Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria.

The ESX-1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX-1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX-1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX-1 activity consistently localize to the cell pole, identified by time-lapse fluoro-microscopy as the non-septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein-protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX-1 apparatus in M. smegmatis. This observation illustrates the cross-species conservation of protein interactions governing assembly of ESX-1, as well as polar localization. Importantly, we describe novel non-esx1-encoded proteins, which affect ESX-1 activity, which colocalize with ESX-1, and which are required for ESX-1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.