Uncovering the Contributions of ESX-1 Substrates to Protein Secretion and Virulence in Mycobacterium marinum

Pathogenic mycobacteria, such as Mycobacterium tuberculosis, infect over 10 million people every year. These numbers have risen over the past few years due to the onset of the COVID-19 pandemic. Pathogenic mycobacteria use the ESX-1 secretion system to avoid degradation. The ESX-1 system transports protein substrates through the mycobacterial cell envelope which lyse the host phagosome. Here, we use genetic, biochemical, and proteomic tools to further understand the secretion and transcriptional mechanisms behind the ESX-1 system. Using the model organism Mycobacterium marinum, we identified the contributions of each ESX-1 substrate to protein secretion and virulence. In doing so, we uncovered a substrate secretion hierarchy for the first time. Our findings support a model in which ESX-1 substrates act as secreted components of the ESX-1 system. Additionally, we identified and characterized a novel ESX-1 transcription factor, EspN, that activates transcription of ESX-1-associated genes. We also found that EspN is important for virulence in vivo. Finally, we constructed a library of ESX-1 substrate double deletion strains to understand the functional relationships between ESX-1 substrates. We identified interactions between substrates that were important in facilitating substrate secretion. We observed unique changes in substrate secretion in a set of double deletion strains, which may suggest that certain ESX-1 substrates are needed to switch between secretion of early, middle, and late substrates. Collectively, our findings deepen our understanding of mycobacterial virulence strategies, which aids in development of targeted therapeutic interventions and diagnostic tools.