Control of bacterial morphology and differentiation

描述（由申请人提供）：细菌经历特定的细胞形状变化，并将各种蛋白质定位于亚细胞位点，这两者对其在环境中的生存都很重要。例如，尿路致病性E.大肠杆菌在感染过程中破坏了先天防御，幽门螺杆菌的螺旋形状对于胃的定植很重要。了解细菌如何实现形态和生长模式的变化，并将其与蛋白质（如毒力因子）的定位相结合，对于我们抑制其持久性，增殖和宿主感染的能力至关重要。本研究的总体目标是研究产生各种细胞形状和生长模式的机制，以及它们如何与蛋白质定位和功能协调。这项研究利用茎合成，一个专门的地带性模式的增长柄杆菌crescentus及其亲属，产生薄的延伸的细胞被膜和最近发现的繁殖的细菌在根瘤菌的极性生长，包括一些人类病原体。该项目有三个主要目标。第一个目的是确定青霉素结合蛋白PbpC在StpX蛋白向茎的募集中的作用。这将通过确定StpX是否在茎伸长的同时插入茎中以及PbpC是否通过蛋白质-蛋白质相互作用或通过其酶活性和茎肽聚糖的修饰将StpX募集到茎中来实现。一种新的高通量显微镜屏幕将被用来识别参与蛋白质靶向的茎和茎合成的基因。第二个目标是 以确定胞壁酶SpmX是如何在Asticcacaulis biprothecum中合成茎所必需的，并指导其亚细胞定位。这将通过研究SpmX的胞壁酶活性并确定其对茎合成和定位、其自身定位以及发育调节剂Div的定位的要求来实现。将构建蛋白质嵌合体，以确定SpmX的结构域如何进化，以产生每个功能的物种特异性元件。还将研究改变发育调节剂的定位对发育结果的影响。第三个目标是确定根瘤菌目中的细菌，包括一些人类病原体，如何在其极点通过带状生长繁殖。这将通过使用根癌农杆菌作为模型来确定该组中的生长机制来实现。将通过候选和高通量方法鉴定指导极性生长所需的基因。将在野生型和特定突变体中分析肽聚糖组成，以确定极性生长是否需要肽聚糖合成和组成，其与侧细胞壁合成不同。最后，将确定在细胞周期中肽聚糖合成被重定向的机制。从这些研究中获得的见解可用于设计策略，以抑制生长，防止关键的形态变化，或改变病原体中重要的蛋白质定位途径，从而提高我们控制它们的能力。 公共卫生相关性：细菌经历特定的细胞形状变化并将各种蛋白质定位于亚细胞位点，这两者对于它们在环境中的生存都很重要，包括病原体的宿主环境。我们将研究产生各种细胞形状和生长模式的机制，以及它们如何协调和影响蛋白质定位和功能。从这些研究中获得的见解可用于设计策略，以抑制生长，防止关键的形态变化，或改变病原体中重要的蛋白质定位途径，从而提高我们控制它们的能力。

DESCRIPTION (provided by applicant): Bacteria undergo specific cell shape changes and localize various proteins to subcellular sites, both of which are important for their survival in te environment. For example, filamentation of uropathogenic E. coli subverts innate defenses during the infection process, and the helical shape of Helicobacter pylori is important for colonization of the stomach. Understanding how bacteria achieve changes in morphology and modes of growth and couple them to the localization of proteins, such as virulence factors, is critical for our ability to inhibit their persistence, proliferation, and host infection. The generl goal of this research is to study the mechanisms that generate various cell shapes and growth patterns and how they are coordinated with protein localization and function. This study takes advantage of stalk synthesis, a specialized zonal mode of growth in Caulobacter crescentus and its relatives that generates thin extensions of the cell envelope and of the recently discovered reproduction of bacteria in the Rhizobiales by polar growth, including some human pathogens. The project has three major aims. The first aim is to determine the role of the penicillin binding protein PbpC in the recruitment of the StpX protein to the stalk. This will be achieved by determining if StpX is inserted into the stalk concurrently with stalk elongation and if PbpC recruits StpX to the stalk by protein-protein interaction or through its enzymatic activity and modification of stalk peptidoglycan. A novel high throughput microscopy screen will be used to identify genes involved in protein targeting to the stalk and in stalk synthesis. The second aim is to determine how the muramidase SpmX is required for stalk synthesis in Asticcacaulis biprosthecum and directs its subcellular location. This will be accomplished by studying the muramidase activity of SpmX and determining its requirement for stalk synthesis and localization, for its own localization, and for the localization of the developmental regulator Div. Protein chimeras will be constructed to determine how the domains of SpmX have evolved to generate species-specific elements of each function. The effect of altering the localization of developmental regulators on developmental outcomes will also be studied. The third aim is to determine how bacteria in the Rhizobiales, including some human pathogens, reproduce by zonal growth at their pole. This will be accomplished by using Agrobacterium tumefaciens as a model to determine the growth mechanism in this group. Genes required to direct polar growth will be identified by candidate and high throughput approaches. Peptidoglycan composition will be analyzed in wild-type and specific mutants to determine if polar growth requires peptidoglycan synthesis and composition that are different than for lateral cell wall synthesis. Finally, the mechanisms by which peptidoglycan synthesis is redirected during the cell cycle will be determined. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them. PUBLIC HEALTH RELEVANCE: Bacteria undergo specific cell shape changes and localize various proteins to subcellular sites, both of which are important for their survival in the environment, including the host environment for pathogen. We will study the mechanisms that generate various cell shapes and growth patterns and how they are coordinated with and influence protein localization and function. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them.