VIBRIO CHOLERAE 01 EPS--STRUCTURE, REGULATION & FUNCTION

描述(改编自申请人的摘要)：流行病学 观察表明，引起亚洲霍乱的O1霍乱弧菌是存在的 作为水生植物区系中的土著成员，在人类 疾病。最近，斯科尔尼克实验室的研究人员分离出了一本新的 胞外多糖(EPS)来源于V。 O1 El Tor霍乱弧菌EPS的化学表征表明，它是 不同于O1内毒素和O139荚膜多糖和 形态研究表明，它通过服务于生物膜的形成 作为一种细菌间基质，使细菌聚集并附着在 无生命的表面。纯化的EPS也保护了平滑的殖民地变体 同一菌株的氯气具有杀菌作用。插入 对四聚糖变异体的突变导致了到目前为止 未知的基因簇，命名为VP，序列具有相同的同源性 来自其他物种的多糖类生物合成基因。另一类 突变体持续插入与双组分调控同源的基因 系统。这些初步数据导致了这样的假设：每股收益 在环境水生生境中，表达赋予了生存优势。 为了检验这一假说，斯库尔尼克博士建议完成 EPS的特性；VPS基因簇的特性；准备基因敲除 选择VPS基因的突变体，并测试这些突变体的能力 在模拟自然水生栖息地的实验微型宇宙中生存。 斯库尔尼克博士的团队还将确定控制 EPS的表达，并研究这些信号如何激活VPS基因转录。 最后，在孟加拉国进行的合作研究中，他们将尝试 了解O1霍乱弧菌在自然感染的水中是否表达EPS 消息来源。综上所述，这些研究应该能解释为什么会出现这种情况 作为一种“重新出现”的传染病病原体，物种继续引起流行病。 赠款建议分为三个具体目标：(1)继续 检查EPS的结构和生物合成 物理/化学结构表征，并通过鉴定基因 VPs基因簇的突变分析和互补 克隆人。(2)利用vps基因研究vps基因的表达调控。 融合以确定VPs基因在不同生长条件下的表达， 包括生物膜形成、小鼠肠道生长和藻类共培养。 通过分离确定介导这一调控的调控基因 具有改变规则的突变体。(3)研究每股收益的功能。 确定Vps突变体在不同条件下对存活率的影响， 并确定了V。 孟加拉国生物膜层中发现霍乱弧菌。确定是否存在 皱纹形态与EPS产量的直接相关性。

DESCRIPTION (Adapted from the applicant's abstract): Epidemiological observations suggest that V. cholerae O1, that cause Asiatic cholera, exists as an indigenous member of the aquatic flora between outbreaks of human disease. Recently investigators in the Schoolnik lab have isolated a novel extracellular polysaccharide (EPS) from the rugose colonial variant of V. cholerae O1 El Tor. Chemical characterization of EPS shows that it is distinct from the O1 LPS and the O139 capsular polysaccharide and morphological studies suggest that it mediates biofilm formation by serving as an interbacterial matrix that causes bacteria to aggregate and attach to inanimate surfaces. Purified EPS also protects the smooth colonial variant of the same strain from the bactericidal effects of chlorine. Insertion mutagenesis of the rugose variant led to the identification of a heretofore unknown cluster of genes, designated vps, with sequences that share homology with polysaccharide biosynthetic genes from other species. Another class of mutants sustained insertions in genes homologous to two-component regulatory systems. These preliminary data have led to the hypothesis that EPS expression confers a survival advantage in environmental aquatic habitats. To test this hypothesis Dr. Schoolnik proposes to complete the structural characterization of EPS; characterize the vps gene cluster; prepare knockout mutants of selected vps genes and test these mutants for their capacity to survive in experimental microcosms that simulate natural aquatic habitats. Dr. Schoolnik's group will also identify physiologic factors that control EPS expression and study how these signals activate vps gene transcription. Finally, in collaborative studies conducted in Bangladesh, they will attempt to learn if V. cholerae O1 expresses EPS in naturally infected water sources. Taken together, these studies should shed some light on why this species continues to cause epidemics as a "re-emerging" infectious agent. The grant proposal is divided into three specific aims:(1) Continue to examine the structure and biosynthesis of EPS by completing the physical/chemical structural characterization, and by identifying the genes of the vps gene cluster by mutational analysis and complementation on clones. (2) Study the regulation of vps gene expression using vps gene fusions to ascertain vps gene expression under different growth conditions, including biofilm formation, murine intestinal growth, and algal co-culture. Identify regulatory genes that mediate this regulation by the isolation of mutants with altered regulation. (3) Study the function of EPS by determining the effects of vps mutants on survival under various conditions, and ascertaining the relative abundance of EPS and O1 LPS present on V. cholerae found in biofilm layers in Bangladesh. Determine if there is a direct correlation between the rugose morphotype and EPS production.