Composition of the type VII secretion system membrane complex

Abstract

SummaryPathogenic mycobacteria require type VII secretion (T7S) systems to transport virulence factors across their complex cell envelope. These bacteria have up to five of these systems, termed ESX-1 to ESX-5. Here, we show that ESX-5 of Mycobacterium tuberculosis mediates the secretion of EsxN, PPE and PE_PGRS proteins, indicating that ESX-5 is a major secretion pathway in this important pathogen. Using the ESX-5 system of Mycobacterium marinum and Mycobacterium bovis BCG as a model, we have purified and analysed the T7S membrane complex under native conditions. blue native-PAGE and immunoprecipitation experiments showed that the ESX-5 membrane complex of both species has a size of~1500 kDa and is composed of four conserved membrane proteins, i.e. EccB 5, EccC5, EccD5 and EccE5. Subsequent limited proteolysis suggests that EccC5 and EccE5 mostly reside on the periphery of the complex. We also observed that EccC5 and EccD5 expression is essential for the formation of a stable membrane complex. These are the first data on a T7S membrane complex and, given the high conservation of its components, our data can likely be generalized to most mycobacterial T7S systems.

Structural biology: Mycobacterial ESX secrets revealed

“…This satisfies earlier studies showing that the membrane-extrinsic region of EccC is monomeric when crystallised 8 and is in agreement with the findings of Beckham et al 5 that this region of EccC can adopt multiple conformations in single particles, suggesting this region is highly flexible in the resting state. Upon substrate binding the ATPase domains of EccC could then oligomerize 8 , and modelling a multimer of the ATPase domains of EssC (from the distantly-related T7 secretion system of Gram-positive firmicutes) suggests that this would form a channel inside the hexamer of approximately 30 Å 9 .…”

“…Further periplasmic density was contributed by EccE which was shown to be located at the periphery of the complex. This careful mapping of individual components within the structure allowed Beckham et al 5 to propose an overall organisation for the secretion machinery where the EccC-EccB-EccD-EccE proteins essentially form four concentric hexameric rings. The structure provides a framework for the design of experiments to assess the roles of individual components within the complex, and to propose models to describe how protein secretion is achieved.…”

“…Collectively, these ESX systems secrete hundreds of proteins; two of the biggest families of ESX substrates are the Pro-Glu (PE) and Pro-ProGlu (PPE) proteins that are particularly abundant in pathogenic mycobacteria and many of these are secreted though ESX-5. ESX systems comprise five essential membrane components -EccB, EccC, EccD, EccE which form a core complex 5 and a peripherallyassociated serine protease mycosin, MycP 6 . EccC is the only component of the core complex with known function.…”

Comparative genome analysis of Streptococcus iniae DX09 reveals new insights into niche adaptation and competitive host colonisation ability

“…ESX-1 is partially missing in the vaccine strain Mycobacterium bovis Bacille Calmette-Guérin [32], indicating a contribution to virulence. Previous investigations have demonstrated a crucial role of T7SS in bacterial survival within the host [21,23,31,32], consistent with transmission electron micrographs of interactions between S. iniae DX09 and spleen cells from diseased catfish. Moreover, considerable evidence is available supporting essential roles of the actinobacterial ESS protein secretion system in virulence [26][27][28][29].…”

“…We have identified an ESS secretion system in S. iniae, which also belongs to the low-GC Firmicutes bacterial group. Pathogenic mycobacteria generally possess up to five different T7SS (ESX-1, ESX-2, ESX-3, ESX-4 and ESX-5), which have possibly evolved via gene duplication [31]. Several studies suggest that ESX-1, the first T7SS identified, strongly influences host-pathogen interactions, in particular, the ability of bacteria to escape the phago-vacuole of infected cells (macrophages and dendritic cells) [23].…”