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Nature Protocols Jun 2013Mycobacterium marinum-infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae...
Mycobacterium marinum-infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 μmol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.
Topics: Animal Husbandry; Animals; Antitubercular Agents; Disease Models, Animal; Fluorometry; Larva; Macrophages; Microscopy, Fluorescence; Mycobacterium Infections, Nontuberculous; Phagocytosis; Zebrafish
PubMed: 23680983
DOI: 10.1038/nprot.2013.068 -
Disease Models & Mechanisms Jul 2014Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged... (Review)
Review
Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish-M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis.
Topics: Animals; Disease Models, Animal; Humans; Immunity, Innate; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis; Zebrafish
PubMed: 24973748
DOI: 10.1242/dmm.016089 -
Nature Communications Jun 2022Many key insights into actin regulation have been derived through examining how microbial pathogens intercept the actin cytoskeleton during infection. Mycobacterium...
Many key insights into actin regulation have been derived through examining how microbial pathogens intercept the actin cytoskeleton during infection. Mycobacterium marinum, a close relative of the human pathogen Mycobacterium tuberculosis, polymerizes host actin at the bacterial surface to drive intracellular movement and cell-to-cell spread during infection. However, the mycobacterial factor that commandeers actin polymerization has remained elusive. Here, we report the identification and characterization of the M. marinum actin-based motility factor designated mycobacterial intracellular rockets A (MirA), which is a member of the glycine-rich PE_PGRS protein family. MirA contains an amphipathic helix to anchor into the mycobacterial outer membrane and, surprisingly, also the surface of host lipid droplet organelles. MirA directly binds to and activates the host protein N-WASP to stimulate actin polymerization through the Arp2/3 complex, directing both bacterial and lipid droplet actin-based motility. MirA is dissimilar to known N-WASP activating ligands and may represent a new class of microbial and host actin regulator. Additionally, the MirA-N-WASP interaction represents a model to understand how the enigmatic PE_PGRS proteins contribute to mycobacterial pathogenesis.
Topics: Actins; Bacterial Proteins; Cell Wall; Glycine; Humans; Mycobacterium marinum; Mycobacterium tuberculosis
PubMed: 35750685
DOI: 10.1038/s41467-022-31333-0 -
Scientific Reports Jan 2019High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but...
High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents.
Topics: Anti-Bacterial Agents; Cell Survival; Fibroblasts; High-Throughput Screening Assays; Mycobacterium marinum; Virulence; Virulence Factors
PubMed: 30631100
DOI: 10.1038/s41598-018-37176-4 -
Scientific Reports Apr 2018Mycobacterium marinum (M. marinum) is a slowly growing nontuberculous mycobacterium. The incidence of M. marinum infections in Denmark is unknown. We conducted a...
Mycobacterium marinum (M. marinum) is a slowly growing nontuberculous mycobacterium. The incidence of M. marinum infections in Denmark is unknown. We conducted a retrospective nationwide study including all culture confirmed cases of M. marinum from 2004 to 2017 in Denmark. All available medical records were reviewed. Demographics, clinical characteristics, and treatment regiments were analyzed. Fifty-five patients were identified, 40 (72.7%) were men with a median age of 50 years. Aquatic exposure was reported by 48 (90.6%) of the patients. Site of infection was upper extremities in 49 (92.5%) patients and 49 (92.5%) had superficial infection. The median time from symptom presentation to diagnosis was 194 days. All patients received antibiotics. Median time of treatment duration among all patients was 112 days. Treatment outcome was classified as improved in 40 (75%), improved with sequela in 4 (7.6%) patients and only 3 patients (3.8%) were classified as failed. Infection with M. marinum is rare and there is a long delay from symptom manifestation to diagnosis. The infection is predominantly related to aquatic exposure. M. marinum should be a differential diagnose in patients with slow-developing cutaneous elements and relevant exposure. Treatment outcomes are overall good and severe sequela are rare.
Topics: Adult; Clarithromycin; Diagnosis, Differential; Ethambutol; Female; Humans; Male; Middle Aged; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Retrospective Studies; Rifampin; Skin Diseases, Bacterial; Treatment Outcome
PubMed: 29712930
DOI: 10.1038/s41598-018-24702-7 -
Biomolecules Jan 2023The ESX-5 secretion system is essential for the viability and virulence of slow-growing pathogenic mycobacterial species. In this study, we identified a 1,2,4-oxadiazole...
