Mycobacterium tuberculosis (Mtb) requires the ESX-5 specialized protein secretion system for both nutrient uptake and virulence, but the ESX-5 effectors that directly influence nutrient acquisition or interactions with host cells are currently undefined. The long-term goal is to understand the mechanisms that enable Mtb to evade host immune defenses to establish and maintain persistent infections. The objective of this proposal is to identify ESX-5 substrates that impact Mtb pathogenesis. The proposed research will test the central hypothesis that ESX-5 secretes many substrates and that proteins encoded within the esx-5 locus directly influence Mtb virulence, independent of a function in nutrient acquisition. This proposal will utilize unique genetic tools to dissect the function of the ESX-5 secretion system. Preliminary data demonstrate substantial alterations in the secreted protein profile of a mutant that hyper-secretes the known ESX-5 substrates.
Specific Aim 1 will make use of this ESX-5 hyper-secretion mutant, growth conditions that stimulate ESX-5 secretion and strains that conditionally express core components of the ESX-5 secretion system to globally identify ESX- 5 substrates by a proteomics approach.
Specific Aim 2 will test the importance of the mycobacteria-specific PE, PPE and Esx proteins that are encoded within the esx-5 locus for Mtb virulence using a series of Mtb deletion mutants and both mouse and macrophage infection models. The results of these studies will significantly enhance our understanding of the Mtb host-pathogen interaction by revealing proteins that are secreted via ESX-5 and identifying individual ESX-5-associated proteins that are required for virulence. Identification of functional ESX-5 effectors will enable detailed molecular characterization of the mechanisms by which these proteins manipulate host cell functions to facilitate design of novel antimicrobials to combat tuberculosis infection.
The proposed research on the Mycobacterium tuberculosis specialized protein secretion system ESX-5 will globally identify ESX-5 substrates that may participate in direct interactions with the host. It will also reveal the importance of the mycobacteria-specific PE, PPE and Esx proteins that are associated with the esx-5 locus in M. tuberculosis virulence. This fundamental knowledge of the interaction between the bacterial pathogen M. tuberculosis and its host is relevant to the NIH mission because it could be applied to develop novel therapeutic interventions that would reduce the burden of tuberculosis disease.