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 C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

Haem oxygenase limits Mycobacterium marinum infection-induced detrimental ferrostatin-sensitive cell death in zebrafish.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary cofactor for host immunity. Haem oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of haem. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish haem oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death.

The Role of Galanin during Bacterial Infection in Larval Zebrafish.

Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.

Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb's main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages' defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1ß-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy.

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

Spreading of a mycobacterial cell-surface lipid into host epithelial membranes promotes infectivity.

Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.

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.

Modeling Tubercular ESX-1 Secretion Using Mycobacterium marinum.

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 marinumM. 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.

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.

Quantification of Natural Growth of Two Strains of Mycobacterium Marinum for Translational Antituberculosis Drug Development.

The zebrafish infected with Mycobacterium marinum (M. marinum) is an attractive tuberculosis disease model, showing similar pathogenesis to Mycobacterium tuberculosis (M. tuberculosis) infections in humans. To translate pharmacological findings from this disease model to higher vertebrates, a quantitative understanding of the natural growth of M. marinum in comparison to the natural growth of M. tuberculosis is essential. Here, the natural growth of two strains of M. marinum, E11 and MUSA , is studied over an extended period using an established model-based approach, the multistate tuberculosis pharmacometric (MTP) model, for comparison to that of M. tuberculosis. Poikilotherm-derived strain E11 and human-derived strain MUSA were grown undisturbed up to 221 days and viability of cultures (colony forming unit (CFU)/mL) was determined by plating at different time points. Nonlinear mixed effects modeling using the MTP model quantified the bacterial growth, the transfer among fast, slow, and non-multiplying states, and the inoculi. Both strains showed initial logistic growth, reaching a maximum after 20-25 days for E11 and MUSA , respectively, followed by a decrease to a new plateau. Natural growth of both E11 and MUSA was best described with Gompertz growth functions. For E11, the inoculum was best described in the slow-multiplying state, for MUSA in the fast-multiplying state. Natural growth of E11 was most similar to that of M. tuberculosis, whereas MUSA showed more aggressive growth behavior. Characterization of natural growth of M. marinum and quantitative comparison with M. tuberculosis brings the zebrafish tuberculosis disease model closer to the quantitative translational pipeline of antituberculosis drug development.

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 .

EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System.

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 pathogenicity viewed through the lens of molecular Koch's postulates.

Thirty years ago Stanley Falkow formulated molecular Koch's postulates as a framework to help dissect the contribution of microbial genes to their pathogenicity (Box 1). Three years later, his advice led me to develop Mycobacterium marinum, a close genetic relative of Mycobacterium tuberculosis, as a model for tuberculosis pathogenesis. Here, I discuss insights into M. tuberculosis pathogenicity from studying M. marinum in the zebrafish, and frame them in terms of molecular Koch's postulates. The highly orchestrated life cycle of M. tuberculosis is achieved in substantial measure not by "traditional" pathogen-exclusive virulence genes acquired along its evolutionary history, but rather by genes that are shared with its environmental ancestors. Together, these genes support its tactics of subterfuge and exploitation to overcome host immunity so as to produce the transmissible disease that ensures the evolutionary survival of this obligate human pathogen.

Aryl Hydrocarbon Receptor Modulation by Tuberculosis Drugs Impairs Host Defense and Treatment Outcomes.

Antimicrobial resistance in tuberculosis (TB) is a public health threat of global dimension, worsened by increasing drug resistance. Host-directed therapy (HDT) is an emerging concept currently explored as an adjunct therapeutic strategy for TB. One potential host target is the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which binds TB virulence factors and controls antibacterial responses. Here, we demonstrate that in the context of therapy, the AhR binds several TB drugs, including front line drugs rifampicin (RIF) and rifabutin (RFB), resulting in altered host defense and drug metabolism. AhR sensing of TB drugs modulates host defense mechanisms, notably impairs phagocytosis, and increases TB drug metabolism. Targeting AhR in vivo with a small-molecule inhibitor increases RFB-treatment efficacy. Thus, the AhR markedly impacts TB outcome by affecting both host defense and drug metabolism. As a corollary, we propose the AhR as a potential target for HDT in TB in adjunct to canonical chemotherapy.

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.

Over-expression of Tgs1 in Mycobacterium marinum enhances virulence in adult zebrafish.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can persist in the host for decades without causing TB symptoms and can cause a latent infection, which is an intricate challenge of current TB control. The DosR regulon, which contains approximately 50 genes, is crucial in the non-replicating persistence of Mtb. tgs1 is one of the most powerfully induced genes in this regulon during Mtb non-replicating persistence. The gene encodes a triacyl glycerol synthase catalyzing synthesis of triacyl glycerol (TAG), which is proposed as an energy source during bacilli persistence. Here, western blotting showed that the Tgs1 protein was upregulated in clinical Mtb strains. To detect its physiological effects on mycobacterium, we constructed serial recombinant M. marinum including over-expressed Tgs1(Tgs1-H), reduced-expressed Tgs1(Tgs1-L), and wild type M. marinum strains as controls. Tgs1 over-expression did not influence M. marinum growth under aerobic shaking and in hypoxic cultures, while growth advantages were observed at an early stage under nutrient starvation. Transmission electron microscopy revealed more lipid droplets in Tgs1-H than the other two strains; the droplets filled the cytoplasm. Two-dimensional thin-layer chromatography revealed more phosphatidyl-myo-inositol mannosides in the Tgs1-H cell wall. To assess the virulence of recombinant M. marinum in the natural host, adult zebrafish were infected with Tgs1-H or wild type strains. Hypervirulence of Tgs1-H was characterized by markedly increased bacterial load and early death of adult zebrafish. Remarkably, zebrafish infected with Tgs1-H developed necrotizing granulomas much more rapidly and in higher amounts, which facilitated mycobacterial replication and dissemination among organs and eventual tissue destruction in zebrafish. RNA sequencing analysis showed Tgs1-H induced 13 genes differentially expressed under aerobiosis. Among them, PE_PGRS54 (MMAR_5307),one of the PE_PGRS family of antigens, was markedly up-regulated, while 110 coding genes were down-regulated in Tgs1-L.The 110 genes included 22 member genes of the DosR regulon. The collective results indicate an important role for the Tgs1 protein of M. marinumin progression of infection in the natural host. Tgs1 signaling may be involved in a previously unknown behavior of M. marinum under hypoxia/aerobiosis.

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.