Mycobacterium tuberculosis is the world's second leading infectious disease killer, with approximately 1.5 million people succumbing to the disease annually. Protein export by the ESX-1 secretion system is essential for the virulence of pathogenic mycobacteria, including M. tuberculosis and nontuberculous mycobacteria (NTMs) such as Mycobacterium marinum. ESX-1 mediates rupture of the phagosomal membrane, allowing mycobacteria to gain access to the cytoplasm of host macrophages. Phagosomal lysis is required for host cell death and mycobacterial cell-to-cell spread. Our previous work demonstrated that the ESX-1 substrates EspE and EspF are required for the membranolytic activity of M. marinum. Our lab recently showed that EspE and EspF are the last set of substrates secreted by the ESX-1 system and require each other for secretion. Moreover, EspE and EspF regulate gene expression in the mycobacterial cytoplasm. Using a targeted genetic approach, we are conducting a structure-function analysis to determine if protein interaction is required for the hemolytic and regulatory activities of EspE and EspF. We show that the disruption of a predicted salt bridge between EspE and EspF abrogates hemolytic activity and EspE secretion. In addition, the protein-protein interface between EspF and EsxA is necessary for hemolytic activity. Disruption of this region leads to a substantial decrease in EspE secretion. This, combined with EsxB secretion data, shows that EspE-EspF interaction is required for secretion, but may not be required for gene regulation. These data provide new insight into the unique ESX-1 substrates EspE and EspF in gene regulation, protein secretion, and virulence.