Control of cell wall synthesis and antibiotic tolerance in mycobacteria

迫切需要了解结核分枝杆菌细胞壁代谢的调节。 因为它导致了抗生素耐受，从而加剧了结核病的后果。这样做的目的是 建议是建立一个分子模型，说明环境信息是如何通过三个蛋白的磷酸化来流动的 用于动态控制分枝杆菌细胞壁代谢的细胞壁调节剂。这一点的中心假设是 建议是，生长过程中细胞壁的中央调节器也在压力下对其进行调节。这一假设是 根据我们的数据，磷酸酶PSTP控制生长和应激反应，并且 磷酸化调节剂CwlM和DivIVA是生长和抗生素生存所必需的。这样做的理由是 研究表明，对细胞壁调节的分子理解将为更好的结核病药物铺平道路。 目的1：确定磷酸酶PSTP如何协调细胞壁代谢。我们的工作假说 是PSTP的磷酸化调节其针对细胞壁因子的活性，并帮助协调过渡 从增长到停滞。我们将：a)确定PSTP磷酸化如何受到Mtb压力的影响；b)确定 PSTP的关键底物，并测定每个底物上不同PSTP磷酸异构体的活性；以及 C)确定PSTP如何促进结核分枝杆菌的抗生素耐受性。 目的2：确定CwlM是如何调节多种肽聚糖酶的。我们的工作假设是 CwlM受磷酸化和循环肽多糖的调节，进而调节肽聚糖的合成。 在多个步骤中。我们将：a)确定改变CwlM在Mtb中磷酸化的条件；b)表征 CwlM和CwlM~P对其相互作用伙伴酶结合和活性的影响；以及c)测量和 表征CwlM的催化活性的功能和调节。 目的3：确定DivIVA如何协调极细胞壁代谢。我们的工作假设是DivIVA 激活细胞壁前体酶以促进生长，并受磷酸化调节。我们将：a) 确定Mtb中DivIVA的磷酸化如何受生长和压力条件的影响；b)确定 其蛋白质相互作用所需的DivIVA，并表征相互作用的磷依赖；以及 C)测量DivIVA和DivIV~P对其交互伙伴活动的影响。 在这项工作完成后，我们希望有一个信号通路的多水平分子模型 它控制着分枝杆菌细胞壁前体的合成。我们将对磷酸酶的调节进行表征 PSTP是主要监管者，也是抗生素和抗耐药药物的候选药物靶点。我们 将描述中间调节因子CwlM和DivIVA之间的蛋白质相互作用的信号作用 以及它们的酶调节靶点；这些相互作用是抗耐药药物的潜在靶点。这部作品 将为新型药物筛选奠定基础。

There is a critical need to understand the regulation of cell wall metabolism in Mycobacterium tuberculosis because it contributes to antibiotic tolerance, which exacerbates tuberculosis outcomes. The objective of this proposal is to build a molecular model for how environmental information flows through phosphorylation of three cell wall regulators to dynamically control cell wall metabolism in mycobacteria. The central hypothesis of this proposal is that the central regulators of the cell wall during growth also regulate it in stress. This hypothesis is based on our data that the phosphatase PstP controls growth as well as stress responses, and that the phosphorylated regulators CwlM and DivIVA are required for growth and antibiotic survival. The rationale for this research is that a molecular understanding of cell wall regulation will pave the way for better TB drugs. Aim 1: Determine how the phosphatase PstP orchestrates cell wall metabolism. Our working hypothesis is that phosphorylation of PstP regulates its activity against cell wall factors and helps coordinate the transition from growth to stasis. We will: a) determine how PstP phosphorylation is affected by stresses in Mtb; b) identify the key substrates of PstP and determine the activity of different PstP phospho-isoforms on each substrate; and c) determine how PstP contributes to antibiotic tolerance in Mtb. Aim 2: Determine how CwlM regulates multiple peptidoglycan enzymes. Our working hypothesis is that CwlM is regulated by phosphorylation and recycled peptidoglycan, and in turn regulates peptidoglycan synthesis at multiple steps. We will: a) identify conditions that alter CwlM’s phosphorylation in Mtb; b) characterize the effects of CwlM and CwlM~P on the binding and activity of its interaction partner enzymes; and c) Measure and characterize the function and regulation of the catalytic activity of CwlM. Aim 3: Determine how DivIVA coordinates polar cell wall metabolism. Our working hypothesis is that DivIVA activates cell wall precursor enzymes to promote growth, and is regulated by phosphorylation. We will: a) determine how DivIVA’s phosphorylation is affected by growth and stress conditions in Mtb; b) identify sites on DivIVA required for its protein interactions, and characterize the phospho-dependence of the interactions; and c) measure the effects of DivIVA and DivIV~P on the activity of their interaction partners. Upon completion of this work we expect to have a multi-level molecular model of the signaling pathways that control cell wall precursor synthesis in mycobacteria. We will characterize the regulation of the phosphatase PstP, which is a master regulator and a candidate drug target for both antibiotics and anti-tolerance drugs. We will describe the signaling role of the protein interactions between the intermediate regulators CwlM and DivIVA and their enzymatic regulatory targets; these interactions are potential targets for anti-tolerance drugs. This work will lay the groundwork for novel drug screens.