Regulation of Bacterial Aggregation During Host Colonization

 描述（由申请方提供）：微生物能够在动物定殖后从环境中的寄生、自由生活的生活方式转变为聚集的生物膜相关状态。在许多生物体中，这种转变与环状二聚GMP（c-di-GMP）水平的增加有关，这是一种细胞内第二信使，在从运动到固着生活方式的转变过程中协调运动基因的下调和生物膜基因的上调。固着的、生物膜嵌入的细菌菌落是慢性细菌发病机制的基础，并且生物膜内的细菌表现出对抗菌处理的抗性增加。已经描述了调节c-di- GMP周转的机制，并且该化合物以各种方式影响输出表型，包括直接结合蛋白质和调节RNA。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，很少有人知道的过程中，与一个完整的动物相互作用刺激细菌信号转导的变化。为了理解宿主-微生物相互作用指导细菌采取固着生活方式的过程，研究一个模型系统是有用的，在该模型系统中，可以通过 遗传学和成像方法。短尾乌贼Euprymna peculopes的发光器官只被一种细菌物种--费氏弧菌（Vibrio fischeri）定殖。定殖是稳健的：它通过天然途径在宿主免疫因子存在下在4小时内发生。生物膜的形成是启动成功的定植所必需的，并且如果细菌不能在宿主粘液中聚集，则它们不会进展通过该初始阶段。我们发现了一种细菌突变体，它显著增加了聚集行为。遗传病变映射到一个杂合组氨酸激酶，VF_A0360，除了赋予增加聚集的突变体还表现出减少鞭毛运动和较高水平的c-di-GMP。总之，这些数据支持这样的假设，即通过VF_A0360的信号转导协调了从环境状态到宿主相关定殖状态的转变，在这里，我们检查了以下具体目标：（1）鉴定由VF_A0360组氨酸激酶调节的基因，并检查它们在进入和离开鱿鱼定殖期间的动力学，和（2）表征VF_A0360通过其调节生物膜形成的磷酸化继电器。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性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.