Recruitment of both the ESCRT and autophagic machineries to ejecting Mycobacterium marinum.

Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non-lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA-ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a "frustrated autophagosome" that is unable to engulf the ejecting bacterium.

Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in Mycobacterium marinum.

The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.

The regulatory functions of ESX-1 substrates, EspE and EspF, are separable from secretion.

Pathogenic mycobacteria are a significant global health burden. The ESX-1 secretion system is essential for mycobacterial pathogenesis. The secretion of ESX-1 substrates is required for phagosomal lysis, which allows the bacteria to enter the macrophage cytoplasm, induce a Type I IFN response, and spread to new host cells. EspE and EspF are dual-functioning ESX-1 substrates. Inside the mycobacterial cell, they regulate transcription of ESX-1-associated genes. Following secretion, EspE and EspF are essential for lytic activity. The link between EspE/F secretion and regulatory function has not been investigated. We investigated the relationship between EspE and EspF using molecular genetics in Mycobacterium marinum, a non-tuberculous mycobacterial species that serves as an established model for ESX-1 secretion and function in Mycobacterium tuberculosis. Our data support that EspE and EspF, which require each other for secretion, directly interact. The disruption of the predicted protein-protein interaction abrogates hemolytic activity and secretion but does not impact their gene regulatory activities in the mycobacterial cell. In addition, we predict a direct protein-protein interaction between the EsxA/EsxB heterodimer and EspF. Our data support that the EspF/EsxA interaction is also required for hemolytic activity and EspE secretion. Our study sheds light on the intricate molecular mechanisms governing the interactions between ESX-1 substrates, regulatory function, and ESX-1 secretion, moving the field forward.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis, is a historical and pervasive disease responsible for millions of deaths annually. The rise of antibiotic and treatment-resistant TB, as well as the rise of infection by non-tuberculous mycobacterial species, calls for a better understanding of pathogenic mycobacteria. The ESX-1 secreted substrates, EspE and EspF, are required for mycobacterial virulence and may be responsible for phagosomal lysis. This study focuses on the mechanism of EspE and EspF secretion from the mycobacterial cell.

Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum.

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Diagnosis of Mycobacterium marinum Infection by Metagenomic next-generation sequencing.

BACKGROUND: In December 2023, our hospital confirmed a case of finger infection with Mycobacterium marinum. The patient sought medical attention at our hospital due to a hard scratch on her left middle finger, which was red, swollen, and ulcerated for one month. PHYSICAL EXAMINATION: A lesion of approximately 1.5 cm x 2 cm in the patient's left middle finger, surrounded by redness and swelling, unclear boundaries, surface rupture, partial scabbing, and no tenderness during compression. She was treated at the previous clinic, common infectious diseases were considered, and was given intravenous infusion treatment: cefotaxime and clarithromycin, and erythromycin ointment was applied externally. She came to our hospital after poor treatment results. The patient has had hypertension for 3 years, no other systemic diseases, no similar medical history among family members, no history of drug or food allergies. METHODS: Clean the wound and remove the scab from the affected area, and use a surgical blade to scrape off necrotic tissue. Send the scraped tissue for pathogen testing: tissue bacterial culture+identification (matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF), tissue acid fast staining, and tissue metagenomic next-generation sequencing (mNGS). Other auxiliary examinations: blood routine, urine routine, blood fat, liver function, and kidney function. RESULTS: Tissue bacterial culture+identification: growth of Mycobacterium marinum; Acid fast staining of tissue: positive; Tissue mNGS: Mycobacterium marinum. Clinical treatment plan: clarithromycin 0.5 g bid po+rifampicin 0.45 g qd po+5-aminolevulinic acid photodynamic therapy (ALA-PDT) qw+boric acid wash wet compress tid. After 14 days of treatment, the area of redness and swelling significantly decreased, and the degree of redness and swelling was significantly reduced compared to admission. The degree of ulcer edge protrusion was also reduced compared to admission. There was a small amount of exudation from the wound, and no necrotic tissue was observed. The patient improved and was discharged. CONCLUSIONS: This article reports a case of finger infection with Mycobacterium marinum. Mycobacterium marinum was quickly and accurately identified by mNGS, and reasonable treatment measures were adopted clinically. The patient improved and was discharged. This study has important reference significance for the clinical diagnosis and treatment of Mycobacterium infection. In addition, mNGS as a novel detection method has considerable prospects for rapid diagnosis of pathogens.

PE/PPE Proteome and ESX-5 Substrate Spectrum in Mycobacterium marinum.

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.

Induction of IL-32 in the immune response of keratinocytes to Mycobacterium marinum infection.

Keratinocytes are the predominant cell type in the skin epidermis, and they not only protect the skin from the influence of external physical factors but also function as an immune barrier against microbial invasion. However, little is known regarding the immune defence mechanisms of keratinocytes against mycobacteria. Here, we performed single-cell RNA sequencing (scRNA-seq) on skin biopsy samples from patients with Mycobacterium marinum infection and bulk RNA sequencing (bRNA-seq) on M. marinum-infected keratinocytes in vitro. The combined analysis of scRNA-seq and bRNA-seq data revealed that several genes were upregulated in M. marinum-infected keratinocytes. Further in vitro validation of these genes by quantitative polymerase chain reaction and western blotting assay confirmed the induction of IL-32 in the immune response of keratinocytes to M. marinum infection. Immunohistochemistry also showed the high expression of IL-32 in patients' lesions. These findings suggest that IL-32 induction is a possible mechanism through which keratinocytes defend against M. marinum infection; this could provide new targets for the immunotherapy of chronic cutaneous mycobacterial infections.

