Conserved ESX-1 Substrates EspE and EspF Are Virulence Factors That Regulate Gene Expression.

Mycobacterium tuberculosis, the cause of human tuberculosis, and Mycobacterium marinum, a nontubercular pathogen with a broad host range, require the ESX-1 secretion system for virulence. The ESX-1 system secretes proteins which cause phagosomal lysis within the macrophage via an unknown mechanism. As reported elsewhere (R. E. Bosserman et al., Proc Natl Acad Sci U S A 114:E10772-E10781, 2017, https://doi.org/10.1073/pnas.1710167114), we recently discovered that the ESX-1 system regulates gene expression in M. marinum This finding was confirmed in M. tuberculosis in reports by C. Sala et al. (PLoS Pathog 14:e1007491, 2018, https://doi.org/10.1371/journal.ppat.1007491) and A. M. Abdallah et al. (PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). We further demonstrated that a feedback control mechanism connects protein secretion to WhiB6-dependent expression of the esx-1 genes via an unknown mechanism. Here, we connect protein secretion and gene expression by showing for the first time that specific ESX-1 substrates have dual functions inside and outside the mycobacterial cell. We demonstrate that the EspE and EspF substrates negatively control esx-1 gene expression in the M. marinum cytoplasm through the conserved WhiB6 transcription factor. We found that EspE and EspF are required for virulence and promote lytic activity independently of the major EsxA and EsxB substrates. We show that the dual functions of EspE and EspF are conserved in the orthologous proteins from M. tuberculosis Our findings support a role for EspE and EspF in virulence that is independent of the EsxA and EsxB substrates and demonstrate that ESX-1 substrates have a conserved role in regulating gene expression.

A New ESX-1 Substrate in Mycobacterium marinum That Is Required for Hemolysis but Not Host Cell Lysis.

The ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis and M. marinum, an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of the M. marinum secretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate in M. marinum We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement of MMAR_2894 for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system in M. marinum and is itself required for the optimal secretion of the known ESX-1 substrates in M. marinum Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to the M. marinum ESX-1 system.IMPORTANCE Both Mycobacterium tuberculosis, the cause of human tuberculosis (TB), and Mycobacterium marinum, a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems in M. tuberculosis and in M. marinum Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system of M. marinum We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because the MMAR_2894 gene is not conserved in M. tuberculosis, our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system in M. marinum, providing new insight into how the M. marinum and M. tuberculosis systems differ.