Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP(3) of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP(3ms) mutant, failed to export the native Esx-3 substrates EsxH(ms) and EsxG(ms) to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxH(ms) and EsxG(ms) were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxG(ms) and EsxH(ms) secretion in the ΔmycP(3ms) mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems.