The Outer Surface of Vibrio cholerae

描述(申请人提供)：革兰氏阴性菌霍乱弧菌被归类为B类食物和水传播的病原体，导致被称为霍乱的急性、严重的腹泻疾病。不幸的是，霍乱仍然严重威胁着发展中国家的健康，每年约有300万至500万例霍乱病例。霍乱弧菌是一种正常生活在水环境中的细菌，属于河口地区自由生活的细菌区系。虽然已知约200个已知的O型血清群，但只有携带脂多糖(LPS)体细胞抗原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，霍乱弧菌的内毒素由三个不同的区域组成，即膜相关脂A区、短核心寡糖和O抗原多糖。尽管类脂A结构域是革兰氏阴性细菌膜的重要组成部分，并且是通过保守的途径合成的，但它是一个高度多样化的分子。病原菌改变其脂多糖的脂类A结构域以响应周围环境。由于脂类A是脂多糖的生物活性部分，这些修饰可以通过改变天然免疫受体复合体TLR4/MD-2来改变脂多糖的识别，从而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抗药性。我们的总体目标是了解细菌表面脂多糖结构的变化如何促进霍乱弧菌在水环境和人类宿主中的生存。这项提案将集中于定义霍乱弧菌脂类A的结构变化，以响应细菌的细胞外环境，并说明这一过程所需的酶机制。将在不同的生长条件下监测霍乱弧菌脂类A的结构变化，这些条件模拟在水环境或小肠中发现的条件。完成以下目标将大大增加我们对导致霍乱的细菌机制的了解，并可能为开发新的治疗方法和改进的疫苗提供目标。本提案的具体目的是：(1)霍乱弧菌脂蛋白A种的结构分析；(2)霍乱弧菌脂蛋白A结构的环境调节；(3)霍乱弧菌脂蛋白A的酶修饰；(4)霍乱弧菌脂多糖介导的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.