Search Results (46)

Carotenoid pigments are widely distributed in nature and play a crucial role in protecting organisms from photodynamic damage. However, the characterization of carotenoid production in clinically relevant mycobacteria has been limited due to the low sensitivity of conventional detection methods. We present a descriptive analysis of carotenoid production in seven mycobacterial isolates from the scotochromogenic, photochromogenic, and non-chromogenic groups. To achieve this, we used a combination of High-performance liquid chromatography with diode-array detection (HPLC-DAD) and Ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) to detect carotenoids pigments. Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (MB) (non-chromogenic mycobacteria) produced β-carotene when cultured in the absence of light, at levels comparable to those of photochromogenic mycobacteria such as M. marinum (MM) and M. kansasii (MK). The highest levels of carotenoids were found in scotochromogenic species M. avium (MAV) and M. gordonae (MGOR). Conversely, M. abscessus (MABS), a non-chromogenic species in which no β-carotene was detected, served as a negative control for matrix effects. As expected, the use of highly sensitive analytical techniques such as HPLC-DAD and UHPLC-MS significantly enhanced the detection of β-carotene compared to visual pigment assessment. These methods allowed the detection of basal β-carotene levels even in mycobacteria classified as non-chromogenic. The proposed analytical approach provides a robust research tool to understand the effects of different stimulus that may alter the cell physiology in terms of pigment production. Full article

Mycobacterium marinum and Mycobacterium leprae are zoonotic mycobacteria causing chronic cutaneous infections that challenge host immunity and tissue integrity. Reactive oxygen species (ROS) play a complex role in the host defense system. While essential for pathogen elimination and intracellular signaling, excessive ROS can lead to immune dysregulation and impaired tissue healing. This review explores M. marinum and M. leprae pathogenesis through the role of ROS in redox imbalances, immunity, and cutaneous wound healing. Physiological ROS levels are vital for T-cell activation and differentiation. However, excessive ROS production, particularly in innate immune cells, can lead to T-cell suppression. M. leprae infection is associated with a significant reduction in key antioxidants such as glutathione (GSH), GSH peroxidase (GSH-Px), and GSH reductase (GR), a reduction that correlates with disease severity. For M. marinum, disrupting the pathogen’s redox balance through thioredoxin reductase (TrxR) inhibition sensitizes bacteria to ROS damage, reducing bacterial load. Overall, redox imbalance is central to the pathogenesis and persistence of cutaneous mycobacterial infections, compromising host defense and impairing tissue repair. Restoring and maintaining proper redox homeostasis, potentially by exploring the role of GSH as an antioxidant, represents a promising adjunct treatment to improve host outcomes in these challenging dermatological conditions. Full article

Fish mycobacteriosis, a chronic progressive disease caused by nontuberculous mycobacteria (NTM), affects marine, brackish, and freshwater fish. Mycobacterium marinum (M. marinum), the most important of the NTM, infects fresh and marine water fish causing necrotizing granulomas and associated morbidity and mortality. M. marinum causes disease in zebrafish in a dose-dependent fashion. The M. marinum-induced disease in the zebrafish is associated with the development of necrotizing granulomas with abundant bacteria in the necrotic areas. Acute infection with high infectious doses of M. marinum infection in zebrafish was characterized by uncontrolled replication of the pathogen and death of all fish within 16 days, while chronic infections were marked by the formation of granulomas in different organs and longer survival in the range of 4–8 weeks. This review therefore synthesizes recent advances in our understanding of M. marinum’s infection of zebrafish, molecular pathogenesis, virulence mechanisms, and immune evasion strategies in zebrafish, while also highlighting the host immune effector responses and the virulence mechanisms of M. marinum. Full article

Objectives: To explore the metabolic changes in zebrafish larvae after infection with Mycobacterium marinum, this study adopted a widely targeted metabolomic approach to analyze the changes in the overall metabolic profiles of zebrafish larvae infected for 5 days. Methods: Data were collected by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Mass spectrometry data were processed using Analyst 1.6.3 and MultiQuant 3.0.3 software, and multivariate statistical analysis was carried out. The KEGG database, HMDB database, and CHEBI database were used to screen and identify differential metabolites, and metabolic pathway enrichment analysis was performed through KEGG pathways. Results: A total of 329 metabolites were detected, among which 61 differential metabolites were screened. Specifically, 41 metabolites, such as kynurenine, isoallolithocholic acid, 2′-deoxyguanosine, indole-3-carboxaldehyde, and L-lactic acid, were downregulated, while 20 metabolites, such as L-palmitoylcarnitine, myristoyl-L-carnitine, dodecanoylcarnitine, 2-isopropyl-malic acid, and 2-methylsuccinic acid, were upregulated. KEGG metabolic pathway enrichment analysis indicated that these differential metabolites were mainly involved in metabolic pathways such as pyrimidine metabolism, nucleotide metabolism, the pentose phosphate pathway, and purine metabolism. Conclusions: This study demonstrated that significant changes occurred in multiple metabolites and metabolic pathways in zebrafish larvae after infection with M. marinum. The research results have improved the understanding of zebrafish as a model organism in the field of Mycobacterium research and laid a solid foundation for subsequent metabolomic-related research using zebrafish. Full article

