Investigating the molecular requirements for ESX-1-lytic activity in pathogenic mycobacteria

研究致病性分枝杆菌 ESX-1 裂解活性的分子需求

• 批准号: 10374860
• 负责人: Patricia A Champion
• 金额: $ 19.56万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-19 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10374860
• 关键词: Attention; Biochemical; Bypass; Cytolysis; Cytoplasm; Data; Detection; Disease; ELF3 gene; Equilibrium; Experimental Designs; Genetic Screening; Genus Mycobacterium; Immune response; Immunity; In Vitro; Infection; Knowledge; Link; Lytic; Mediating; Membrane; Mission; Molecular; Molecular Genetics; Mycobacterium marinum; Mycobacterium tuberculosis; Nonlytic; Outcome; Pathogenesis; Pathogenicity; Phagocytes; Phagosomes; Prevention; Production; Proteins; Public Health; Research; Resources; Seminal; System; Testing; Tuberculosis; United States National Institutes of Health; Variant; Virulence; genetic approach; human disease; human pathogen; innovation; insight; lysin; mycobacterial; pathogen

PROJECT SUMMARY The mechanism of membrane lysis by the ESAT-6-system-1 (ESX-1) secretion system in pathogenic mycobacteria remains elusive. The objective of this application is to define the ESX-1-associated lysin. The hypothesis is that the conserved, pathogen-specific substrate, EspE promotes ESX-1 lytic activity by both M. tb and M. marinum. To test this hypothesis, the following specific aims will be investigated. Under the first aim, the applicant proposes to reroute the secretion of the EspE substrate and characterize the lytic activity and virulence of the resulting strains. The objective of this aim is to define if the EspE substrate is necessary and sufficient for ESX-1 lytic function and virulence. The working hypothesis is that rerouting EspE secretion will relieve the requirement for the ESX-1 system in lysis and virulence. The hypothesis will be tested using molecular and genetic approaches to bypass the requirement of ESX-1 for EspE secretion from M. marinum and M. tb and to characterize the lytic activity and virulence of resulting strains. Under the second aim, the applicant proposes to isolate and characterize nonlytic variants of the EspE substrate in M. marinum and M. tb. The objective of this aim is to define how EspE promotes virulence in M. marinum and in M. tb. The working hypothesis is that EspE is required for virulence because it promotes lysis. This hypothesis will be tested using molecular and biochemical approaches to characterize the impact of the resulting EspE variants on ESX-1 mediated secretion in vitro, lysis and virulence of both M. marinum and M. tb. The applicant expects that Aims 1 and 2 will contribute seminal insight into how EspE and ESX-1 contribute to mycobacterial pathogenesis at the molecular level, and demonstrate that EspE is functionally conserved in the human pathogen, M. tb. Moreover, completion of the proposal will result in a pipeline to test if additional ESX-1 substrates function as lysins in pathogenic mycobacteria. These contributions will be significant, moving the field vertically by drawing attention to substrates other than EsxA and EsxB in understanding ESX-1-dependent lysis. This application is innovative because it focuses on the previously understudied EspE substrate as a lysin. The experimental design is innovative because it will produce a pipeline that can be used to test the requirement of any ESX-1 substrate for lysis and virulence in pathogenic mycobacteria.

项目总结 ESAT-6-System-1(ESX-1)分泌系统在致病过程中的膜溶解机制 分枝杆菌仍然难以捉摸。本申请的目的是定义ESX-1相关的赖氨酸。这个 假设ESPE是一种保守的、病原体特异的底物，通过结核分枝杆菌和结核分枝杆菌 和马丁尼先生。为了验证这一假设，我们将调查以下具体目标。在第一个目标下， 申请人建议改变ESPE底物的分泌路线，并表征裂解活性和毒力 所产生的菌株。此目标的目的是确定ESPE衬底是否是必要和充分的 ESX-1的裂解功能和毒力。工作假设是，改变ESPE分泌的路线将缓解 对ESX-1系统在裂解和毒力方面的要求。这一假说将用分子和 绕过ESX-1对海洋分枝杆菌和结核分枝杆菌ESPE分泌要求的遗传途径 鉴定产生的菌株的裂解活性和毒力。在第二个目标下，申请人建议 分离和鉴定海洋分枝杆菌和结核分枝杆菌ESPE底物的非裂解变异体。这样做的目的是 目的是确定ESPE如何促进海洋分枝杆菌和结核分枝杆菌的毒力。工作假设是ESPE 是毒力所必需的，因为它促进了裂解。这一假说将用分子和 用生物化学方法表征ESPE变异对ESX-1介导的分泌的影响 在体外，测定了海洋分枝杆菌和结核分枝杆菌的裂解和毒力。申请人预期目标1和目标2将作出贡献。 对ESPE和ESX-1如何在分子水平上促进分枝杆菌发病的开创性见解，以及 证明ESPE在人类病原体结核分枝杆菌中是功能保守的。此外，完成 该提案将导致一项流水线，以测试额外的ESX-1底物是否在致病菌中起到溶蛋白的作用 分枝杆菌。这些贡献将是显著的，通过引起人们对衬底的关注来垂直移动场 在理解ESX-1依赖的裂解方面不同于EsxA和EsxB。此应用程序具有创新性，因为它 重点介绍了以前未被研究的ESPE底物作为一种裂解酶。实验设计具有创新性。 因为它将生产一条管道，该管道可用于测试任何ESX-1底物对裂解和 致病分枝杆菌的毒力。

项目成果

Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.

海分枝杆菌中 ESX-1 相关转录因子 EspM 的功能分析。

• DOI: 10.1128/jb.00233-22
• 发表时间: 2022
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Sanchez,KevinG;Prest,RebeccaJ;Nicholson,KathleenR;Korotkov,KonstantinV;Champion,PatriciaA
• 通讯作者: Champion,PatriciaA