Tracking the hierarchical secretion of ESX-1 substrates through the mycobacterial cell envelope

Project Summary Mycobacteria, including human pathogens such as Mycobacterium tuberculosis and the non-tuberculous mycobacterium Mycobacterium marinum, require Type VII ESX secretion systems for optimal growth, survival, and virulence. Despite being the focus of extensive genetic and biochemical efforts, the mechanism of ESX- mediated protein secretion, especially through the cell wall, remains elusive. In general, the exploration of secretion pathways within the cell wall of mycobacteria is constrained by the current limited ability to localize proteins to specific subcellular compartments. The objective of this exploration is to overcome this obstacle towards elucidating protein secretion by the ESX-1 system. The rationale is that the tools and the knowledge gained will allow us to (1) track components of the ESX-1 system to the cell wall and/or cell surface and (2) thereby test a model for ESX-mediated secretion through the cell envelope by establishing the hierarchy of export to these subcellular compartments. The innovation of this proposal is to apply cell wall-specific protein tagging, surface-specific protein detection, and site-specific protein photocrosslinking methods that have not yet been applied to ESX-1-mediated secretion. Prior research, including initial studies, provide evidence that these approaches will be successful in achieving the stated goals. The proposed studies are expected to establish functional and molecular relationships among ESX-1 substrates that determine their transit through the cell envelope and thereby enable future studies into the detailed mechanism of ESX-1 secretion. These results are anticipated thereby to advance fundamental knowledge of a unique pathway critical to mycobacterial pathogenesis.