Mycobacterium marinum is an opportunistic pathogen prevalent in aquatic environments, causing significant morbidity in fish, including Coho salmon (Oncorhynchus kisutch), a species increasingly cultured in Chinese salmonid aquaculture. This study investigated the immune response of Coho salmon to M. marinum infection and the bacterial proliferation dynamics in the liver and kidney. Transcriptome analysis revealed 5028 differentially expressed genes (DEGs) in the kidney and 3419 DEGs in the liver at 6 weeks post-infection. Gene Ontology and KEGG enrichment analysis highlighted pathways such as cytokine–cytokine receptor interaction, metabolic pathways, and Toll-like receptor signaling in the kidney, while the DEGs in the liver were enriched in metabolic pathways, immune system processes, and stress and defense responses. The temporal expression profiling of 15 immune-related genes, including acute-phase proteins (serum amyloid A-5 and hepcidin), cytokines (TNF-α, IL-1β, IL-17A), chemokines (CXCL13 and CCL19), pattern recognition receptors (Toll-like receptor 13), and other immune-related genes, showed significant upregulation against M. marinum infection, with stronger responses in the liver. Furthermore, it was found that there was a progressive proliferation of M. marinum in the infected liver and kidney from approximately 2.5 log₁₀ cfu/g at week 2 to about 6 log₁₀ cfu/g by 6 weeks, with a significantly higher load in the liver. These findings provide critical insights into the immune mechanisms of Coho salmon against M. marinum and the pathogen’s tissue-specific proliferation, offering a foundation for developing targeted control strategies against M. marinum in aquaculture. Full article

Background/Objectives: Over the past two centuries, tuberculosis (TB) has been responsible for approximately one billion deaths and continues to represent a significant global health challenge. Despite extensive research efforts, fully effective strategies for the prevention or eradication of TB remain elusive, highlighting the urgent demand for novel vaccines with enhanced safety profiles and efficacy. Lipoproteins, integral surface proteins of mycobacteria, are frequently associated with virulence and display notable immunogenicity, rendering them promising candidates for vaccine development. This study investigates the potential of the mycobacterial lipoprotein, LprO, as a vaccine antigen against TB. Methods: A pcDNA-lprO DNA vaccine was constructed, and its immunogenicity was evaluated using a murine model. Its protective efficacy was further assessed using a Mycobacterium marinum (M. marinum)-infected zebrafish model. Additionally, a recombinant BCG vaccine strain, BCG Japan::pNBV1-lprO, was generated. Its immunogenicity was tested in mice, and its safety was evaluated in SCID mice. Both vaccine candidates were further assessed in regard to their protective efficacy in a murine Mycobacterium tuberculosis (M. tb) infection model. Results: The pcDNA-lprO vaccine increased the M. tb-specific IFN-γ-secreting lymphocytes in murine spleens and prolonged the survival of zebrafish infected with M. marinum. The recombinant BCG Japan::pNBV1-lprO vaccine elicited M. tb-specific Th1-type immune responses in mice compared to the standard BCG Japan strain. Both vaccines effectively reduced the bacterial burden of M. tb in murine lungs, offering superior protection relative to the control groups. Conclusions: These findings establish LprO as a compelling candidate for TB vaccine development, with both LprO-based DNA and recombinant BCG vaccines demonstrating robust protective effects against TB. Full article

PE_PGRS domain proteins represent a family of proteins found in pathogenic and non-pathogenic mycobacteria such as M. smegmatis. This conserved family is characterized by two distinct regions denoted as the variable PGRS domain defined by glycine-rich repeats, and a PE domain consisting of two antiparallel alpha-helices. There are many indications that PE_PGRS proteins are involved in immunopathogenesis and virulence by evading or triggering the host immune response. However, there is not yet any information on their degree of specialization or redundancy. Computational analysis and structural annotation using AlphaFold3 combined with other tools reveals an exceptionally powerful and unprecedented ability to undergo phase separation by the PGRS domain. This suggests that PGRS’s glycine-rich, multivalent, low-complexity composition supports phase separation while adopting a structured conformation, contrary to the disordered nature typical of such domains. While previously never reported, the hypothesized role of PGRS in virulence indicates a novel window into the seemingly ubiquitous role of phase separation in cellular compartmentalization and molecular dynamics. This review aims to summarize the current understanding of the PE_PGRS family and its various biological roles in the context of bioinformatic analyses of some interesting representatives of M. marinum that are under control by host sterols. Based on the structural bioinformatics analysis, we discuss future approaches to uncover the mechanistic role of this intriguing family of mycobacterial proteins in both pathogenic and non-pathogenic mycobacteria. Full article

