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

Summary 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.