The Outer Surface of Vibrio cholerae

描述（由申请人提供）：革兰氏阴性菌霍乱弧菌被列为B类食物和水传播病原体，引起急性、严重的腹泻疾病，即霍乱。不幸的是，霍乱对发展中国家仍然是一个严重的健康威胁，每年大约发生300万至500万例病例。霍乱弧菌是水生环境的正常居民，属于河口地区的自由生活菌群。虽然已知约200个已确认的O血清群，但只有携带脂多糖（LPS）体抗原O1或O139的霍乱弧菌菌株与霍乱大流行有关。与大多数革兰氏阴性菌一样，霍乱弧菌的LPS由三个不同的区域组成：膜相关脂质A结构域、短核寡糖和o抗原多糖。尽管脂质A结构域是革兰氏阴性菌膜的重要组成部分，并通过保守途径合成，但它是一个高度多样化的分子。致病菌根据其周围环境改变其脂质A结构域。由于脂质A是脂多糖的生物活性部分，这些修饰可以通过改变先天免疫受体复合物TLR4/MD-2对脂多糖的识别，对疾病产生深远的影响。此外，脂质A结构的改变可以影响外膜通透性屏障，以及细菌对宿主抗菌肽的耐药性。我们的总体目标是了解位于细菌表面的LPS结构的改变如何促进霍乱弧菌在水生环境和人类宿主中的生存。这一建议将侧重于定义霍乱弧菌脂质A的结构变化，以响应细菌的细胞外环境，并在这一过程所需的酶机制。霍乱弧菌脂质A的结构变化将在不同的生长条件下进行监测，这些条件可以模拟水生环境或小肠中的生长条件。完成以下目标将大大增加我们对导致霍乱的细菌机制的理解，并可能为开发新疗法和改进疫苗提供目标。当前提案的具体目的是：(i)霍乱弧菌脂质A种的结构分析；（ii）霍乱弧菌脂质A结构的环境调控；（iii）酶修饰霍乱弧菌脂质A；（iv）霍乱弧菌LPS介导toll样受体介导的免疫激活。

DESCRIPTION (provided by applicant): The Gram-negative bacterium Vibrio cholerae is classified as a Category B food- and water-borne pathogen, causing the acute, severe, diarrheal disease known as cholera. Unfortunately, cholera still remains a serious health threat to developing countries with approximately 3-5 million cases occurring annually. V. cholerae is a normal inhabitant of aquatic environments, belonging to the free-living bacterial flora in estuarine areas. Although approximately 200 recognized O serogroups are known, only V. cholerae strains bearing the lipopolysaccharide (LPS) somatic antigens O1 or O139 have been associated with cholera pandemics. As is the case with most Gram-negative bacteria, the LPS of V. cholerae is composed of three distinct regions the membrane associated lipid A domain, a short core oligosaccharide, and the O-antigen polysaccharide. Although the lipid A domain is an essential component of Gram-negative bacterial membranes and is synthesized via a conserved pathway, it is a highly diverse molecule. Pathogenic bacteria modify the lipid A domain of their LPS in response to their surrounding environment. Since lipid A is the bioactive portion of LPS, these modifications can have a profound impact on disease, by altering LPS recognition via the innate immune receptor complex, TLR4/MD-2. Additionally, alteration of the lipid A structure can impact the outer membrane permeability barrier, and bacterial resistance to host antimicrobial peptides. Our overall objective is to understand how alterations in the structure of LPS located on the bacterial surface promote survival of V. cholerae both in the aquatic environment and in the human host. This proposal will focus on defining structural alterations of V. cholerae lipid A in response to the bacterium's extracellular environment and on the enzymatic mechanisms required for this process. Structural alterations of V. cholerae lipid A will be monitored under diverse growth conditions that mimic conditions found either in the aquatic environment or in the small intestine. Completion of the aims below will significantly increase our understanding of the bacterial mechanisms contributing to cholera and possibly provide targets for the development of novel therapies and improved vaccines. The specific aims of the current proposal are: (i) structural analysis of V. cholerae lipid A species; (ii) environmental regulation of V. cholerae lipid A structure; (iii) enzymatic modification of V. cholerae lipid A; and (iv) Toll-like receptor mediated immune activation by V. cholerae LPS.