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.

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.

is a nontuberculous pathogen of poikilothermic fish and an opportunistic human pathogen. Like tuberculous mycobacteria, the M strain requires the ESX-1 (ESAT-6 system 1) secretion system for virulence in host cells. EsxB and EsxA, two major virulence factors exported by the ESX-1 system, are encoded by the genes within the ESX-1 locus. Deletion of the genes abrogates ESX-1 export and attenuates in and models of infection. Interestingly, there are several duplications of the and genes (, ,, , and) in the M genome located outside the ESX-1 locus. We sought to understand if this region, known as ESX-6, contributes to ESX-1-mediated virulence. We found that deletion of the_ gene alone or the entire ESX-6 locus did not impact ESX-1 export or function, supporting the idea that the genes present at the ESX-1 locus are the primary contributors to ESX-1-mediated virulence. Nevertheless, overexpression of the locus complemented ESX-1 function in the Δ strain, signifying that the two loci are functionally equivalent. Our findings raise questions about why duplicate versions of the genes are maintained in the M genome and how these proteins, which are functionally equivalent to virulence factors, contribute to mycobacterial biology. is the causative agent of the human disease tuberculosis (TB). There are 10.4 million cases and 1.7 million TB-associated deaths annually, making TB a leading cause of death globally. Nontuberculous mycobacteria (NTM) cause chronic human infections that are acquired from the environment. Despite differences in disease etiology, both tuberculous and NTM pathogens use the ESX-1 secretion system to cause disease. The nontubercular mycobacterial species, , has additional copies of specific ESX-1 genes. Our findings demonstrate that the duplicated genes do not contribute to virulence but can substitute for virulence factors in These findings suggest that the duplicated genes may play a specific role in NTM biology.

Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.