Changes in cell shape have been shown to be an integral part of the mycobacterial life cycle; however, systematic investigations into its patterns of pleomorphic behaviour in connection with stages or conditions of growth are scarce. We have studied the complete growth cycle of Mycobacterium monacense cultures, a Non-Tuberculous Mycobacterium (NTM), in solid as well as in liquid media. We provide data showing changes in cell shape from rod to coccoid and occurrence of refractive cells ranging from Phase Grey to phase Bright (PGB) in appearance upon ageing. Changes in cell shape could be correlated to the bi-phasic nature of the growth curves for M. monacense (and the NTM Mycobacterium boenickei) as measured by the absorbance of liquid cultures while growth measured by colony-forming units (CFU) on solid media showed a uniform exponential growth. Based on the complete M. monacense genome we identified genes involved in cell morphology, and analyses of their mRNA levels revealed changes at different stages of growth. One gene, dnaK_3 (encoding a chaperone), showed significantly increased transcript levels in stationary phase cells relative to exponentially growing cells. Based on protein domain architecture, we identified that the DnaK_3 N-terminus domain is an MreB-like homolog. Endogenous overexpression of M. monacense dnaK_3 in M. monacense was unsuccessful (appears to be lethal) while exogenous overexpression in Mycobacterium marinum resulted in morphological changes with an impact on the frequency of appearance of PGB cells. However, the introduction of an anti-sense “gene” targeting the M. marinum dnaK_3 did not show significant effects. Using dnaK_3-lacZ reporter constructs we also provide data suggesting that the morphological differences could be due to differences in the regulation of dnaK_3 in the two species. Together these data suggest that, although its regulation may vary between mycobacterial species, the dnaK_3 might have a direct or indirect role in the processes influencing mycobacterial cell shape. Full article

Background: Mycobacterium chelonae is a ubiquitous, rapidly growing, nontuberculous mycobacteria that primarily affects immunocompromised patients. The most common presentation is an atypical, chronic skin and soft tissue infection. Due to its high resistance rate, early diagnosis based on clinical suspicion, risk factor assessment, and exposure history is crucial for initiating appropriate multi-drug treatment. Methods: We report two cases of M. chelonae skin and soft tissue infections, one presenting with localized disease and the other with disseminated involvement. One case had a specific exposure to fish-related activities, a risk factor more commonly associated with Mycobacterium marinum infections rather than M. chelonae. Results: One of the cases involved osteomyelitis and tenosynovitis which are rare presentations of M. chelonae infection. While the limbs are the most commonly affected sites in disseminated M. chelonae infections, involvement of the lower extremities, as seen in one of our cases, is rarely reported. Treatment posed challenges due to antibiotic resistance and patient tolerance. However, in both cases where follow up was possible, prolonged multi-drug therapy led to complete resolution of the lesions. Conclusions: These cases highlight the importance of considering M. chelonae in chronic skin and soft tissue infections, especially in patient with relevant exposures or immunosupression. Uncommon presentations require a high index of suspicion. Given the challenges of treatment resistance and patient tolerance, prolonged multi-drug therapy remains essential for successful outcomes. Full article

Background/Objectives: Tuberculosis (TB) is one of the leading infectious causes of death worldwide, highlighting the importance of identifying new anti-TB agents. In previous research, our team identified antimycobacterial activity in Kielmeyera membranacea leaf extract; therefore, this study aims to conduct further exploration of its potential. Methods: Classical chromatography was applied for fractionation and spectrometric techniques were utilized for chemical characterization. For in vitro tests, samples were assessed against Mycobacterium tuberculosis and Mycobacterium marinum. The toxicity and efficacy of active samples were evaluated in vivo using different zebrafish models. Chemogenomics studies were applied to predict the isolated active compound’s potential mode of action. Results: We performed fractionation of K. membranacea ethanolic extract (EE) and then its dichloromethane fraction (DCM), and the biflavonoid podocarpusflavone A (PCFA) was isolated and identified as a promising active compound. The EE and PCFA were found to be non-toxic to zebrafish larvae and were able to inhibit M. tuberculosis growth extracellularly. Additionally, PCFA demonstrated antimycobacterial activity within infected macrophages, especially when combined with isoniazid. In addition, the EE, DCM, and PCFA have shown the ability to inhibit M. marinum’s growth during in vivo zebrafish larvae yolk infection. Notably, PCFA also effectively countered systemic infection established through the caudal vein, showing a similar inhibitory activity profile to rifampicin, both at 32 µM. A reduction in the transcriptional levels of pro-inflammatory cytokines confirmed the infection resolution. The protein tyrosine phosphatase B (PtpB) of M. tuberculosis, which inhibits the macrophage immune response, was predicted as a theoretical target of PCFA. This finding is in agreement with the higher activity observed for PCFA intracellularly and in vivo on zebrafish, compared with the direct action in M. tuberculosis. Conclusions: Here, we describe the discovery of PCFA as an intracellular inhibitor of M. tuberculosis and provide evidence of its in vivo efficacy and safety, encouraging its further development as a combination drug in novel therapeutic regimens for TB. Full article

