ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the gene. Finally, we found that the loss of WhiB6 resulted in a virulent strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in are fine-tuned by negative feedback control, linking the expression of the gene to the presence, not the functionality, of the ESX-1 membrane complex.

Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor (MMAR_5438, Rv3863, now espM). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1-associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum. Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum, a pathogen of ectothermic animals; M. tuberculosis, the human-pathogenic species that causes tuberculosis; and M. smegmatis, a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown.

Mycobacterium tuberculosis, the cause of human tuberculosis, and Mycobacterium marinum, a non-tubercular 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 in R.E. Bosserman et al (Proc Natl Acad Sci U S A doi:10.1073/pnas.1710167114), we recently discovered that the ESX-1 system regulates gene expression in M. marinum This finding has been confirmed in M. tuberculosis in reports by C. Sala et al (PLoS Pathog 14 (12):e1007491. doi: 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.

SUMMARY Pathogenic mycobacteria cause chronic and acute diseases ranging from human tuberculosis (TB) to nontubercular infections. Mycobacterium tuberculosis causes both acute and chronic human tuberculosis. Environmentally acquired nontubercular mycobacteria (NTM) cause chronic disease in humans and animals. Not surprisingly, NTM and M. tuberculosis often use shared molecular mechanisms to survive within the host. The ESX-1 system is a specialized secretion system that is essential for virulence and is functionally conserved between M. tuberculosis and Mycobacterium marinum. M. marinum is an NTM found in both salt water and freshwater that is often used to study mycobacterial virulence. Since the discovery of the secretion system in 2003, the use of both M. tuberculosis and M. marinum has defined the conserved molecular mechanisms underlying protein secretion and the lytic and regulatory activities of the ESX-1 system. Here, we review the trajectory of the field, including key discoveries regarding the ESX-1 system. We highlight the contributions of M. marinum studies and the conserved and unique aspects of the ESX-1 secretion system.