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

项目摘要 ESAT-6-system-1（ESX-1）分泌系统在致病性大肠杆菌中的膜溶解机制 分枝杆菌仍然难以捉摸。本申请的目的是定义ESX-1相关溶素。的 假设是保守的病原体特异性底物EspE促进M. TB 和M. marinum。为了检验这一假设，将研究以下具体目标。在第一个目标下， 申请人建议重新确定EspE底物的分泌途径，并表征裂解活性和毒力 所产生的菌株。这一目标的目的是确定EspE底物是否是必要的和足够的， ESX-1裂解功能和毒力。工作假设是，重新路由EspE分泌将缓解 ESX-1系统在裂解和毒力方面的要求。该假设将使用分子和 遗传方法绕过ESX-1对M. marinum和M. TB和TO 表征所得菌株的裂解活性和毒力。根据第二个目标，申请人建议 分离并表征M中EspE底物的非裂解变体。marinum和M. TB.的目的 目的是确定EspE如何促进M. marinum和M. TB.工作假设是EspE 是毒力所必需的，因为它能促进细胞溶解。这一假设将使用分子和 表征所得EspE变体对ESX-1介导的分泌的影响的生物化学方法 体外测定了两株M. marinum和M. TB.申请方希望目标1和2将有助于 对EspE和ESX-1如何在分子水平上促进分枝杆菌发病机制的开创性见解， 证明EspE在人类病原体M中功能保守。TB.此外，完成 一项提案将导致一个管道，以测试是否额外的ESX-1底物作为溶素在致病性 分枝杆菌这些贡献将是显著的，移动领域垂直提请注意基板 而不是EsxA和EsxB。这个应用程序是创新的，因为它 专注于以前未充分研究的EspE底物作为溶素。实验设计具有创新性 因为它将产生可用于测试任何ESX-1底物的裂解要求的管道， 致病分枝杆菌的毒力。

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.