The incidence of Mycobacterium marinum infection is on the rise; however, the existing drug treatment cycle is lengthy and often requires multi-drug combination. Therefore, there is a need to develop new and effective anti-M. marinum drugs. Cochliomycin A, a 14-membered resorcylic acid lactone with an acetonide group at C-5′ and C-6′, exhibits a wide range of antimicrobial, antimalarial, and antifouling activities. To further explore the effect of this structural change at C-5′ and C-6′ on this compound’s activity, we synthesized a series of compounds with a structure similar to that of cochliomycin A, bearing ketal groups at C-5′ and C-6′. The R/S configuration of the diastereoisomer at C-13′ was further determined through an NOE correlation analysis of CH₃ or CH₂ at the derivative C-13′ position and the H-5′ and H-6′ by means of a 1D NOE experiment. Further comparative ¹H NMR analysis of diastereoisomers showed the difference in the chemical shift (δ) value of the diastereoisomers. The synthetic compounds were screened for their anti-microbial activities in vitro. Compounds 15–24 and 28–35 demonstrated promising activity against M. marinum, with MIC₉₀ values ranging from 70 to 90 μM, closely approaching the MIC₉₀ of isoniazid. The preliminary structure–activity relationships showed that the ketal groups with aromatic rings at C-5′ and C-6′ could enhance the inhibition of M. marinum. Further study demonstrated that compounds 23, 24, 29, and 30 had significant inhibitory effects on M. marinum and addictive effects with isoniazid and rifampicin. Its effective properties make it an important clue for future drug development toward combatting M. marinum resistance. Full article

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis. The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated. Full article

The emergence of drug-resistant mycobacteria has rendered many clinical drugs and regimens ineffective, imposing significant economic and healthcare burden on individuals and society. Repurposing drugs intended for treating other diseases is a time-saving, cost-effective, and efficient approach for identifying excellent antimycobacterial candidates or lead compounds. This study is the first to demonstrate that rupatadine (RTD), a drug used to treat allergic rhinitis, possesses excellent activity against mycobacteria without detectable resistance, particularly Mycobacterium tuberculosis and Mycobacterium marinum, with a minimal inhibitory concentration as low as 3.13 µg/mL. Furthermore, RTD exhibited moderate activity against nonreplicating M. tuberculosis with minimal inhibitory concentrations lower than drugs targeting the cell wall, suggesting that RTD has great potential to be modified and used for the treatment of nonreplicating M. tuberculosis. Additionally, RTD exhibits partial synergistic effects when combined with clofazimine, pretomanid, and TB47 against M. tuberculosis, providing the theoretical foundation for the development of treatment regimens. Transcriptomic profiling leads us to speculate that eight essential genes may be the targets of RTD or may be closely associated with mycobacterial resistance to RTD. In summary, RTD may be a promising hit for further antimycobacterial drug or regimen optimization, especially in the case of nonreplicating mycobacteria. Full article

With the emergence of drug-resistant strains, the treatment of tuberculosis (TB) is becoming more difficult and there is an urgent need to find new anti-TB drugs. Mycobacterium marinum, as a model organism of Mycobacterium tuberculosis, can be used for the rapid and efficient screening of bioactive compounds. The 14-membered resorcylic acid lactones (RALs) have a wide range of bioactivities such as antibacterial, antifouling and antimalarial activity. In order to further study their bioactivities, we initially constructed a 14-membered RALs library, which contains 16 new derivatives. The anti-M. marinum activity was evaluated in vitro. Derivatives 12, 19, 20 and 22 exhibited promising activity with MIC₉₀ values of 80, 90, 80 and 80 μM, respectively. The preliminary structure–activity relationships showed that the presence of a chlorine atom at C-5 was a key factor to improve activity. Further studies showed that 12 markedly inhibited the survival of M. marinum and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 12 is a bioactive compound capable of enhancing the potency of existing positive drugs, and its effective properties make it a very useful leads for future drug development in combating TB resistance. Full article

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus. Full article