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Journal of Fish Diseases Oct 2019The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is...
The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.
Topics: Animal Feed; Animals; Artemia; Diet; Female; Fish Diseases; Male; Mycobacterium Infections, Nontuberculous; Mycobacterium chelonae; Mycobacterium marinum; Paramecium caudatum; Prevalence; Rotifera; Zebrafish
PubMed: 31418901
DOI: 10.1111/jfd.13071 -
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 -
PloS One 2020Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently...
Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay-a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.
Topics: Cell Line; Cell Membrane; Humans; Lipids; Macrophages; Mycobacterium marinum; Permeability; Phagosomes
PubMed: 32701962
DOI: 10.1371/journal.pone.0233252 -
PLoS Pathogens May 2023Infections caused by members of the mycobacterium tuberculosis complex [MTC] and nontuberculous mycobacteria [NTM] can induce widespread morbidity and mortality in...
Infections caused by members of the mycobacterium tuberculosis complex [MTC] and nontuberculous mycobacteria [NTM] can induce widespread morbidity and mortality in people. Mycobacterial infections cause both a delayed immune response, which limits rate of bacterial clearance, and formation of granulomas, which contain bacterial spread, but also contribute to lung damage, fibrosis, and morbidity. Granulomas also limit access of antibiotics to bacteria, which may facilitate development of resistance. Bacteria resistant to some or all antibiotics cause significant morbidity and mortality, and newly developed antibiotics readily engender resistance, highlighting the need for new therapeutic approaches. Imatinib mesylate, a cancer drug used to treat chronic myelogenous leukemia [CML] that targets Abl and related tyrosine kinases, is a possible host-directed therapeutic [HDT] for mycobacterial infections, including those causing TB. Here, we use the murine Mycobacterium marinum [Mm] infection model, which induces granulomatous tail lesions. Based on histological measurements, imatinib reduces both lesion size and inflammation of surrounding tissue. Transcriptomic analysis of tail lesions indicates that imatinib induces gene signatures indicative of immune activation and regulation at early time points post infection that resemble those seen at later ones, suggesting that imatinib accelerates but does not substantially alter anti-mycobacterial immune responses. Imatinib likewise induces signatures associated with cell death and promotes survival of bone marrow-derived macrophages [BMDMs] in culture following infection with Mm. Notably, the capacity of imatinib to limit formation and growth of granulomas in vivo and to promote survival of BMDMs in vitro depends upon caspase 8, a key regulator of cell survival and death. These data provide evidence for the utility of imatinib as an HDT for mycobacterial infections in accelerating and regulating immune responses, and limiting pathology associated with granulomas, which may mitigate post-treatment morbidity.
Topics: Humans; Animals; Mice; Imatinib Mesylate; Pyrimidines; Piperazines; Benzamides; Anti-Bacterial Agents; Granuloma
PubMed: 37200402
DOI: 10.1371/journal.ppat.1011387 -
European Journal of Medicinal Chemistry Nov 2023A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium...
A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) in an in vitro assay were identified. SAR analysis showed that the most active compounds, which included a phenyl group bearing fluoro substituent(s) at C2, a methoxy function at C3, and a benzyl-heteroatom moiety at C6, exhibited in vitro MIC values generally around 0.63-1.26 μM against Mtb and Mm. However, these compounds were inactive against Mtb in vivo (mice), and investigations revealed very short metabolic half-lives (<10 min) when incubated with mouse liver microsomes. Multiple observations of side products produced from oxidative cleavage of the imidazole moiety during the chemical synthesis work suggested that this is a likely metabolic pathway leading to the lack of observed activity in vivo.
Topics: Animals; Mice; Mycobacterium tuberculosis; Antitubercular Agents; Mycobacterium marinum; Pyridazines; Microbial Sensitivity Tests
PubMed: 37524009
DOI: 10.1016/j.ejmech.2023.115637 -
Journal of Veterinary Research Mar 2022Nontuberculous mycobacteria (NTM) are increasingly recognised as causative agents of opportunistic infections in humans for which effective treatment is challenging....
