A periplasmic global regulator, ExoR, for bacterial invasion of host cells

描述(申请人提供)：植入的医疗器械的细菌感染和表面定植是并将继续是主要的健康威胁，特别是随着多重耐药病原体的发展。细菌病原体依靠其膜传感器来检测宿主细胞的存在，以便在正确的时间产生毒力因子来入侵宿主细胞或在医疗器械的表面定植。阻断细菌对宿主的感知可能是阻止细菌感染的一种新方法。作为我们长期努力了解细菌在宿主细胞入侵过程中的感知和信号的一部分，我们正在研究苜蓿中华根瘤菌Exor蛋白控制自由活细胞向入侵准备细胞转换的分子机制。我们以前已经证明，Exor蛋白和EXOS/ChvI双组分调控系统都反向调节宿主细胞入侵所需的多糖(琥珀糖)和细菌运动所需的鞭毛的产生。我们目前的假设是Exor蛋白的活性形式(ExoRm)与EXOS结合并使其处于关闭状态，改变了EXOS/ChvI系统调控的基因的表达。周质中ExoRm的量通过反馈调节和ExoRm的蛋白分解产生ExoRc20来维持。ExoRm蛋白水解率的变化被细胞用来感知环境刺激，控制自由生活和依附生活之间的切换。该模型基于过去两年的以下主要发现。首先，Exor位于EXOS/ChvI双组分调控系统的上游，负向调节EXOS的活性；其次，Exor通过反馈调节进行自我调节；第三，Exor在周质中被消化，产生ExoRc20。我们计划1)通过分别表达不同形式的Exor、ExoRp、ExoRm和ExoRc20来确定不同Exor蛋白的功能；并通过检测它们的生物学功能及其与ExOS蛋白的直接相互作用的能力；2)通过表征Exor蛋白分解的分子机制来确定Exor蛋白分解的分子机制；通过鉴定环境刺激来调节Exor蛋白分解，并鉴定紫花苜蓿Exor蛋白水解酶。我们的新发现可以立即应用于了解56种与苜蓿链霉菌有密切同源的动植物病原体的致病性。我们的新发现将有助于开发控制细菌感染的新方法。 与公共卫生相关：大多数致病菌在感知到宿主的存在后，都会生产特殊产品来增强它们感染宿主细胞的能力。我们的研究将导致开发新的方法来控制细菌感染，方法是阻断它们感知宿主存在的能力。

DESCRIPTION (provided by applicant): Bacterial infections and surface colonization of implanted medical devices are and will continue to be major health threats especially with the development of multiple drug resistant pathogens. Bacterial pathogens rely on their membrane sensors to detect the presence of host cells in order to produce virulence factors at the right time to invade host cells or colonize the surface of medical devices. Blocking bacterial sensing of hosts could be a new approach to stop bacterial infection. As part of our long term effort to understand bacterial sensing and signaling during host cell invasion, we are studying the molecular mechanism of Sinorhizobium meliloti ExoR protein in controlling the switch from free living cells to invasion ready cells. We have shown previously that both the ExoR protein and the ExoS/ChvI two-component regulatory system regulates inversely the production of a polysaccharide (succinoglycan) required for host cell invasion, and flagella, required for bacterial motility. Our current hypothesis is that the active form of the ExoR protein (ExoRm) binds ExoS and keeps it in the OFF state, altering the expression of the genes regulated by ExoS/ChvI system. The amount of ExoRm in the periplasm is maintained through feedback regulation and the proteolysis of ExoRm to produce ExoRc20. The changes in ExoRm proteolysis rate is being used by the cells to sense environmental stimuli control the switch between free living and attached living. This model is based on the following key discoveries made in the last two years. First, ExoR functions upstream of ExoS/ChvI two-component regulatory system and negatively regulates the activity of ExoS; Second, ExoR autoregulates through feedback regulation; Third, ExoR is digested in periplasm to generate ExoRc20. We plan to 1) determine the function of different forms of ExoR proteins by expressing different forms of ExoR, ExoRp, ExoRm, and ExoRc20, individually; and by examining their biological functions and their ability to interact directly with ExoS protein; 2) determine the molecular mechanisms of ExoR proteolysis and its regulation by characterizing the molecular mechanism of ExoR proteolysis; identifying environmental stimuli modulating ExoR proteolysis, and identifying the S. meliloti ExoR protease. Our new discoveries can be applied immediately to the understanding of the pathogenicities of 56 animal and plant pathogens that have close homologs of S. meliloti ExoR. Our new discoveries will contribute to the development of new approaches to control bacterial infections. PUBLIC HEALTH RELEVANCE: After sensing the presence of their hosts, most pathogenic bacteria produce special products to enhance their abilities to infect host cells. Our study will lead to the development of new approaches to control bacterial infections by blocking their abilities to sense of the presence of their hosts.