Regulation of Vibrio Biofilm Formation by H-NS Repression and Anti-repression.

描述（由申请人提供）：霍乱是一种由血清群O 1和O 139的霍乱弧菌引起的水传播疾病，其特征是排出大量的水样便。在亚洲和非洲的流行地区，这一疾病仍然是一个公共卫生问题，每年有500万病例和130 000人死亡。消灭霍乱的一个主要障碍是霍乱弧菌在水生环境中的持续存在，这是由于其形成生物膜的能力。生物膜中的弧菌对环境胁迫（包括杀生物剂和消毒剂）具有更强的抵抗力。此外，生物膜中的弧菌表现出高感染性表型，其促进疾病在爆发中的快速传播。因此，更好地了解调节生物膜形成的因素对于预防和控制霍乱非常重要。负责弧菌多糖（VPS）胞外基质（生物膜的主要成分）生物合成的基因位于两个操纵子中，其中vpsA和vpsL分别是操纵子I和II的第一基因。这些基因受群体感应、第二信使环二鸟苷酸（c-di-GMP）和转录因子AphA、VpsR和VpsT调节。我们最近发现组蛋白样类核结构蛋白（H-NS）直接抑制参与VPS基质生物合成的基因的表达。H-NS转录沉默中的一个共同主题是存在抗阻遏物，其破坏H-NS与DNA启动子的相互作用。本申请的目的是测试c-di-GMP通过激活作用于vps启动子的H-NS抗阻遏物的表达/活性来增强霍乱弧菌中生物膜形成的新假设。为此，我们将表征H-NS和c-di-GMP-受体蛋白VpsT之间的相互作用，使用遗传和分子生物学方法的组合调节vpsA和vpsL启动子（目的1）。在目标2中，我们将鉴定能够拮抗vpsT启动子处的H-NS阻遏的转录因子，并使用染色质免疫沉淀（ChIP）来证明升高的c-di-GMP细胞内含量在vpsT启动子处启动抗阻遏级联反应，该级联反应随后传递到下游vpsA和vpsL启动子。最后，在目标3中，我们将使用共聚焦显微镜和小鼠竞争性定殖测定来表征霍乱弧菌突变体中表达的生物膜的分子结构、组成和性质。

DESCRIPTION (provided by applicant): Cholera, a waterborne disease caused by Vibrio cholerae of serogroups O1 and O139, is characterized by the passage of voluminous, rice-watery stools. In endemic areas in Asia and Africa, this disease continues to be a public health concern, with a prevalence of 5 million cases and 130,000 deaths per year. A major obstacle to the eradication of cholera is the persistence of V. cholerae in aquatic environments, which is facilitated by its capacity to form biofilms. Vibrios in biofilms are more resistant to environmentl stresses, including biocides and disinfectants. Further, Vibrios in a biofilm exhibit a hyperinfective phenotype that facilitates the rapid dissemination of the disease in outbreaks. Thus, a better understanding of the factors that regulate biofilm formation is important for the prevention and control of cholera. The genes responsible for the biosynthesis of the Vibrio polysaccharide (VPS) extracellular matrix, a major component of biofilms, are located in two operons, in which vpsA and vpsL are the first genes of operons I and II, respectively. These genes are regulated by quorum sensing, the second messenger cyclic diguanylate (c-di-GMP), and by the transcription factors AphA, VpsR and VpsT. We recently showed that the histone-like nucleoid structuring protein (H-NS) directly represses the expression of genes involved in the biosynthesis of the VPS matrix. A common theme in H-NS transcriptional silencing is the presence of anti-repressors that disrupt its interaction with DNA promoters. The objective of this application is to test the novel hypothesis that c-di-GMP enhances biofilm formation in V. cholerae by activating the expression/activity of H-NS anti-repressors acting at vps promoters. To this end, we will characterize the interaction between H-NS and the c-di-GMP-receptor protein VpsT in the regulation of the vpsA and vpsL promoters using a combination of genetic and molecular biology approaches (Aim 1). In Aim 2, we will identify the transcription factor(s) capable of antagonizing H-NS repression at the vpsT promoter and use chromatin immunoprecipitation (ChIP) to demonstrate that elevated c-di-GMP intracellular content initiates an anti-repression cascade at the vpsT promoter that is subsequently transmitted to the downstream vpsA and vpsL promoters. Finally, in Aim 3, we will use confocal microscopy and a mouse competitive colonization assay to characterize the molecular architecture, composition and properties of the biofilm expressed in V. cholerae hns mutants.