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Mycobacterium tuberculosis and its close relative Mycobacterium marinum infect macrophages and induce the formation of granulomas, organized macrophage-rich immune aggregates. These mycobacterial 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 phagosomal compartments, within which the bacteria reside. This phagosomal 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 establish is needed for ESX-1-mediated virulence early in infection. Unexpectedly, 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 uncoupling mutants highlight an enigmatic functional domain of ESAT-6 and provide key tools to investigate the mechanism of phagosomal damage and virulence.Significance StatementTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes.Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C-terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.