Biology subjects

Tobin, D. M.

Publications and source records attributed to Tobin, D. M..

12 recordsLinked to original sources

bioRxiv2026

Polyketide synthase 12 is an in vivo essential source of novel mycolyl lipids in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a major pathogen worldwide that infects and transmits only among humans, yet nearly all in vivo virulence research occurs in non-human hosts. To overcome this central challenge in tuberculosis research, we leveraged a dataset of more than 50,000 sequenced isolates to identify Mtb genes that that are functionally preserved during natural infection, disease causation, and transmission between humans. This whole-genome ranking identified polyketide synthase 12 (pks12 ) as an exceptionally in vivo essential gene in human tuberculosis, which we validated in zebrafish and mouse models. Though Pks12 produces a mycoketide lipid in only trace amounts, pks12 deletion severely altered the host-facing surface and arabinoglycan architecture of Mtb. This amplified effect was explained through the discovery of mannosyl-{beta}-1-phosphomycoketide monomycolate (MPMMM), which is synthesized from the known Pks12 product at higher abundance by antigen 85 mycolyltransferases. Thus, we used a new host-facing genomic-metabolomic-phenotypic approach to discover the functions of an in vivo essential Mtb gene, which controls the physical structure of the Mtb-host interface.

microbiology↗

bioRxiv2026

pH-responsive substrate switching in mycobacterial Type VII ESX secretion.

During infection, pathogenic mycobacteria reside within phagosomes of varying acidity based on the macrophage activation state. The ESX-1 secretion system [early secreted antigen 6 kilodaltons (ESAT-6) system-1] delivers protein virulence factors essential for phagosome lysis, facilitating infection. The mechanisms underlying ESX-1 lytic activity in heterogeneous environments remain unknown. Here we show that the canonical Type VII secretion system, ESX-1, orchestrates substrate switching in response to different environments. Growing Mycobacterium marinum at acidic pH resulted in substrate switching in vitro. Substrate switching was accompanied by significant changes to the levels of ESX-1 substrate transcripts, and to the levels of both ESX-1 substrates and chaperones at the protein level. We showed that specific ESX-1 transcripts were significantly upregulated in vivo, and that distinct substrate sets are required in an acidic infection model. Significance StatementPathogenic mycobacteria cause chronic and acute disease. Mycobacterial pathogens promote infection by transporting bacterial proteins into the host using ESX/Type VII secretion systems. The ESX-1 system secretes proteins into the phagosome that release the bacteria into the cytoplasm and promote bacterial survival in the macrophage. We show that Mycobacterium marinum, an animal pathogen and model for studying ESX-1 and tuberculosis, switches which ESX-1 proteins are secreted in response to acidic pH, an infection relevant signal. We demonstrate that protein secretion reflects changes in substrate transcripts and in substrate and chaperone protein levels. Finally, we leveraged two infection models to support that ESX-1 substrate switching likely occurs during infection. Our findings support a model in which mycobacterial pathogens use different proteins to lyse macrophage phagosomes of different pH.

microbiology↗

bioRxiv2025

Mapping unsolved lipidomes accelerates lipid discovery in major bacterial pathogens

Unlike gene-first approaches to understanding bacterial pathogenesis, molecule-forward discovery can uncover unexpected chemical diversity. Here, new lipidomic analytical methods and quality metrics defined the large scope of unknown lipids in the worlds deadliest pathogen, Mycobacterium tuberculosis (Mtb). This map allowed rapid discovery of Mtb lysyldiacylglycerol linked to the biosynthetic gene lysX, which controls in vivo infection outcomes in moth larvae, mice, guinea pigs, and here, zebrafish. A broader search for orthologous lysyltransferase domains identified the Staphylococcus aureus virulence gene mprF, where the same lipoamino acid was shown to be a previously unknown biosynthetic product. Thus, lipidomic mapping showed that the cell envelope composition of well-studied bacterial pathogens remains substantially unsolved and offers a new way to generate lists of discoverable lipids to accelerate molecular discovery.

microbiology↗

bioRxiv2025

Granuloma Dual RNA-Seq Reveals Composite Transcriptional Programs Driven by Neutrophils and Necrosis within Tuberculous Granulomas

Mycobacterial granulomas lie at the center of tuberculosis (TB) pathogenesis and represent a unique niche where infecting bacteria survive in nutrient-restricted conditions and in the face of a host immune response. The granulomas necrotic core, where bacteria reside extracellularly in humans, is difficult to assess in many experimentally tractable models. Here, using necrotic mycobacterial granulomas in adult zebrafish, we develop dual RNA-seq across different host genotypes to identify the transcriptional alterations that enable bacteria to survive within this key microenvironment. Through pharmacological and genetic interventions, we find that neutrophils within mature, necrotic granulomas promote bacterial growth, in part through upregulation of the bacterial devR regulon. We identify conserved suites of bacterial transcriptional programs induced only in the context of this unique necrotic extracellular niche, including bacterial modules related to K+ transport and rpf genes. Analysis of Mycobacterium tuberculosis strains across diverse lineages and human populations suggests that granuloma-specific transcriptional modules are targets for bacterial genetic adaptation in the context of human infection. Summary sentenceDual host-pathogen transcriptional profiling defines granuloma-specific programs during mycobacterial infection.

immunology↗

bioRxiv2025

Cathepsin Z is a conserved susceptibility factor underlying tuberculosis severity

