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.
The proposed research is relevant to public health because the ESX-1 system is directly linked to the prevention, detection and treatment of the human disease Tuberculosis. The proposed research is relevant to the mission of the NIH because it involves the pursuit of fundamental knowledge that will result in new scientific resources to promote the prevention of human disease.
