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The type VII secretion system ESX-1 (ESAT-6 secretion system 1) is required for virulence in Mycobacterium tuberculosis and M. marinum . Here, we identify the clinical drug candidate ebselen as an inhibitor of ESX-1 secretion in both organisms. We find that ebselen inhibits the activity of the ESX-1 AAA+ ATPase EccA1, which is required for full ESX-1 secretion and virulence. EccA1 ATPase activity is inhibited through covalent modification of an N-terminal cysteine residue that is outside its ATPase domain. Our findings suggest the potential of ebselen as an anti-virulence drug complement to standard antituberculous therapy regimens, particularly for drug resistant infection. Our findings also provide new insight into the function of the EccA1 protein domains in its ATPase activity and ESX-1 secretion.

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay— a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.