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Pathogenic mycobacteria, suchMycobacterium tuberculosisandMycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of themycobacteriumto the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates.