Search Results (54)

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

Fish meal (FM) and fish oil (FO) are vital components commonly used in feed formulations. However, their supply, which generally comes from capture fisheries, is being exhausted, necessitating the exploration of sustainable alternatives. In a two-part study, the first part evaluated the FM and FO derived from smoked salmon by-product (SSBP) over a 12-week accelerated shelf-life test, comparing their lipid oxidation, amino acid, and fatty acid profiles to those of commercial whitefish meal and oil. In the second part, the SSBP FM and FO were then included in three experimental feeds at 25%, 50%, and 100% inclusion levels. These feeds were tested on juvenile striped bass (Morone saxatilis) cultured in a recirculating aquaculture system (RAS). The results indicated that the quality of SSBP FM and FO was lower than the commercial product (less amino acids (23.98% vs. 60.30%) and omega-3 fatty acids (9.46% vs. 26.6%), respectively). SSBP FO exhibited high initial peroxide value (21.00 ± 0.00 meq/kg oil), with gradually increasing total oxidation value and p-Anisidine value during storage. Regarding the feeding trial, all fish showed signs of Mycobacterium marinum infection after one month. While there was no significant difference in feed palatability (p > 0.8559), the feed conversion ratio was less efficient for the 100% SSBP feed (1.44 ± 0.14) compared to commercial feed (1.36 ± 0.13), but these differences were not statistically significant. This study suggests that SSBP FM and FO can be used as supplements at lower levels (25% and 50%) without negatively affecting growth, feed efficiency, or survival. Our findings may be useful for enabling beneficial collaborations between smoked salmon processors, feed manufacturers, and striped bass farmers, therefore contributing to sustainability in aquaculture practices. 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

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

Tuberculosis (TB) remains the major cause of mortality and morbidity, causing approximately 1.3 million deaths annually. As a highly successful pathogen, Mycobacterium tuberculosis (Mtb) has evolved numerous strategies to evade host immune responses, making it essential to understand the interactions between Mtb and host cells. G-protein-coupled receptor 84 (GPR84), a member of the G-protein-coupled receptor family, contributes to the regulation of pro-inflammatory reactions and the migration of innate immune cells, such as macrophages. Its role in mycobacterial infection, however, has not yet been explored. We found that GPR84 is induced in whole blood samples from tuberculosis patients and Mycobacterium marinum (Mm)-infected macrophage models. Using a Mm-wasabi infection model in mouse tails, we found that GPR84 is an important determinant of the extent of tissue damage. Furthermore, from our studies in an in vitro macrophage Mm infection model, it appears that GPR84 inhibits pro-inflammatory cytokines expression and increases intracellular lipid droplet (LD) accumulation, thereby promoting intracellular bacterial survival. Our findings suggest that GPR84 could be a potential therapeutic target for host-directed anti-TB therapeutics. 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

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host–pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE–PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins. 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

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb), 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. Mycobacterium marinum (Mm) and Mtb are both species of mycobacteria. In zebrafish, Mm proliferates and forms chronic granulomatous infections, which are similar to Mtb 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 Mm-infected cells and zebrafish. Our results demonstrated that SA inhibits Mm-infected pulmonary epithelial cells and inhibits the proliferation of Mm in Mm-infected zebrafish, suggesting that SA provides an antibacterial effect during Mm 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 Mm-infection-induced macrophages. SA inhibits the levels of MDA in Mm-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 Mm 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. Full article

Hydroxamic acid (HA) derivatives display antibacterial and antifungal activities. HA with various numbers of carbon atoms (C₂, C₆, C₈, C₁₀, C₁₂ and C₁₇), complexed with different metal ions, including Fe(II/III), Ni(II), Cu(II) and Zn(II), were evaluated for their antimycobacterial activities and their anti-biofilm activities. Some derivatives showed antimycobacterial activities, especially in biofilm growth conditions. For example, 20–100 µM of HA10Fe2, HA10FeCl, HA10Fe3, HA10Ni2 or HA10Cu2 inhibited Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium marinum biofilm development. HA10Fe2, HA12Fe2 and HA12FeCl could even attack pre-formed Pseudomonas aeruginosa biofilms at higher concentrations (around 300 µM). The phthiocerol dimycocerosate (PDIM)-deficient Mycobacterium tuberculosis H37Ra was more sensitive to the ion complexes of HA compared to other mycobacterial strains. Furthermore, HA10FeCl could increase the susceptibility of Mycobacterium bovis BCG to vancomycin. Proteomic profiles showed that the potential targets of HA10FeCl were mainly related to mycobacterial stress adaptation, involving cell wall lipid biosynthesis, drug resistance and tolerance and siderophore metabolism. This study provides new insights regarding the antimycobacterial activities of HA and their complexes, especially about their potential anti-biofilm activities. Full article