Genetic Determinants of Virulence in Vibrio cholerae

这项拟议工作的长期目标是定义分子组成和调节机制 霍乱弧菌的定植和毒力蛋白分泌到有足够知识的程度 智能地将这些信息纳入改进的霍乱疫苗策略和抗菌剂 旨在抑制这些事件的治疗方法。该提案的大部分涉及对分子的分析 毒素共调节菌毛形成和介导肠道定植的机制。一些人 构建TCP和其他类型4 PILI的过程中的步骤链接和/或与以下过程相关 由II型分泌系统分泌毒素和其他毒力决定簇。从而进一步了解 4型菌毛的生物发生机制应导致潜在的抗菌剂的特征 参与多种致病途径的靶标。我们将详细研究菌毛生物发生的各个方面。 这些实验将由我们目前可用的TCP基因结构和 免疫反应剂。对于Tcp的作用机制，我们将利用场发射技术 扫描电子显微镜结合特殊工程的TCPA错义突变和 TcpB缺失突变体以解决导致菌毛超扭曲形成的生物物理机制， 促进代表肠道微菌落形成基础的细菌联合。 将对TcpF及其在殖民中的作用进行进一步的描述。这一分析得到了 TcpF晶体结构的最新解决方案和蛋白质功能结构域的定位 这个结构。上皮与霍乱弧菌相互作用的其他步骤将使用碳水化合物来定义 约束性分析。综上所述，拟议研究的结果将提供详细的信息 关于霍乱弧菌定植的机制，从成分定义的水平来看， 了解它们是如何详细阐述到细胞表面和细胞以外的。 相关性(请参阅说明)： 由肠道感染细菌引起的腹泻疾病继续造成严重的发病率和死亡率。 在世界范围内，包括在美国境内。尽管多年来对霍乱弧菌进行了复杂的研究，但其病原体 霍乱的毒剂，目前还没有有效的疫苗。弧菌4型菌毛为毒素共调节菌毛(ToxoxinCoregoredPilus，TCPs)。 霍乱弧菌是主要的定植因素，代表了4型菌毛的生物发生和功能的范例 在整个肠道细菌领域。建议的研究与预防和治疗 霍乱以及由革兰氏阴性肠道细菌感染引起的其他一些肠道疾病 在世界范围内，它解决了国家卫生研究院任务的主要目标。

The long range goal of the proposed work is to define the molecular components and mechanisms mediating Vibrio cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which toxin coregulated pilus (TCP) is formed and mediates intestinal colonization. Some steps in the process by which TCP and other type 4 pili are built are linked and/or related to the process of toxin and other virulence determinant secretion by type II secretion systems. Thus further understanding of the mechanisms of type 4 pilus biogenesis should lead to the characterization of potential antimicrobial targets involved in multiple virulence pathways. We will examine the aspects of pilus biogenesis in detail. These experiments will be facilitated by our currently available collections of tcp genetic constructs and immunoreagents. Regarding the mechanism of TCP function, we will utilize the technique of field emission scanning electron microscopy in combination with specifially engineered tcpA missense mutations and a tcpB deletion mutant to address the biophysical mechanisms that lead to pilus supertwist formation, promoting the bacterial associations that represent the basis of microcolony formation in the intestine. Further characterization of TcpF and its role in colonization will be undertaken. This analysis is aided by the recent solution of the crytsal structure of TcpF and the mapping of a functional domain of the protein within this structure. Additional steps in the epithelial interaction with V. cholerae will be defined using carbohydrate binding analyses. Taken together, the results of the proposed studies will provide detailed information regarding the mechanisms of V. cholerae colonization from the levels of defining the components, understanding how they are elaborated to the cell surface and beyond. RELEVANCE (See Instructions): Diarrheal diseases caused by enteric infectious bacteria continue to cause significant morbidity and mortality world-wide, including within the US. Despite years of sophisticated research on V. cholerae, the causative agent of cholera, there is still no effective vaccine. The type 4 pilus, toxin coregulated pilus (TCP), of V. cholerae is the major colonization factor and represents a paradigm for type 4 pilus biogenesis and function throughout the realm of enteric bacteria. The proposed studies are relevant to the prevention and cure of cholera as well as a number of other enteric diseases caused by gram-negative enteric bacterial infections world-wide, which addresses major goals of the NIH mission.