Determine host surface interactions of MARTX toxin of foodborne Vibrio vulnificus

项目摘要/摘要 海洋致病菌创伤弧菌是一种生活在沿海水域的高度致死性的革兰氏阴性细菌。它 通过进食生海鲜引起肠道感染，通过开放性伤口引起伤口感染。感染 迅速发展为败血症，病死率很高。这种细菌的经济成本是最高的 与海鲜有关的疾病。由于已检测到多重耐药菌株，感染发生率为 由于气候变化，阐明创伤弧菌致病机制的研究越来越多 对支持新的治疗开发具有重要意义。多功能自动处理毒素重复序列 动物研究表明，(MARTX)毒素是创伤弧菌的主要毒力因子 与死亡同在。该项目的目标是确定MARTX在宿主细胞中的致病机制。 醉酒所必需的表面。解决这个问题可以为以下方面提供关键信息 设计能够阻止MARTX与其目标细胞结合的抗体。这个目标特别是 与NIAID有关，因为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。具体地说，第一个目标是确定创伤弧菌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确定了宿主细胞 膜蛋白激酶是最有希望的受体候选者。在这个目标中，我们将结合使用 基因操作、体外细胞分析和基于FRET的流式细胞术验证该激酶的作用 作为MARTXVv受体。第二个目标将确定MARTXVv的哪些区域可以与细胞表面结合 基于之前对MARTXVv片段的研究，使用包括蛋白质纯化、细胞为基础的技术 结合分析和荧光显微镜检查。总的来说，这些数据将定义宿主受体和受体 毒素的结合域揭示了毒素与细胞表面寄主之间的关键相互作用，第一 MARTXVV中毒的一步。拟议工作的完成将提供所需的培训机会 支持成为研究细菌毒素的首席研究员的长期职业目标。具体地说， 培训将包括1)开发研究宿主细胞系统中毒素功能的专业知识，2)开发 遗传筛选、免疫荧光和蛋白质生物化学的实验技术和3)发展 学术独立研究人员所需的一般技能，如撰写拨款和科学交流。 西北大学范伯格医学院为 实现这些目标。发起人Karla Satchell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来获得深入的毒素生物学研究知识。这个 本部门提供大量的科学交流机会，包括周刊俱乐部和 研究报告。此外，该计划配备了优秀的核心设施，并配备了熟练的顾问来 帮助开发使用先进仪器进行成像、流式细胞术和蛋白质纯化的技术。

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.