Structure-Function Analyses of the Virulence-Associated Mycobacterial Secretion System ESX-1

Tuberculosis (TB), an airborne disease caused by Mycobacterium tuberculosis, remains the world’s leading cause of death from infectious disease. While there are effective drugs to prevent and treat TB disease, these regimens are many months long with catastrophic costs to more than half of people with TB. In addition, the BCG vaccine, the only widely used vaccine against TB, has a multitude of issues. However, this attenuated vaccine strain of M. bovis was key in advancing TB research, as the primary reason for attenuation of this strain was loss of the Type VII secretion system ESX-1. Pathogenic mycobacteria use ESX-1 for survival in the host macrophage through lysis of the phagosomal membrane, allowing intracellular replication and cell to cell spread. However, the mechanisms surrounding phagosomal lysis by ESX-1 are unknown. In these studies, we used molecular genetics in combination with computational modeling to better understand the mechanisms behind the genetic regulation and virulence of ESX-1. We showed that although the EspM transcription factor contains two DNA binding domains, only one is required for negative regulation of ESX-1 at the whiB6 promoter. Also, the secreted substrates EspE and EspF, which are required for virulence, likely interact, and this interaction is required for their secretion. Further, the EspE/EspF heterodimer is targeted for secretion by EspF interaction with EsxA. However, the roles of EspE and EspF in virulence and regulation are separable, as the repression of whiB6 is independent of EspE/EspF interaction. We also performed initial structure-function analyses and characterization of EspN, a novel regulator of ESX-1 that is required for infection in vivo. Overall, this work contributed to the field's understanding of mycobacterial pathogenesis during early infection by structure-function analysis of ESX-1.