Phosphorylation and acetylation of secreted bacterial proteins: a new regulatory

描述(由申请人提供)：这项工作的长期目标是了解结核分枝杆菌(Mtb)在细胞生长和休眠期间细胞壁是如何调节的，并确定在这两种条件下针对细胞壁酶的候选药物，从而制造有效的抗生素。这项建议中描述的研究的目的是在结核分枝杆菌和相关物种中描述一种以前未描述的分泌细菌蛋白的调节机制。到目前为止的工作已经表明，一种重要的细胞伸长因子CwlM被磷酸化，这种磷酸化改变了它调节细胞伸长的活性。这项拟议的工作将通过分析CwlM耗尽时的肽聚糖结构和其他伸长因子的定位来探讨CwlM的细胞和分子功能，并将通过免疫共沉淀来确定CwlM的调控靶点以确定蛋白质相互作用。CwlM的一个破坏细胞伸长的磷化等位基因已经被鉴定出来。这项拟议的工作将测量蛋白质的稳定性和这种突变蛋白的分泌，并将分析肽聚糖结构和延伸因子在突变菌株中的定位，以了解磷酸化如何影响CwlM的活性。此外，还将鉴定CwlM的同源激酶。为了了解应激如何改变细胞壁结构和生长动力学，本工作将研究应激条件如何改变CwlM的磷酸化和活性。这项工作将进一步了解分泌的蛋白质--特别是那些重塑细胞壁的蛋白质--是如何受到调节的，以及它们的活动是如何与细胞质事件相协调的。了解这些过程对于建立一个模型，研究在营养生长和导致休眠的胁迫期间细胞壁是如何重塑的，以及识别细胞壁的药物靶点至关重要。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how the cell wall of M. tuberculosis (Mtb) is regulated during cell growth and dormancy, and to identify drug candidates that will target cell wall enzymes in both of these conditions, thus making effective antibiotics. The objective of the research described in this proposal is to characterize, in Mtb and a related species, a previously-undescribed regulatory mechanism for secreted bacterial proteins. Work to date has shown that an essential cell elongation factor, CwlM, is phosphorylated and that this phosphorylation alters its activity in regulating cell elongation. The proposed work will probe the cellular and molecular function of CwlM by analyzing peptidoglycan structure and the localization of other elongation factors when CwlM is depleted, and will identify the regulatory targets of CwlM by using co-immunoprecipitation to identify protein interactors. A phosphoablative allele of CwlM that impairs cell elongation has been identified. The proposed work will measure the protein stability, and secretion of this mutant protein, and will analyze peptidoglycan structure and elongation factor localization in the mutant strain in order to understand how phosphorylation affects the activity of CwlM. In addition, the cognate kinase for CwlM will be identified. In an effort to understand how stress changes cell wall structure and growth dynamics, this work will study how stressful conditions alter the phosphorylation and activity of CwlM. This work will further the understanding of how secreted proteins - and especially those that remodel the cell wall - are regulated and how their activity is coordinated with cytoplasmic events. Understanding these processes is critical to building a model for how the cell wall is remodeled during both vegetative growth and the stress that leads to dormancy, and for identifying cell wall drug targets.