Regulation of Bacterial Aggregation During Host Colonization

 描述(申请人提供)：微生物能够在动物定居后，从环境中浮游的、自由生活的生活方式转变为聚集的、与生物膜相关的状态。在许多生物体中，这种转变与环状二聚体GMP(c-di-GMP)水平的增加有关，c-di-GMP是细胞内的第二信使，在从运动到静止的生活方式转变过程中协调运动基因的下调和生物膜基因的上调。固着、生物膜嵌入的细菌菌落是慢性细菌发病的基础，生物膜内的细菌对抗菌治疗表现出更高的耐药性。调节c-di-GMP周转的机制已被描述，该化合物以各种方式影响输出表型，包括与蛋白质和调节RNA的直接结合。以前的工作依赖于细菌培养、真核细胞培养和动物模型中的复杂系统。然而，关于与完整的动物相互作用刺激细菌信号转导变化的过程，人们知之甚少。为了理解宿主-微生物相互作用引导细菌采取固定生活方式的过程，研究一个模型系统是有用的，在这个模型系统中，可以解剖各个阶段。 遗传学和成像方法。短尾鱿鱼的发光器官，真乌贼的发光器官，只由一种细菌物种--费氏弧菌--定居。定殖性很强：在宿主免疫因素存在的情况下，通过原生路线在四小时内发生。生物膜的形成是启动成功的定植所必需的，如果细菌不能在宿主粘液中聚集，它们就不会经过这个初始阶段。我们发现了一种细菌突变体，它可以显著增加聚集行为。遗传损伤被定位到杂合组氨酸激酶VF_A0360，除了导致聚集增加外，突变体还表现出鞭毛运动性降低和c-di-GMP水平升高。综上所述，这些数据支持这样的假设，即通过VF_A0360进行的信号转导协调了从环境状态到宿主相关的定植状态的转变，在这里，我们考察了以下具体目标：(1)识别受VF_A0360组氨酸激酶调控的基因，并检测它们在进入和离开鱿鱼定植过程中的动态；(2)表征VF_A0360调节生物膜形成的磷酸继电器。通过询问这一信号通路，我们将深入了解细菌信号转导如何在有益和致病的定植过程中适应宿主环境。

 DESCRIPTION (provided by applicant): Microorganisms are able to switch from a planktonic, freeliving lifestyle in the environment to an aggregated, biofilm-associated state upon animal colonization. Across many organisms, this transition is associated with increased levels of cyclic dimeric GMP (c-di-GMP), an intracellular second messenger that coordinates the downregulation of motility genes and the upregulation of biofilm genes during the transition from the motile to the sessile lifestyle. Sessile, biofilm embedded bacterial colonies underlie chronic bacterial pathogenesis and bacteria within biofilms exhibit increased resistance to antibacterial treatments. Mechanisms regulating c-di- GMP turnover have been described, and the compound influences output phenotypes in various ways, including direct binding to proteins and to regulatory RNA. Previous work has relied on sophisticated systems in bacterial culture, eukaryotic cell culture, and animal models. However, little is known about the processes by which interaction with an intact animal stimulates a change in bacterial signal transduction. To understand the processes by which host-microbe interaction directs bacteria to adopt a sessile lifestyle, it is useful to study a model system in which individual stages can be dissected through genetics and imaging approaches. The light organ of the bobtail squid, Euprymna scolopes, is colonized exclusively by one bacterial species, Vibrio fischeri. Colonization is robust: it occurs within four hours via the native route and in the presence of host immune factors. Biofilm formation is required for initiating a successful colonization, and bacteria do not progress past this initial phase if they fail to aggregate in host mucus. We identified a bacterial mutant that dramatically increases aggregation behavior. The genetic lesion mapped to a hybrid histidine kinase, VF_A0360, and in addition to conferring increased aggregation the mutant also exhibited reduced flagellar motility and higher levels of c-di-GMP. Together, these data support the hypothesis that signal transduction through VF_A0360 coordinates the transition from the environmental state to the host-associated colonized state, and here we examine the following specific aims: (1) Identify genes regulated by the VF_A0360 histidine kinase and examine their dynamics during entry to and exit from squid colonization, and (2) Characterize the phosphorelay through which VF_A0360 regulates biofilm formation. By interrogating this signaling pathway we will gain insight into how bacterial signal transduction adjusts to the host environment during beneficial and pathogenic colonization.