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Antimicrobial Agents and Chemotherapy Mar 2021Screening strategies for antituberculosis compounds using are time consuming and require biosafety level 3 (BSL3) facilities, which makes the development of...
Screening strategies for antituberculosis compounds using are time consuming and require biosafety level 3 (BSL3) facilities, which makes the development of high-throughput assays difficult and expensive. , a close genetic relative of , 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 as a model for antituberculosis drug identification by heterologous expression of two common drug activators, EthA and KatG. These two activators were overexpressed in , and the strains were tested against ethionamide, isoniazid, and a library of established antimycobacterial compounds from TB Alliance to compare drug susceptibility. Both and using zebrafish larvae, these genetically modified strains showed significantly higher susceptibility against ethionamide and isoniazid, which require activation by EthA and KatG. More importantly, a strain overexpressing both and 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 Four of these compounds were selected for further analysis, and three of them showed obvious EthA-dependent activity against Overall, our developed strains are valuable tools for high-throughput discovery of potential novel antituberculosis prodrugs.
Topics: Animals; Antitubercular Agents; Bacterial Proteins; Isoniazid; Mutation; Mycobacterium marinum; Mycobacterium tuberculosis; Prodrugs; Tuberculosis, Multidrug-Resistant; Zebrafish
PubMed: 33495223
DOI: 10.1128/AAC.01445-20 -
MSphere Jun 2022In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins)...
In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. , an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in . Ku70/80 proteins are also required for the healthy proliferation of cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in . This improved DSB repair fidelity and efficiency in and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins. In this study, we found that Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into . Understanding DSB repair mechanisms of may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.
Topics: Animals; DNA; DNA End-Joining Repair; DNA Ligase ATP; DNA-Binding Proteins; Humans; Leishmania; Mammals; Mycobacterium marinum
PubMed: 35695492
DOI: 10.1128/msphere.00156-22 -
American Journal of Veterinary Research Feb 2000To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum.
OBJECTIVE
To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum.
SAMPLE POPULATION
Isolates of M. marinum obtained from fish and humans.
PROCEDURE
Electrocompetent cells were prepared from isolates of M. marinum grown to various growth phases at several temperatures and with or without the addition of ethionamide or cycloheximide. Mycobacterial cells were transformed by electroporation with a replicative Escherichia coli-mycobacteria shuttle vector (pYUB18) as well as suicide vectors (pYUB285 and pUS252) that carried transposable elements (IS1096 and IS6110, respectively). Mutants from both isolates of M. marinum were recovered on 7H10 agar plates supplemented with kanamycin. Transformation and transposition efficiencies for various protocols were compared. Southern hybridization analysis was performed on mycobacterial mutants to confirm transposition events.
RESULTS
Competent cells prepared at room temperature (23-25 C) from organisms in late-exponential growth phase yielded higher transposition efficiency, compared with cells prepared at 4 C or from organisms in early- or mid-exponential growth phase. Naturally developing kanamycin-resistant colonies of M. marinum were not detected. Only the IS1096-derived transposition was able to efficiently mutate M. marinum. Southern hybridization of M. marinum mutants revealed random integration of IS 1096 into the M. marinum genome.
CONCLUSIONS
Transposition and transformation efficiencies were comparable, suggesting that the limiting factor in transposition is the transformation step. Most of the experiments resulted in transposition of IS1096; however, better approaches are needed to improve transposition efficiency.
Topics: Animals; Electroporation; Escherichia coli; Fish Diseases; Fishes; Genetic Vectors; Genome, Bacterial; Humans; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Plasmids; Transformation, Bacterial
PubMed: 10685681
DOI: 10.2460/ajvr.2000.61.125 -
MBio Oct 2023N-terminal acetylation is a protein modification that broadly impacts basic cellular function and disease in higher organisms. Although bacterial proteins are...
N-terminal acetylation is a protein modification that broadly impacts basic cellular function and disease in higher organisms. Although bacterial proteins are N-terminally acetylated, little is understood how N-terminal acetylation impacts bacterial physiology and pathogenesis. Mycobacterial pathogens cause acute and chronic disease in humans and in animals. Approximately 15% of mycobacterial proteins are N-terminally acetylated, but the responsible enzymes are largely unknown. We identified a conserved mycobacterial protein required for the N-terminal acetylation of 23 mycobacterial proteins including the EsxA virulence factor. Loss of this enzyme from reduced macrophage killing and spread of to new host cells. Defining the acetyltransferases responsible for the N-terminal protein acetylation of essential virulence factors could lead to new targets for therapeutics against mycobacteria.
Topics: Humans; Animals; Virulence; Mycobacterium marinum; Acetylation; Mycobacterium tuberculosis; Bacterial Proteins; Virulence Factors; Acetyltransferases
PubMed: 37772840
DOI: 10.1128/mbio.00987-23 -
Cell Reports Dec 2022During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans,...
