The Fundamentals of Type VII secretion in Mycobacteria: An ESX-1 Perspective

A unique feature of mycobacteria, which includes the major human pathogen M. tuberculosis, is their impermeable cell envelope, containing two membranes. The mycobacterial outer membrane functionally resembles the outer membrane of Gram-negative bacteria but is differently composed of specific long-chain fatty acids, called mycolic acids, and other (glyco)lipids. The mycolic acids in the inner leaflet are covalently linked to a distinctive arabinogalactan-peptidoglycan polymer. The overall structure of this complex cell envelope contributes to the low permeability and in turn the intrinsic tolerance of mycobacteria to antibiotics. In order to secrete proteins over this impermeable cell envelope, mycobacteria have acquired type VII secretion systems (T7SSs), called ESX-1 to ESX-5. These systems are crucial for mycobacterial virulence (ESX-1) and viability (ESX-3 and ESX-5). They are encoded by continuous gene clusters, which include genes that encode conserved membrane and cytosolic components and (a subset of) substrates. Substrates can be categorized into four distinctive protein families i.e. Esx/WxG100, PE, PPE and Esp. While each T7SS secretes its own subset of Esx, PE and PPE substrates, the Esp proteins appear to be exclusively secreted by the ESX-1 system. An interesting characteristic of the mycobacterial T7SS substrates is that they are secreted as folded heterodimers. A conserved secretion motif (YxxxD/E) is present in one protein partner, although this signal does not determine through which system the heterodimer is secreted. The conserved EspG chaperones bind substrates of the PPE family to keep them in a soluble state, and this interaction conveys a role in ESX system-specific substrate recognition and secretion. In addition, the ESX-1 system has dedicated chaperones of the ESX-1 specific Esp substrates. This thesis aimed to investigate the mechanism of type VII secretion in mycobacteria, with a focus on the ESX system-specific aspects of this process. We have used the model organism Mycobacterium marinum to investigate the function of the mycosin protease of the ESX-1 and ESX-5 system, one of the ESX membrane complex components, as well as specific ESX-1 substrates that have a role in the secretion process or contribute to the pathogenic life cycle of mycobacteria. The results in this thesis describe and elaborate on the mechanism of system-specific secretion and substrate co-dependence for secretion by focusing on specific ESX-1 membrane components and substrates. Elucidating the inner workings of this system and its substrates is essential to understand the molecular mechanism of mycobacterial infections and for future target-based drug design and vaccine strategies.