Novel Regulatory Functions of ESX-1 Associated Proteins in Pathogenic Mycobacteria

Tuberculosis (TB) remains a formidable global health threat, with 10 million new cases and 1.5 million deaths reported in 2018 alone. TB disease is caused by the bacteria Mycobacterium tuberculosis (M. tuberculosis). Additionally, infections with Non-tuberculous Mycobacteria (NTM) are rapidly becoming a public health threat, especially to those who are immunocompromised. During infection, pathogenic mycobacteria are taken into the phagosome by host macrophages. They use the ESX-1 secretion system to permeabilize the phagosomal membrane, and evade death by fusion with the lysosome. This critical step in virulence is highly conserved between M. tuberculosis and the NTM pathogen M. marinum, which has been used for decades as a model system to study ESX-1 secretion and virulence. The ESX-1 secretion system consists of a core membrane complex, which resides in the cytoplasmic membrane of the mycobacteria. It is responsible for transporting small ESX-1 protein substrates from the inside of the mycobacterial cell, across the cytoplasmic membrane, to the exterior of the cell. This process is both exceedingly important and tightly regulated. The survival of the pathogenic mycobacteria relies upon secretion of the correct substrates, in the appropriate amounts at exactly the right time. To do this, the bacteria must connect membrane complex assembly with gene transcription, and substrate secretion. WhiB6 is a transcription factor which activates expression of ESX-1 substrate genes. We found that the WhiB6 transcription factor responds to the assembly of the core ESX-1 complex at the cytoplasmic membrane, a critical checkpoint. Additionally, a transcription factor, EspM, binds the promoter of the whiB6 gene, and is required for repression of whiB6 gene transcription in the absence of the core membrane complex. This effectively connects the assembly of the core membrane complex to gene expression in M. marinum. Further, we discovered that EspE and EspF, known ESX-1 secreted substrates, negatively regulate whiB6 gene expression. They are also required for virulence in M. marinum and M. tuberculosis, effectively connecting ESX-1 secretion and virulence with gene expression and fine-tuning of ESX-1 substrate production.