The Mycobacterium marinum ESX-1 system mediates phagosomal permeabilization and type I interferon production via separable mechanisms.

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.

Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represses whiB6 expression by specifically binding the whiB6 promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspM in vitro. We demonstrate that multimerization in vitro can be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerization in vitro. Our data support models where EspM monomers or hexamers contribute to the regulation of whiB6 expression. IMPORTANCE Pathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.

Modification of a PE/PPE substrate pair reroutes an Esx substrate pair from the mycobacterial ESX-1 type VII secretion system to the ESX-5 system.

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

Genome-scale analyses of transcriptional start sites in Mycobacterium marinum under normoxic and hypoxic conditions.

BACKGROUND: Hypoxic stress plays a critical role in the persistence of Mycobacterium tuberculosis (Mtb) infection, but the mechanisms underlying this adaptive response remain ill defined. MATERIAL AND METHODS: In this study, using M. marinum as a surrogate, we analyzed hypoxic responses at the transcriptional level by Cappable-seq and regular RNA-seq analyses. RESULTS: A total of 6808 transcriptional start sites (TSSs) were identified under normoxic and hypoxic conditions. Among these TSSs, 1112 were upregulated and 1265 were downregulated in response to hypoxic stress. Using SigE-recognized consensus sequence, we identified 59 SigE-dependent promoters and all were upregulated under hypoxic stress, suggesting an important role for SigE in this process. We also compared the performance of Cappable-seq and regular RNA-seq using the same RNA samples collected from normoxic and hypoxic conditions, and confirmed that Cappable-seq is a valuable approach for global transcriptional regulation analyses. CONCLUSIONS: Our results provide insights and information for further characterization of responses to hypoxia in mycobacteria, and prove that Cappable-seq is a valuable approach for global transcriptional studies in mycobacteria.

Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum.

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.

Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5.

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC5 , possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC5 . This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC5 , showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5 .

Heterologous Expression of ethA and katG in Mycobacterium marinum Enables the Rapid Identification of New Prodrugs Active against Mycobacterium tuberculosis.

Screening strategies for antituberculosis compounds using Mycobacterium tuberculosis are time consuming and require biosafety level 3 (BSL3) facilities, which makes the development of high-throughput assays difficult and expensive. Mycobacterium marinum, a close genetic relative of M. tuberculosis, possesses several advantages as a suitable model for tuberculosis drug screening. However, despite the high genetic similarity, there are some obvious differences in susceptibility to some tuberculosis drugs between these two species, especially for the prodrugs ethionamide and isoniazid. In this study, we aimed to improve M. marinum as a model for antituberculosis drug identification by heterologous expression of two common drug activators, EthA and KatG. These two activators were overexpressed in M. marinum, and the strains were tested against ethionamide, isoniazid, and a library of established antimycobacterial compounds from TB Alliance to compare drug susceptibility. Both in vitro and in vivo using zebrafish larvae, these genetically modified M. marinum strains showed significantly higher susceptibility against ethionamide and isoniazid, which require activation by EthA and KatG. More importantly, a strain overexpressing both ethA and katG was potentially more susceptible to approximately 20% of the antituberculosis hit compounds from the TB Alliance library. Most of these compounds were activated by EthA in M. marinum Four of these compounds were selected for further analysis, and three of them showed obvious EthA-dependent activity against M. tuberculosis Overall, our developed M. marinum strains are valuable tools for high-throughput discovery of potential novel antituberculosis prodrugs.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes.

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

Protease domain and transmembrane domain of the type VII secretion mycosin protease determine system-specific functioning in mycobacteria.

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.

The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages.

As important virulence factors of Mycobacterium tuberculosis, EsxA and EsxB not only play a role in phagosome rupture and M. tuberculosis cytosolic translocation but also function as modulators of host immune responses by modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection downregulated miR-148a-3p (now termed miR-148) in macrophages in an ESX-1-dependent manner. The upregulation of miR-148 reduced mycobacterial intracellular survival. Here, we investigated miR-147-3p (now termed miR-147), a negative regulator of inflammatory cytokines (e.g., interleukin-6 [IL-6] and IL-10), in mycobacterial infection. We infected murine RAW264.7 macrophages with Mycobacterium marinum, a surrogate model organism for M. tuberculosis, and found that the esxBA-knockout strain (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that induced by the M. marinum wild-type (WT) strain or by the M. marinum ΔesxBA complemented strain, M. marinum ΔesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other codependently secreted factors) is the negative regulator of miR-147. miR-147 was also downregulated by directly incubating the macrophages with the purified recombinant EsxA or EsxB protein or the EsxBA heterodimer, which further confirms the role of the EsxBA proteins in the downregulation of miR-147. The upregulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced M. marinum intracellular survival. Interestingly, inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on M. marinum intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by data indicating that miR-147 upregulation inhibits the Toll-like receptor 4/NF-κB pathway.

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.