Control of bacterial morphology and differentiation

描述(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置，这两者对它们在TE环境中的生存都是重要的。例如，致尿性大肠杆菌的丝状化在感染过程中颠覆了固有的防御系统，而幽门螺杆菌的螺旋形状对胃的定植很重要。了解细菌如何实现形态和生长模式的变化，并将它们与蛋白质的定位相结合，如毒力因子，对于我们抑制它们的持久性、增殖和宿主感染的能力至关重要。这项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的定位和功能相协调。这项研究利用了柄合成，这是一种特殊的带状生长模式，在新月形杆菌及其近亲中，它产生了细胞膜的细小延伸，以及最近发现的根瘤菌通过极地生长繁殖的细菌，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蛋白被募集到茎中的作用。这将通过确定StpX是否与茎伸长同时插入茎中，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对茎多聚糖的修饰将StpX招募到茎中来实现这一点。一种新的高通量显微镜屏幕将被用来识别涉及蛋白质靶向茎和茎合成的基因。第二个目标是 目的：确定溶菌胺酶SpmX是如何在双股鞭毛虫的茎合成中所必需的，并指导其亚细胞定位。这将通过研究SpmX的溶菌胺酶活性并确定其对茎的合成和定位、其自身的定位以及发育调节因子Div的定位来实现。将构建蛋白质嵌合体，以确定SpmX的结构域是如何进化的，以产生每种功能的特定物种元件。还将研究改变发育调节因子的本地化对发育结果的影响。第三个目标是确定根瘤菌中的细菌，包括一些人类病原体，是如何通过极地的带状生长繁殖的。这将通过使用农杆菌作为模型来实现，以确定这一组中的生长机制。指导极地生长所需的基因将通过候选和高通量方法进行鉴定。将对野生型和特定突变体的肽聚糖组成进行分析，以确定极地生长是否需要不同于侧壁合成的肽聚糖合成和组成。最后，将确定在细胞周期中肽聚糖合成被重新定向的机制。从这些研究中获得的见解可以用来设计抑制生长的策略，防止关键的形态变化，或者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.