The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence

Mycobacterium tuberculosis and its close relative Mycobacterium marinum infect macrophages and induce the formation of granulo-mas, organized macrophage-rich immune aggregates. These myco-bacterial pathogens can accelerate and co-opt granuloma formation for their benefit, using the specialized secretion system ESX-1, a key virulence determinant. ESX-1-mediated virulence is attributed to the damage it causes to the membranes of macrophage phagoso-mal compartments, within which the bacteria reside. This phagoso-mal damage, in turn, has been attributed to the membranolytic activity of ESAT-6, the major secreted substrate of ESX-1. However, mutations that perturb ESAT-6's membranolytic activity often result in global impairment of ESX-1 secretion. This has precluded an understanding of the causal and mechanistic relationships between ESAT-6 membranolysis and ESX-1-mediated virulence. Here, we identify two conserved residues in the unstructured C-terminal tail of ESAT-6 required for phagosomal damage, granuloma formation, and virulence. Importantly, these ESAT-6 mutants have near-normal levels of secretion, far higher than the minimal threshold we estab-lish is needed for ESX-1-mediated virulence early in infection. Unex-pectedly, these loss-of-function ESAT-6 mutants retain the ability to lyse acidified liposomes. Thus, ESAT-6's virulence functions in vivo can be uncoupled from this in vitro surrogate assay. These uncou-pling mutants highlight an enigmatic functional domain of ESAT-6 and provide key tools to investigate the mechanism of phagosomal damage and virulence.

Automated summary

This study reveals the causal relationship between ESAT-6 membranolysis and ESX-1-mediated virulence in Mycobacterium tuberculosis and Mycobacterium marinum. Mutations in ESAT-6's C-terminal tail residues disrupt phagosomal damage and granuloma formation, while still retaining the ability to lyse acidified liposomes.