Smooth to Rugose phase variation in Vibrio cholerae

描述（由申请人提供）：霍乱弧菌引起霍乱，是水生环境的自然居民。季节性霍乱暴发发生在疾病流行的地方，并可在世界范围内传播。霍乱弧菌引起流行病的能力与其在水生栖息地的生存能力有关。已经提出，霍乱弧菌形成生物膜的能力（即，基质封闭、表面相关的群落）对于其在流行病之间的水生生境中的生存至关重要，并且有利于流行病期间的宿主到宿主的传播。该提案的目的是提高我们对生物膜基质组分、生物膜形成的机制和调节、环状二聚鸟苷一磷酸（c-diGMP）信号传导的机制及其在霍乱弧菌生物学中的重要性的理解。在目标1中，我们将专注于生物膜基质组分的表征。我们将确定弧菌多糖（VPS）的结合能力和生物膜中的基质蛋白的定位模式，并测试我们的假设，这些蛋白质结合VPS，以稳定的矩阵。我们将确定vps基因簇中哪些基因是生物膜形成所需的。我们还将研究一个假定的VPS裂解酶的酶学性质。最后，我们将确定已知的生物膜决定因素在霍乱弧菌发病机制中的作用。在目标2中，我们将剖析生物膜形成的调节。生物膜调控网络由两个正转录调控因子（VpsR和VpsT）、一个负转录调控因子（HapR）和我们最近鉴定的传感器组氨酸激酶（VpsS）组成。我们将确定VpsS是否磷酸化VpsR，并确定控制VpsS的重要信号。我们将表征的vps和基质蛋白基因的顺式作用的功能，并阐明是否转录调节VpsR，VpsT和HapR直接与这些启动子相互作用。在目标3中，我们将阐明c-diGMP信号传导控制生物膜形成的分子机制。我们将确定调节生物膜形成的c-diGMP信号蛋白是否依赖于蛋白质-蛋白质相互作用。为了鉴定c-di-GMP信号传导系统的靶蛋白，我们将寻找与c-diGMP信号传导蛋白相互作用的蛋白，以及在皱纹变体内的c-diGMP受体蛋白。然后，我们将评估这些c-diGMP信号蛋白如何影响霍乱弧菌的发病机制。更好地了解生物膜形成的机制，c-diGMP信号传导，以及这两个过程在霍乱弧菌生物学中的重要性，将证明有助于预测和控制霍乱流行的未来策略的发展，并将有助于识别新的药物靶点，用于在感染期间对抗病原体。公共卫生相关性：生物膜，表面附着的微生物群落，在环境，工业和临床环境中引起重大问题。霍乱弧菌是霍乱病的病原体，形成自然发生的生物膜，对环境生存和病原体的传播至关重要。该提案旨在提高我们对生物膜形成的理解，这将使我们能够确定在肠道和水生生态系统中对抗这种致命病原体的目标，并有助于开发预测和/或控制霍乱流行的工具。

DESCRIPTION (provided by applicant): Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. Seasonal cholera outbreaks occur where the disease is endemic and can spread worldwide. V. cholerae's ability to cause epidemics is tied to its ability to survive in aquatic habitats. It has been proposed that V. cholerae's ability to form biofilms (i.e., matrix-enclosed, surface-associated communities) is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The objective of this proposal is to improve our understanding of biofilm matrix components, the mechanisms and regulation of biofilm formation, the mechanism of cyclic dimeric guanosine monophosphate (c- diGMP) signaling, and their importance in the biology of V. cholerae. In Aim 1, we will focus on characterization of biofilm matrix components. We will determine Vibrio polysaccharide (VPS) binding capacities and localization patterns of the matrix proteins in biofilms, and test our hypothesis that these proteins bind to VPS in order to stabilize the matrix. We will determine which genes within the vps gene clusters are required for biofilm formation. We will also investigate the enzymatic properties of a putative VPS lyase. Finally, we will ascertain the contribution of known biofilm determinants in V. cholerae pathogenesis. In Aim 2, we will dissect regulation of biofilm formation. Biofilm regulatory network consists of two positive transcriptional regulators (VpsR and VpsT), a negative transcriptional regulator (HapR), and our recently-identified sensor histidine kinase (VpsS). We will determine whether VpsS phosphorylates VpsR and identify important signals that control VpsS. We will characterize the cis-acting features of the vps and matrix-protein genes and elucidate whether the transcriptional regulators VpsR, VpsT and HapR interact directly with these promoters. In Aim 3, we will elucidate the molecular mechanisms by which c-diGMP signaling controls biofilm formation. We will determine whether c-diGMP signaling proteins that modulate biofilm formation rely on protein-protein interactions. To identify the target proteins of the c-di-GMP signaling systems, we will search for proteins interacting with c-diGMP signaling proteins, as well as for c-diGMP receptor proteins within the rugose variants. We will then assess how these c-diGMP signaling proteins affect V. cholerae pathogenesis. Better understanding of the mechanism of biofilm formation, c-diGMP signaling, and the importance of both of these processes in V. cholerae biology will prove useful for the development of future strategies for predicting and controlling cholera epidemics, and will facilitate identification of novel drug targets for combating the pathogen during infection. PUBLIC HEALTH RELEVANCE: Biofilms, surface attached microbial communities, cause significant problems in environmental, industrial, and clinical settings. Vibrio cholerae, the causative agent of the disease cholera, forms naturally-occurring biofilms that are critical for environmental survival and the transmission of the pathogen. This proposal aims to improve our understanding of biofilm formation, which will allow us to define targets to combat this deadly pathogen in both intestinal and aquatic ecosystems, and also help in the development of tools for prediction and/or control of cholera epidemics.