INTRODUCTION
Nontuberculous mycobacteria (NTM) are increasingly recognised as causative agents of opportunistic infections in humans for which effective treatment is challenging. There is very little information on the prevalence of NTM drug resistance in Poland. This study was aimed to evaluate the susceptibility to antibiotics of NTM, originally isolated from diseased ornamental fish.
MATERIAL AND METHODS
A total of 99 isolates were studied, 50 of them rapidly growing mycobacteria (RGM) (among which three-quarters were , , and and the rest , , , , , , and ). The other 49 were slowly growing mycobacteria (SGM) isolates (among which only one was and the bulk and ). Minimum inhibitory concentrations for amikacin (AMK), kanamycin (KAN), tobramycin (TOB), doxycycline (DOX), ciprofloxacin (CIP), clarithromycin (CLR), sulfamethoxazole (SMX), isoniazid (INH) and rifampicin (RMP) were determined.
RESULTS
The majority of the isolates were susceptible to KAN (95.95%: RGM 46.46% and SGM 49.49%), AMK (94.94%: RGM 45.45% and SGM 49.49%), CLR (83.83%: RGM 36.36% and SGM 47.47%), SMX (79.79%: RGM 30.30% and SMG 49.49%), CIP (65.65%: RGM 24.24% and SGM 41.41%), and DOX (55.55%: RGM 9.06% and SGM 46.46%). The majority were resistant to INH (98.98%: RGM 50.50% and SGM 48.48%) and RMP (96.96%: RGM 50.50% and SGM 46.46%).
CONCLUSION
The drug sensitivity of NTM varies from species to species. KAN, AMK, CLR and SMX were the most active against RGM isolates, and these same four plus DOX and CIP were the best drugs against SGM isolates.
PubMed: 35582485
DOI: 10.2478/jvetres-2022-0011 -
Microorganisms Feb 2024Tuberculosis (TB) is caused by infection with (), which has a unique resistance to many antimicrobial agents. TB has emerged as a significant worldwide health issue...
Tuberculosis (TB) is caused by infection with (), which has a unique resistance to many antimicrobial agents. TB has emerged as a significant worldwide health issue because of the rise of multidrug-resistant strains causing drug-resistant TB (DR-TB). As a result, the development of new drugs or effective strategies is crucial for patients with TB. () and are both species of mycobacteria. In zebrafish, proliferates and forms chronic granulomatous infections, which are similar to infections in lung tissue. Syringaldehyde (SA) is a member of the phenolic aldehyde family found in various plants. Here, we investigated its antioxidative and antibacterial properties in -infected cells and zebrafish. Our results demonstrated that SA inhibits -infected pulmonary epithelial cells and inhibits the proliferation of in -infected zebrafish, suggesting that SA provides an antibacterial effect during infection. Further study demonstrated that supplementation with SA inhibits the production of malondialdehyde (MDA) and reactive oxygen species (ROS) and increases the levels of reduced glutathione (GSH) in -infection-induced macrophages. SA inhibits the levels of MDA in -infected zebrafish, suggesting that SA exerts antioxidative effects in vivo. Additionally, we found that SA promotes the expression of NRF2/HO-1/NQO-1 and the activation of the AMPK-α1/AKT/GSK-3β signaling pathway. In summary, our data demonstrated that SA exerts antioxidative and antibacterial effects during infection both in vivo and in vitro and that the antioxidative effects of SA may be due to the regulation of NRF2/HO-1/NQO-1 and the AMPK-α1/AKT/GSK-3β signaling pathway.
PubMed: 38399751
DOI: 10.3390/microorganisms12020348 -
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 -
MBio Mar 2021Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more...
Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as and To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including , in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many species, including the less experimentally tractable pathogens , , , , , , and Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.
Topics: Computational Biology; Gene Library; Genes, Bacterial; Mycobacterium; Mycobacterium smegmatis; Research
PubMed: 33653882
DOI: 10.1128/mBio.02401-20