A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria

The ESX-5 secretion system of Mycobacterium tuberculosis is important for bacterial virulence and for the secretion of the large PE/PPE protein family, whose genes constitute 10% of the M. tuberculosis genome. A four-gene region of the ESX-5 system is duplicated three times in the M. tuberculosis genome, but the functions of these duplicates are unknown. Here we investigated one of these duplicates: the region carrying the esxI, esxJ, ppe15, and pe8 genes (ESX-5a). An ESX-5a deletion mutant in the model system M. marinum background was deficient in the secretion of some members of the PE/PPE family of proteins. Surprisingly, we also identified other proteins that are not members of this family, thus expanding the range of ESX-5 secretion substrates. In addition, we demonstrated that ESX-5a is important for the virulence of M. marinum in the zebrafish model. Furthermore, we showed the role of the M. tuberculosis ESX-5a region in inflammasome activation but not host cell death induction, which is different from the case for the M. tuberculosis ESX-5 system. In conclusion, the ESX-5a region is nonredundant with its ESX-5 paralog and is necessary for secretion of a specific subset of proteins in M. tuberculosis and M. marinum that are important for bacterial virulence of M. marinum. Our findings point to a role for the three ESX-5 duplicate regions in the selection of substrates for secretion via ESX-5, and hence, they provide the basis for a refined model of the molecular mechanism of this type VII secretion system.