Validating a potential interaction between error-prone polymerases and SSB as a therapeutic target for Mycobacterium tuberculosis

验证易错聚合酶和 SSB 之间潜在的相互作用作为结核分枝杆菌的治疗靶点

• 批准号: 10189804
• 负责人: Joseph J. Loparo
• 金额: $ 8.45万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-08 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189804
• 关键词: Ablation; Affect; Air; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Architecture; Bacteria; Binding; Biological Assay; Biological Models; Cell Death; Cells; Crystallization; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Developing Countries; Docking; Double Strand Break Repair; Drug resistance; Drug resistance in tuberculosis; Engineering; Epidemic; Escherichia coli; Exposure to; Family; Genetic; Genetic Recombination; Genetic Transcription; Goals; Homologous Gene; Homology Modeling; Human; In Vitro; Laboratories; Lesion; Mediating; Modeling; Mutagenesis; Mutate; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nature; Patient-Focused Outcomes; Pharmacology; Point Mutation; Polymerase; Process; Proteins; Resistance; Resistance development; Role; SS DNA BP; Single-Stranded DNA; Site; Source; Stress; Structural Models; Structure; Surface; Synapses; Testing; Time; Tuberculosis; Uncertainty; Work; base; cell killing; chemotherapy; course development; emerging antibiotic resistance; experience; genetic regulatory protein; global health; improved; mutant; novel; overexpression; pol Gene Products; protein complex; protein protein interaction; replication stress; resistant strain; standard care; targeted treatment; therapeutic development; therapeutic target; tuberculosis treatment

Project Summary Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a serious global health threat that kills over a million people per year worldwide. While antibiotic treatments have existed for some time, they are often not successful. The necessity for an extended treatment course and the development of resistance to commonly prescribed antibiotics during treatment are major challenges that need to be overcome to improve patient outcomes. Chromosomal mutations due to the action of error-prone DNA polymerases are a major driver of mutagenesis in Mtb and therefore the emergence of antibiotic resistance. Error-prone polymerases are involved in a DNA damage tolerance process known as translesion synthesis. Typically, error-prone polymerases are tightly regulated so that they are not active during DNA replication. However, they become activated under stress conditions, such as those experienced during antibiotic treatment. In recent work from our laboratory, we demonstrated that within the model bacterium Escherichia coli, the activation of the error- prone polymerase Pol IV requires an interaction with single-stranded DNA binding protein (SSB), which acts to locally concentrate Pol IV near sites of DNA damage. Importantly, ablation of this interaction substantially reduced Pol IV-mediated translesion synthesis and mutagenesis. In this proposal we will test whether the three Pol IV homologs in Mtb interact with MtSSB. Next, we will identify mutations that ablate this putative interaction without affecting other molecular interactions or polymerase activity. Finally, we will introduce these mutations into Mycobacterium smegmatis, a non-pathogenic model system of Mtb, and test whether these strains are sensitized to DNA damage agents. If successful, these studies will identify a novel molecular interaction to potentially target therapeutically to suppress the emergence of antibiotic resistance in Mtb.

项目摘要 由结核分枝杆菌(Mtb)引起的结核病仍然是一个严重的全球健康问题。 每年导致全球100多万人死亡的威胁。虽然抗生素治疗已经存在了一些 一段时间后，他们往往不会成功。延长疗程的必要性和发展 治疗过程中对常用抗生素的耐药性是需要克服的主要挑战 以改善患者的预后。 容易出错的DNA聚合酶的作用导致的染色体突变是导致 结核分枝杆菌中的突变，因此出现了抗生素耐药性。容易出错的聚合酶是 参与称为跨损伤合成的DNA损伤耐受过程。通常，容易出错 聚合酶受到严格的调控，因此它们在DNA复制过程中不活跃。然而，它们变成了 在应激条件下被激活，例如在抗生素治疗期间经历的那些。在最近从 我们的实验室，我们证明了在模型细菌大肠杆菌中，错误的激活- POLE聚合酶Pol IV需要与单链DNA结合蛋白(SSB)相互作用，SSB作用于 在DNA损伤部位附近局部浓缩POL IV。重要的是，这种相互作用的消融基本上 减少Pol IV介导的跨损伤合成和诱变。在这项提案中，我们将测试 Mtb中的三个Pol IV同源物与MtSSB相互作用。接下来，我们将确定导致这一推测的突变 相互作用而不影响其他分子相互作用或聚合酶活性。最后，我们将介绍这些 突变到污垢分枝杆菌，一个非致病的结核分枝杆菌模型系统，并测试这些 菌株对DNA损伤剂敏感。如果成功，这些研究将识别出一种新的分子 相互作用，潜在的治疗靶点，以抑制结核分枝杆菌抗生素耐药性的出现。