The regulatory functions of ESX-1 substrates, EspE and EspF, are separable from secretion.

Pathogenic mycobacteria are a significant global health burden. The ESX-1 secretion system is essential for mycobacterial pathogenesis. The secretion of ESX-1 substrates is required for phagosomal lysis, which allows the bacteria to enter the macrophage cytoplasm, induce a Type I IFN response, and spread to new host cells. EspE and EspF are dual-functioning ESX-1 substrates. Inside the mycobacterial cell, they regulate transcription of ESX-1-associated genes. Following secretion, EspE and EspF are essential for lytic activity. The link between EspE/F secretion and regulatory function has not been investigated. We investigated the relationship between EspE and EspF using molecular genetics in , a non-tuberculous mycobacterial species that serves as an established model for ESX-1 secretion and function in . Our data support that EspE and EspF, which require each other for secretion, directly interact. The disruption of the predicted protein-protein interaction abrogates hemolytic activity and secretion but does not impact their gene regulatory activities in the mycobacterial cell. In addition, we predict a direct protein-protein interaction between the EsxA/EsxB heterodimer and EspF. Our data support that the EspF/EsxA interaction is also required for hemolytic activity and EspE secretion. Our study sheds light on the intricate molecular mechanisms governing the interactions between ESX-1 substrates, regulatory function, and ESX-1 secretion, moving the field forward.IMPORTANCETuberculosis (TB), caused by , is a historical and pervasive disease responsible for millions of deaths annually. The rise of antibiotic and treatment-resistant TB, as well as the rise of infection by non-tuberculous mycobacterial species, calls for a better understanding of pathogenic mycobacteria. The ESX-1 secreted substrates, EspE and EspF, are required for mycobacterial virulence and may be responsible for phagosomal lysis. This study focuses on the mechanism of EspE and EspF secretion from the mycobacterial cell.