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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 -
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 -
Frontiers in Microbiology 2018is a close relative of that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. Sliding motility correlates with biofilm...
is a close relative of that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. Sliding motility correlates with biofilm formation and virulence in most bacteria. In this study, we used a sliding motility assay to screen 2,304 transposon mutants of NTUH-M6885 and identified five transposon mutants with decreased sliding motility. Transposons that interrupted the type VII secretion system (T7SS) ESX-1-related genes, (), (), and (), were present in 3 mutants. We performed reverse-transcription polymerase chain reaction to verify genes from to , which were found to belong to a single transcriptional unit. Deletion mutants of , , (), and () displayed significant attenuation regarding sliding motility and biofilm formation. NTUH-M6885 possesses a functional ESX-1 secretion system. However, deletion of or resulted in slightly decreased secretion of EsxB (which is also known as CFP-10). Thus, the ESX-1 secretion system mediates sliding motility and is crucial for biofilm formation. These data provide new insight into biofilm formation.
PubMed: 29899738
DOI: 10.3389/fmicb.2018.01160 -
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 -
PLoS Neglected Tropical Diseases Jul 2021Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However,...
Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera. We show that SPR719 is active against these NTM species with a MIC range of 0.125-4 μg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rifampicin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.
Topics: Anti-Bacterial Agents; DNA Gyrase; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Mutation; Mycobacterium; Mycobacterium marinum; Mycobacterium ulcerans
PubMed: 34310615
DOI: 10.1371/journal.pntd.0009636 -
Anais Brasileiros de Dermatologia 2022The number of skin infections caused by atypical mycobacteria has increased in recent decades. They usually appear after contact with wounds and interruptions in the... (Review)
Review
The number of skin infections caused by atypical mycobacteria has increased in recent decades. They usually appear after contact with wounds and interruptions in the integrity of the skin. The present report describes a case of cutaneous infection by Mycobacterium marinum, in a young, immunocompetent patient, with a prolonged evolution, diagnosed through a skin lesion culture (from a spindle biopsy of the skin). The patient was treated with multidrug therapy, including clarithromycin, doxycycline, and rifampicin, due to the lesion extent, with satisfactory results. A brief review of the literature is also provided.
Topics: Anti-Bacterial Agents; Cellulitis; Drug Therapy, Combination; Humans; Leprostatic Agents; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Nontuberculous Mycobacteria; Skin Diseases, Bacterial; Skin Diseases, Infectious
PubMed: 35428530
DOI: 10.1016/j.abd.2021.03.013 -
Cureus Nov 2022is a non-tuberculous mycobacteria present in natural and non-chlorinated bodies of water. It is a known fish pathogen but can also cause human disease. It usually...
is a non-tuberculous mycobacteria present in natural and non-chlorinated bodies of water. It is a known fish pathogen but can also cause human disease. It usually causes cutaneous lesions but in rare cases may originate more invasive diseases with the involvement of deep structures. We describe three cases of patients with cutaneous infection by evaluated in a tertiary care center, two with confirmed infection and one with a presumptive diagnosis based on clinical and epidemiological features. A brief bibliographic review of infections is then presented to support the theme. We aim to alert one to the difficulties in establishing the correct diagnosis of this infection, emphasize the importance of a high degree of suspicion for its identification, and review the therapeutic management options.
PubMed: 36579262
DOI: 10.7759/cureus.31787 -
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 -
MBio Apr 2023The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium...
The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6CMHCII monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6CMHCII monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.
Topics: Animals; Mice; Neutrophils; Bacterial Proteins; Type VII Secretion Systems; Mycobacterium tuberculosis; Mycobacterium marinum; Inflammation; Cell Differentiation
PubMed: 37017530
DOI: 10.1128/mbio.02764-22 -
Scientific Reports May 2021Microorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions....
Microorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foci distributed at scattered locations in the M. marinum cytoplasm; the number, intensity and distribution of such foci changed in cells under stressed conditions. Finally, we provide bioinformatics data that 17 (of 244) mycobacteria, which lack the RsbRST genes, carry homologs of Bacillus cereus genes rsbK and rsbM indicating the existence of alternative σ activation pathways among mycobacteria.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Multiprotein Complexes; Mycobacterium marinum; Phosphoproteins; Phosphorylation; Sigma Factor; Signal Transduction; Stress, Physiological
PubMed: 33980893
DOI: 10.1038/s41598-021-89069-8