During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans, non-human primates, and zebrafish models of infection, pathogenic mycobacteria produce and modify the specialized lipid trehalose 6,6'-dimycolate (TDM) in the bacterial cell envelope to drive host angiogenesis toward the site of forming granulomas, leading to enhanced bacterial growth. Here, we use the zebrafish-Mycobacterium marinum infection model to define the signaling basis of the host angiogenic response. Through intravital imaging and cell-restricted peptide-mediated inhibition, we identify macrophage-specific activation of NFAT signaling as essential to TDM-mediated angiogenesis in vivo. Exposure of cultured human cells to Mycobacterium tuberculosis results in robust induction of VEGFA, which is dependent on a signaling pathway downstream of host TDM detection and culminates in NFATC2 activation. As granuloma-associated angiogenesis is known to serve bacterial-beneficial roles, these findings identify potential host targets to improve tuberculosis disease outcomes.
Topics: Animals; Humans; Zebrafish; Macrophages; Mycobacterium tuberculosis; Tuberculosis; Signal Transduction; Granuloma; Mycobacterium marinum; NFATC Transcription Factors
PubMed: 36516756
DOI: 10.1016/j.celrep.2022.111817 -
Trends in Microbiology Jan 2020Zebrafish (Danio rerio) larvae are widely recognized for studying host-pathogen interactions in vivo because of their optical transparency, genetic manipulability, and... (Review)
Review
Zebrafish (Danio rerio) larvae are widely recognized for studying host-pathogen interactions in vivo because of their optical transparency, genetic manipulability, and translational potential. The development of the zebrafish immune system is well understood, thereby use of larvae enables investigation solely in the context of innate immunity. As a result, infection of zebrafish with natural fish pathogens including Mycobacterium marinum has significantly advanced our understanding of bacterial pathogenesis and vertebrate host defense. However, new work using a variety of human pathogens (bacterial, viral, and fungal) has illuminated the versatility of the zebrafish infection model, revealing unexpected and important concepts underlying infectious disease. We propose that this knowledge can inform studies in higher animal models and help to develop treatments to combat human infection.
Topics: Animals; Communicable Diseases; Disease Models, Animal; Drug Resistance, Bacterial; Host-Pathogen Interactions; Humans; Immunity, Innate; Larva; Macrophages; Mycobacterium marinum; Zebrafish
PubMed: 31604611
DOI: 10.1016/j.tim.2019.08.005 -
Frontiers in Immunology 2022Arabinogalactan (AG) participates in forming the cell wall core of mycobacteria, a structure known as the mAGP complex. Few studies have reported the virulence of...
Arabinogalactan (AG) participates in forming the cell wall core of mycobacteria, a structure known as the mAGP complex. Few studies have reported the virulence of inartificial AG or its interaction with the host immune system. Using clustered regularly interspaced short palindromic repeats interference gene editing technology, conditional mutants were constructed with a low expression of or (EmbA_KD or GlfT2_KD), which are separately involved in the biosynthesis of AG arabinose and galactose domains. High-performance gel permeation chromatography and high-performance liquid chromatography assays confirmed that the EmbA_KD strain showed a remarkable decrease in AG content with fragmentary arabinose chains, and the GlfT2_KD strain displayed less reduction in content with cut-down galactose chains. Based on transmission and scanning electron microscopy observations, the cell walls of the two mutants were found to be dramatically thickened, and the boundaries of different layers were more distinct. Phenotypes including the over-secretion of extracellular substances and enhanced spreading motility with a concomitant decreased resistance to ethambutol appeared in the EmbA_KD strain. The EmbA_KD and GlfT2_KD strains displayed limited intracellular proliferation after infecting murine J774A.1 macrophages. The disease progression infected with the EmbA_KD or GlfT2_KD strain significantly slowed down in zebrafish/murine tail infection models as well. Through transcriptome profiling, macrophages infected by EmbA_KD/GlfT2_KD strains showed enhanced oxidative metabolism. The cell survival measured using the CCK8 assay of macrophages exposed to the EmbA_KD strain was upregulated and consistent with the pathway enrichment analysis of differentially expressed genes in terms of cell cycle/apoptosis. The overexpression of C/EBPβ and the increasing secretion of proinflammatory cytokines were validated in the macrophages infected by the EmbA_KD mutant. In conclusion, the AG of appears to restrain the host innate immune responses to enhance intracellular proliferation by interfering with oxidative metabolism and causing macrophage death. The arabinose chains of AG influence the virulence and pathogenicity to a greater extent.
Topics: Animals; Arabinose; Galactans; Galactose; Immunity, Innate; Mice; Mycobacterium marinum; Virulence; Zebrafish
PubMed: 36090984
DOI: 10.3389/fimmu.2022.879775 -
Ugeskrift For Laeger Nov 2016
Topics: Female; Fingers; Gastrointestinal Agents; Humans; Infliximab; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Skin Diseases, Bacterial
PubMed: 27908321
DOI: No ID Found -
Journal of Clinical Microbiology Nov 2012Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis,...
Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.
Topics: Adolescent; Adult; Aged; Animals; Biota; Child; Child, Preschool; DNA, Bacterial; Female; Fish Diseases; Fishes; Genetic Variation; Genotype; Humans; Interspersed Repetitive Sequences; Male; Middle Aged; Molecular Typing; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Young Adult
PubMed: 22952269
DOI: 10.1128/JCM.01274-12 -
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