Tuberculosis (TB) outcomes vary widely, from asymptomatic infection to mortality, yet most animal models do not recapitulate human phenotypic and genotypic variation. The genetically diverse Collaborative Cross mouse panel models distinct facets of TB disease that occur in humans and allows identification of genomic loci underlying clinical outcomes. We previously mapped a TB susceptibility locus on mouse chromosome 2. Here, we identify cathepsin Z (Ctsz) as a lead candidate underlying this TB susceptibility and show that Ctsz ablation leads to increased bacterial burden, CXCL1 overproduction, and decreased survival in mice. Ctsz disturbance within murine macrophages enhances production of CXCL1, a known biomarker of TB severity. From a Ugandan household contact study, we identify significant associations between CTSZ variants and TB disease severity. Finally, we examine patient-derived TB granulomas and report CTSZ localization within granuloma-associated macrophages, placing human CTSZ at the host-pathogen interface. These findings implicate a conserved CTSZ-CXCL1 axis in humans and genetically diverse mice that mediates TB disease severity.

genetics↗

bioRxiv2024

The antagonistic transcription factors, EspM and EspN, regulate the ESX-1 secretion system in M. marinum

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum, ESX-1 (ESAT-6 system 1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 genes does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum. In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes both during laboratory growth and during macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis. ImportancePathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

microbiology↗

bioRxiv2023

The EspN transcription factor is an infection-dependent regulator of the ESX-1 system in M. marinum

Bacterial pathogens use protein secretion systems to translocate virulence factors into the host and to control bacterial gene expression. The ESX-1 (ESAT-6 system 1) secretion system facilitates disruption of the macrophage phagosome during infection, enabling access to the cytoplasm, and regulates widespread gene expression in the mycobacterial cell. The transcription factors contributing to the ESX-1 transcriptional network during mycobacterial infection are not known. We showed that the EspM and WhiB6 transcription factors regulate the ESX-1 transcriptional network in vitro but are dispensable for macrophage infection by Mycobacterium marinum. In this study, we used our understanding of the ESX-1 system to identify EspN, a critical transcription factor that controls expression of the ESX-1 genes during infection, but whose effect is not detectable under standard laboratory growth conditions. Under laboratory conditions, EspN activity is masked by the EspM repressor. In the absence of EspM, we found that EspN is required for ESX-1 function because it activates expression of the whiB6 transcription factor gene, and specific ESX-1 substrate and secretory component genes. Unlike the other transcription factors that regulate ESX-1, EspN is required for M. marinum growth within and cytolysis of macrophages, and for disease burden in a zebrafish larval model of infection. These findings demonstrate that EspN is an infection-dependent regulator of the ESX-1 transcriptional network, which is essential for mycobacterial pathogenesis. Moreover, our findings suggest that ESX-1 expression is controlled by a genetic switch that responds to host specific signals. ImportancePathogenic mycobacteria cause acute and long-term diseases, including human tuberculosis. The ESX-1 system transports proteins that control the host response to infection and promotes bacterial survival. Although ESX-1 transports proteins, it also controls gene expression in the bacteria. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes, and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

microbiology↗

bioRxiv2022

mTOR-regulated Mitochondrial Metabolism Limits Mycobacterium-induced Cytotoxicity

Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanitys most lethal pathogen, is successful in only a minority of infected individuals.

immunology↗

bioRxiv2021

Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish

Mycobacterial granuloma formation involves significant stromal remodeling and the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as anti-angiogenic or vascular normalizing therapies are beneficial host-directed therapies in pre-clinical models of tuberculosis. Here we demonstrate that vascular normalization through vascular endothelial-protein tyrosine phosphatase inhibition decreases granuloma hypoxia, the opposite effect of hypoxia-inducing anti-angiogenic therapy. Vascular normalization leads to increased T cell and decreased neutrophil recruitment to granulomas, correlates of a protective immune response against mycobacterial infection.

immunology↗

bioRxiv2020

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

Tissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

genetics↗

bioRxiv2018

Intestinal Serum Amyloid A suppresses systemic neutrophil activation and bactericidal activity in response to microbiota colonization

The intestinal microbiota influence diverse aspects of host physiology, including the development and function of myeloid lineages. Numerous host and microbial factors are known to poise neutrophils and other granulocytes for response to pathogens and danger signals, yet the mechanisms by which the intestinal microbiota regulate this process are largely unknown. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions are poorly defined. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory mRNAs and bactericidal activity. Saas effects on neutrophils depends on microbiota colonization, suggesting this protein mediates the microbiotas influence on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we generated transgenic zebrafish over-expressing saa in the intestine. Transgenic intestinal saa expression was sufficient to partially complement the neutrophil phenotypes in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.

immunology↗

bioRxiv2018

Inhibition of thrombocyte activation restores protective immunity to mycobacterial infection

Infection-induced thrombocytosis is a clinically important complication of tuberculosis (TB). Recent studies have separately highlighted a correlation of platelet activation with TB severity and utility of aspirin as a host-directed therapy for TB that modulates the inflammatory response. Here we investigate the possibility that the beneficial effects of aspirin are related to an anti-platelet mode of action. We utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin or platelet-specific glycoprotein IIb/IIIa inhibitors reduced mycobacterial burden demonstrating a detrimental role for infection-induced thrombocyte activation. We found platelet inhibition reduced thrombocyte-macrophage interactions and restored indices of macrophage-mediated immunity to mycobacterial infection. Pathological thrombocyte activation and granuloma formation were found to be intrinsically linked illustrating a bidirectional relationship between host hemostasis and TB pathogenesis. Our study illuminates platelet activation as an efficacious target of anti-platelets drugs including aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.\n\nKey PointsO_LIInhibition of thrombocyte activation improves control of mycobacterial infection.\nC_LIO_LIInhibition of thrombocyte activation reduces thrombocyte-macrophage interactions and improves indices of macrophage immune function against mycobacterial infection.\nC_LI

immunology↗