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MSphere Apr 2024(), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS)...
UNLABELLED
(), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The gene in and () plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of in resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of , a universal stress sensor. The absence of also resulted in reduced intracellular levels of NAD, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria.
IMPORTANCE
This study unveils the pivotal role played by the gene in and in in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, -hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of . Moreover, changes in crucial cofactors responsible for electron transfer highlighted 's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.
Topics: Mycobacterium tuberculosis; Homeostasis; Oxidation-Reduction; Energy Metabolism; Oxidative Stress; Bacterial Proteins; Reactive Oxygen Species; Antitubercular Agents; Ergothioneine; Inositol; Mycobacterium marinum; Gene Deletion; Cysteine; Glycopeptides
PubMed: 38564709
DOI: 10.1128/msphere.00061-24 -
Marine Drugs Mar 2024With 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. , as a...
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. , as a model organism of , 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- activity was evaluated in vitro. Derivatives , , and exhibited promising activity with MIC 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 markedly inhibited the survival of and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 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.
Topics: Mycobacterium marinum; Antibodies; Antimalarials; Antitubercular Agents; Lactones
PubMed: 38535476
DOI: 10.3390/md22030135 -
Microbiology Resource Announcements Apr 2024N-terminal acetylation in is correlated with pathogenic activity. We used genomics and bottom-up proteomics to identify protein Emp1 as the sole acetyltransferase...
N-terminal acetylation in is correlated with pathogenic activity. We used genomics and bottom-up proteomics to identify protein Emp1 as the sole acetyltransferase responsible for acetylation of EsxA, a known virulence factor. Using custom data analysis, we screened the proteome to identify 22 additional putative substrates of Emp1.
PubMed: 38477461
DOI: 10.1128/mra.01263-23 -
MBio Apr 2024Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including and , the ESAT-6...
Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including and , the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the or gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by . In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.
Topics: Animals; Transcription Factors; Bacterial Proteins; Type VII Secretion Systems; Zebrafish; Tuberculosis; Mycobacterium tuberculosis; Mycobacterium marinum
PubMed: 38445877
DOI: 10.1128/mbio.03357-23 -
ACS Chemical Biology Mar 2024Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid...
Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical strains. PGL is also found in , where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor -hydroxybenzoic acid (HB) with an azide group (3-azido HB). When fed to mycobacteria, 3-azido HB was incorporated into the cell surface, which could then be visualized the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido HB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing and strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.
Topics: Animals; Glycolipids; Virulence Factors; Zebrafish; Mycobacterium tuberculosis; Mycobacterium marinum
PubMed: 38442242
DOI: 10.1021/acschembio.3c00724 -
Archives of Biochemistry and Biophysics Apr 2024The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a...
The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.
Topics: Mycobacterium marinum; Cytochrome P-450 Enzyme System; Protein Structure, Secondary; Macrolides; Heme; Crystallography, X-Ray
PubMed: 38430969
DOI: 10.1016/j.abb.2024.109950 -
IScience Mar 2024Pathogenic mycobacteria orchestrate the complex cell populations known as granuloma that is the hallmark of tuberculosis. Foam cells, a lipid-rich cell-type, are...
Pathogenic mycobacteria orchestrate the complex cell populations known as granuloma that is the hallmark of tuberculosis. Foam cells, a lipid-rich cell-type, are considered critical for granuloma formation; however, the causative factor in foam cell formation remains unclear. Atherosclerosis is a chronic inflammatory disease characterized by the abundant accumulation of lipid-laden-macrophage-derived foam cells during which cholesterol 25-hydroxylase (CH25H) is crucial in foam cell formation. Here, we show that . (), a relative of . , induces foam cell formation, leading to granuloma development following CH25H upregulation. Moreover, the -driven increase in CH25H expression is associated with the presence of phthiocerol dimycocerosate, a determinant for virulence and integrity. CH25H- mice showed decreased foam cell formation and attenuated pathology. Atorvastatin, a recommended first-line lipid-lowering drug, promoted the elimination of and concomitantly reduced CH25H production. These results define a previously unknown role for CH25H in controlling macrophage-derived foam cell formation and Tuberculosis pathology.
PubMed: 38420591
DOI: 10.1016/j.isci.2024.109204 -
Microbiology Resource Announcements Apr 2024, a slow-growing Actinobacterium, typically induces tuberculosis-like disease in fish. Here, we report a new reference sequence for ATCC 927, along with its DNA...
, a slow-growing Actinobacterium, typically induces tuberculosis-like disease in fish. Here, we report a new reference sequence for ATCC 927, along with its DNA methylome. This aims to maximize the research potential of this type strain and facilitates investigations into the pathomechanisms of human tuberculosis.
PubMed: 38415640
DOI: 10.1128/mra.01016-23 -
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 -
PloS One 2024Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly,...
Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-β secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-β levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-β to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-β from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-β response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.
Topics: Humans; Mycobacterium tuberculosis; Mycobacterium marinum; Signal Transduction; Macrophages; DEAD Box Protein 58; Tuberculosis
PubMed: 38394154
DOI: 10.1371/journal.pone.0281564