Smooth to Rugose phase variation in Vibrio cholerae

描述（由申请人提供）：霍乱弧菌引起霍乱疾病，是水生环境的天然居民。季节性霍乱暴发发生在霍乱地方性流行并可在全球传播的地方。霍乱弧菌引起流行病的能力与其在水生生境中生存的能力有关。有人提出，霍乱弧菌形成生物膜（即基质封闭的表面相关群落）的能力对其在流行病期间在水生栖息地的生存至关重要，并且有利于流行病期间宿主对宿主的传播。本研究的目的是提高我们对生物膜基质成分、生物膜形成机制和调控、环二聚鸟苷单磷酸（c- diGMP）信号传导机制及其在霍乱弧菌生物学中的重要性的认识。在目标1中，我们将重点关注生物膜基质成分的表征。我们将确定弧菌多糖（VPS）的结合能力和基质蛋白在生物膜中的定位模式，并验证我们的假设，即这些蛋白与VPS结合以稳定基质。我们将确定vps基因簇中的哪些基因是生物膜形成所必需的。我们还将研究推定的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控。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）和我们最近发现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。我们将描述vps和基质蛋白基因的顺式作用特征，并阐明转录调控因子VpsR、VpsT和HapR是否直接与这些启动子相互作用。在Aim 3中，我们将阐明c-diGMP信号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-蛋白相互作用。为了确定c-di-GMP信号系统的靶蛋白，我们将在rugoose变异体中寻找与c-diGMP信号蛋白相互作用的蛋白，以及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，将有助于制定未来预测和控制霍乱流行的策略，并将有助于在感染过程中识别对抗病原体的新药物靶点。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。霍乱弧菌是霍乱的病原体，它形成自然形成的生物膜，对环境生存和病原体传播至关重要。这一建议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.