The ESX-5 secretion system is essential for the viability and virulence of slow-growing pathogenic mycobacterial species. In this study, we identified a 1,2,4-oxadiazole derivative as a putative effector of the ESX-5 secretion system. We confirmed that this 1,2,4-oxadiazole and several newly synthesized derivatives inhibited the ESX-5-dependent secretion of active lipase LipY by (). Despite reduced lipase activity, we did not observe a defect in LipY secretion itself. Moreover, we found that several other ESX-5 substrates, especially the high molecular-weight PE_PGRS MMAR_5294, were even more abundantly secreted by treated with several 1,2,4-oxadiazoles. Analysis of grown in the presence of different oxadiazole derivatives revealed that the secretion of LipY and the induction of PE_PGRS secretion were, in fact, two independent phenotypes, as we were able to identify structural features in the compounds that specifically induced only one of these phenotypes. Whereas the three most potent 1,2,4-oxadiazoles displayed only a mild effect on the growth of or in culture, these compounds significantly reduced bacterial burden in -infected zebrafish models. In conclusion, we report a 1,2,4-oxadiazole scaffold that dysregulates ESX-5 protein secretion.
Topics: Animals; Bacterial Proteins; Mycobacterium marinum; Zebrafish; Virulence; Mycobacterium tuberculosis; Type VII Secretion Systems; Lipase
PubMed: 36830581
DOI: 10.3390/biom13020211 -
Developmental and Comparative Immunology Mar 2019Owing to the high incidence of multi-drug resistance and challenges posed by the complex and long duration of treatments, Mycobacterium tuberculosis (Mtb) infections... (Review)
Review
Owing to the high incidence of multi-drug resistance and challenges posed by the complex and long duration of treatments, Mycobacterium tuberculosis (Mtb) infections remain a significant clinical burden, which would benefit from development of novel immuno-therapeutic-based treatment strategies. Among early immune effectors, invariant or innate-like (i)T cells are attracting attention because of their potential regulatory activity, which can shape anti-mycobacterial immune responses. Unlike conventional T cells, iT cells express a semi-invariant T cell receptor, and respond rapidly and robustly to molecular patterns presented by MHC class I-like molecules. To date, functional studies of iT cells in vivo has been problematic and the role of iT cells in anti-Mtb responses remains unclear. Here, after reviewing the recent literature on anti-mycobacterial iT cell immunity, we describe a novel alternative model system in the amphibian Xenopus laevis tadpoles during infection with Mycobacterium marinum (Mm). X. laevis tadpoles rely mostly on a few distinct prominent innate-like (i)T cell subsets, whose development and function are governed by distinct MHC class I-like molecules. Thus, X. laevis tadpoles provide a convenient and cost-effective in vivo model uniquely suited to investigate the roles of iT cells during mycobacterial infections. We have developed reverse genetics and MHC tetramer technology to characterize this MHC-like/iT system in tadpoles. Our study in X. laevis provides evidence of a conserved convergent function of iT cells in host defenses against mycobacteria between mammals and amphibians.
Topics: Animals; Histocompatibility Antigens Class I; Immunity, Cellular; Immunity, Innate; Models, Animal; Mycobacterium marinum; Mycobacterium tuberculosis; Natural Killer T-Cells; T-Lymphocytes; Tuberculosis; Xenopus
PubMed: 30521838
DOI: 10.1016/j.dci.2018.12.002 -
Diseases of Aquatic Organisms Nov 2013Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to...
Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), and M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low-dose group. These results demonstrate that P. caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.
Topics: Aging; Animals; Ciliophora Infections; Fish Diseases; Larva; Mycobacterium Infections; Mycobacterium chelonae; Mycobacterium marinum; Paramecium caudatum; Refrigeration; Zebrafish
PubMed: 24192000
DOI: 10.3354/dao02649 -
The Journal of Experimental Medicine Nov 2003Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living...
Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.
Topics: Actins; Animals; Biopolymers; Mice; Mice, Inbred Strains; Mycobacterium marinum; Phagosomes
PubMed: 14597736
DOI: 10.1084/jem.20031072 -
BMC Genomics Apr 2021Hypoxic stress plays a critical role in the persistence of Mycobacterium tuberculosis (Mtb) infection, but the mechanisms underlying this adaptive response remain ill...
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.
Topics: Humans; Hypoxia; Mycobacterium marinum; Promoter Regions, Genetic; Sequence Analysis, RNA; Transcription Initiation Site
PubMed: 33823801
DOI: 10.1186/s12864-021-07572-8