Control of cell wall synthesis and antibiotic tolerance in mycobacteria

分枝杆菌细胞壁合成和抗生素耐受性的控制

• 批准号: 10772460
• 负责人: Cara Cheney Boutte
• 金额: $ 6.54万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10772460
• 关键词: Affect; Antibiotics; Binding; Cell Polarity; Cell Wall; Cell model; Data; Dependence; Drug Controls; Drug Screening; Drug Targeting; Drug Tolerance; Effectiveness; Enzyme Precursors; Enzymes; Event; Foundations; Future; Genus Mycobacterium; Goals; Growth; In Vitro; Infection; Left; Measures; Metabolic; Metabolism; Modeling; Molecular; Mycobacterium smegmatis; Mycobacterium tuberculosis; Outcome; Patient-Focused Outcomes; Peptidoglycan; Permeability; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Process; Protein Dephosphorylation; Protein Isoforms; Proteins; Regimen; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Stress; Testing; Tuberculosis; Work; antibiotic tolerance; biological adaptation to stress; design; drug candidate; effective therapy; genetic regulatory protein; hatching; improved; men; molecular modeling; mycobacterial; novel therapeutics; treatment strategy; tuberculosis drugs

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.

目前迫切需要了解结核分枝杆菌细胞壁代谢的调控 因为它有助于抗生素耐药性，从而加剧结核病的后果。的目的 一项新的提议是建立一个分子模型，研究环境信息如何通过磷酸化三个 细胞壁调节剂以动态控制分枝杆菌中的细胞壁代谢。这个问题的核心假设是 这表明，生长期间细胞壁的中心调节因子在胁迫下也对其进行调节。这种假设是 基于我们的数据，磷酸酶PstP控制生长以及应激反应， 磷酸化调节剂CwIM和DivIVA是生长和抗生素存活所必需的。这样做的理由 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。我们的工作假设 PstP的磷酸化调节其对抗细胞壁因子的活性， 从增长到停滞我们将：a）确定Mt B中应激如何影响PstP磷酸化; B）鉴定 PstP的关键底物，并确定不同PstP磷酸化同种型对每种底物的活性;和 c）确定PstP如何有助于Mtb中的抗生素耐受性。 目的2：确定CwIM如何调节多种肽聚糖酶。我们的假设是 CwlM受磷酸化和再循环肽聚糖的调节，并反过来调节肽聚糖的合成 在多个步骤。我们将：a）鉴定改变Mt B中CwIM磷酸化的条件; B）表征Mt中CwIM磷酸化的条件。 CwIM和CwIM-P对其相互作用配偶体酶的结合和活性的影响;以及 表征CwlM的催化活性的功能和调节。 目的3：确定DivIVA如何协调极性细胞壁代谢。我们假设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DivIVA的磷酸化如何受到Mt B中生长和应激条件的影响; B）鉴定Mt上的位点 DivIVA所需的蛋白质相互作用，并表征相互作用的磷酸依赖性;和 c）测量DivIVA和DivIV~P对其相互作用伴侣的活性的影响。 在完成这项工作后，我们希望有一个多层次的分子模型的信号通路 控制分枝杆菌细胞壁前体合成的基因。我们将描述磷酸酶的调节 PstP是抗生素和抗耐药性药物的主要调节剂和候选药物靶标。我们 将描述中间调节物CwlM和DivIVA之间的蛋白质相互作用的信号传导作用 以及它们的酶调节靶点;这些相互作用是抗耐受药物的潜在靶点。这项工作 将为新药筛选奠定基础。

项目成果

Localized Production of Cell Wall Precursors May Be Critical for Regulating the Mycobacterial Cell Wall.

细胞壁前体的本地化生产对于调节分枝杆菌细胞壁可能至关重要。

• DOI: 10.1128/jb.00125-22
• 发表时间: 2022
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Boutte,CaraC

Phosphorylation on PstP Regulates Cell Wall Metabolism and Antibiotic Tolerance in Mycobacterium smegmatis.

PstP 的磷酸化调节耻垢分枝杆菌的细胞壁代谢和抗生素耐受性。

• DOI: 10.1128/jb.00563-20
• 发表时间: 2021
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Shamma,Farah;Papavinasasundaram,Kadamba;Quintanilla,SamanthaY;Bandekar,Aditya;Sassetti,Christopher;Boutte,CaraC
• 通讯作者: Boutte,CaraC

Mycobacterial serine/threonine phosphatase PstP is phosphoregulated and localized to mediate control of cell wall metabolism.

• DOI: 10.1111/mmi.14951
• 发表时间: 2022-07
• 期刊: MOLECULAR MICROBIOLOGY
• 影响因子: 3.6
• 作者: Shamma, Farah;Rego, E. Hesper;Boutte, Cara C.
• 通讯作者: Boutte, Cara C.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

极性蛋白 Wag31 既激活又抑制两极和隔膜的细胞壁代谢。

• DOI: 10.3389/fmicb.2022.1085918
• 发表时间: 2022
• 期刊: FRONTIERS IN MICROBIOLOGY
• 影响因子: 5.2
• 作者: Habibi Arejan, Neda;Ensinck, Delfina;Diacovich, Lautaro;Patel, Parthvi Bharatkumar;Quintanilla, Samantha Y.;Emami Saleh, Arash;Gramajo, Hugo;Boutte, Cara C.
• 通讯作者: Boutte, Cara C.

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis.

• DOI: 10.1371/journal.pone.0280336
• 发表时间: 2023
• 期刊: PLOS ONE
• 影响因子: 3.7
• 作者: Quintanilla, Samantha C.;Arejan, Neda Habibi;Patel, Parthvi;Boutte, Cara
• 通讯作者: Boutte, Cara