Monday, July 27, 2015 at 1:00 pm, Rm 5310, Life Sciences Centre, 2350 Health Science Mall
Mycobacterium tuberculosis (TB) uses the ESX-1 type VII secretion system to export proteins to its cell surface, which permeabilize the host macrophage phagosomal membrane, allowing the bacterium to escape and spread to new cells. The structure of the type VII membrane complex and how it mediates this function is unknown, but it is hypothesized that some of the secreted proteins form an extracellular appendage that facilitates membrane lysis or direct secretion of virulence factors into the host cytoplasm. This thesis investigates the structural relationship between one of these secreted proteins, EspB, and a protease that processes it, MycP1. The x-ray crystallographic structures of both proteins are determined. EspB is shown to form a multimer with heptameric stoichiometry, and an EM reconstruction of this multimer is generated and used to create a model of the oligomer using symmetric Rosetta docking. The final model is supported by mass spectrometry-based detection of chemically cross-linked peptides from adjacent subunits. We use mass spectrometry to determine how EspB is proteolytically processed during secretion and discuss the effect of this processing event on the EspB ultrastructure. Finally, the structure of one of the membrane apparatus proteins, EccB1 is determined, revealing structural homology to a phage lysin. The combination of x-ray crystallography, EM, modeling, and mass-spectrometry provides an exciting first glimpse at the structure and function of the type VII secretion system – a critical factor in the TB pathogenesis cycle.