVv can bind to the cellular surface based on previous studies on MARTXVv fragments, using techniques including protein purification, cell-based binding assays and fluorescence microscopy. Collective, these data will define the host receptor and receptor binding domain of the toxin revealing critical interactions between the toxin and host at cellular surface, the first step of MARTXVv intoxication. Completion of the proposed work will provide training opportunities required to support the long-term career goal of becoming a principal investigator studying bacterial toxins. Specifically, the training will include 1) developing expertise in studying toxin functions in the host cell system, 2) developing experimental techniques in genetic screens, immunofluorescence, and protein biochemistry and 3) developing general skills required for academic independent researchers such as grant writing and scientific communication. Northwestern University Feinberg School of Medicine provides an outstanding training environment for accomplishing these goals. The sponsor Dr. Karla Satchell is an expert in bacterial toxins and protein structure. The trainee will gain a depth of knowledge in studying toxin biology by having weekly meetings with her. The department provides plenty of opportunities for scientific communication including weekly journal club and research presentations. Further, the program is equipped with excellent core facilities with skillful consultants to help develop techniques using advanced instruments in imagining, flow cytometry, and protein purification.

项目总结/摘要 海洋病原体创伤弧菌是一种高致死性革兰氏阴性细菌，栖息于沿海沃茨。它 通过食用生海鲜引起肠道感染，通过开放性伤口引起伤口感染。感染 迅速发展为具有高致死率的败血症。这种细菌的经济成本是最高的 海鲜相关疾病由于已检测到多种抗生素耐药菌株， 由于气候变化，阐明创伤弧菌致病机制的研究越来越多， 重要的是支持新的治疗发展。多功能毒素重复序列自动处理仪 在动物研究中已显示，MARTX毒素是创伤弧菌相关的主要毒力因子， 与死亡。该项目的目标是确定MARTX在宿主细胞中的致病机制 这是中毒所必需的。解决这个问题可以提供关键信息， 设计能够阻断MARTX与其靶细胞结合的抗体。这一目标尤其 鉴于该研究所的使命是支持研究，以更好地了解，治疗和预防， 传染病具体而言，第一个目标将确定创伤弧菌MARTXVv毒素的宿主受体。 我们使用无偏全基因组CRISPR-Cas9筛选方法的初步数据鉴定了一种宿主细胞 膜蛋白激酶作为最有前途的受体候选者。为此，我们将结合使用 基因操作、体外细胞分析和基于FRET的流式细胞术来验证这种激酶的作用 作为MARTXVv受体。第二个目标是确定MARTXVv的哪些区域可以结合到细胞表面 基于先前对MARTXVv片段的研究，使用包括蛋白质纯化、基于细胞的 结合测定和荧光显微术。集体，这些数据将定义宿主受体和受体 毒素的结合结构域揭示了毒素和宿主在细胞表面的关键相互作用， MARTXVv中毒的步骤。完成拟议的工作将提供所需的培训机会， 支持成为研究细菌毒素的主要研究者的长期职业目标。具体而言是 培训将包括1）发展研究宿主细胞系统中毒素功能的专业知识，2）发展 遗传筛选、免疫荧光和蛋白质生物化学方面的实验技术; 学术独立研究人员所需的一般技能，如赠款写作和科学交流。 西北大学范伯格医学院提供了一个优秀的培训环境， 实现这些目标。赞助商Karla Saturday博士是细菌毒素和蛋白质结构方面的专家。 学员将通过每周与她会面，深入学习毒素生物学知识。 该系提供大量的科学交流机会，包括每周的期刊俱乐部， 研究介绍。此外，该计划配备了优秀的核心设施和熟练的顾问， 帮助开发使用先进仪器的成像、流式细胞术和蛋白质纯化技术。