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Journal of Microbiology (Seoul, Korea) Nov 2022Mycobacterium tuberculosis (M. tuberculosis) is a highly pathogenic intracellular pathogen that causes tuberculosis (TB), the leading cause of mortality from single...
Mycobacterium tuberculosis (M. tuberculosis) is a highly pathogenic intracellular pathogen that causes tuberculosis (TB), the leading cause of mortality from single infections. Redox homeostasis plays a very important role in the resistance of M. tuberculosis to antibiotic damage and various environmental stresses. The antioxidant sulforaphane (SFN) has been reported to exhibit anticancer activity and inhibit the growth of a variety of bacteria and fungi. Nonetheless, it remains unclear whether SFN exhibits anti-mycobacterial activity. Our results showed that the SFN against M. tuberculosis H37Ra exhibited bactericidal activity in a time and dose-dependent manner. The anti-tubercular activity of SFN was significantly correlated with bacterial reactive oxygen species (ROS) levels. In addition, SFN promoted the bactericidal effect of macrophages on intracellular bacteria in a dose-dependent manner, mediated by increasing intracellular mitochondrial ROS levels and decreasing cytoplasmic ROS levels. Taken together, our data revealed the previously unrecognized antimicrobial functions of SFN. Future studies focusing on the mechanism of SFN in macrophages against M. tuberculosis are essential for developing new host-directed therapeutic approaches against TB.
Topics: Humans; Mycobacterium tuberculosis; Reactive Oxygen Species; Mycobacterium smegmatis; Tuberculosis, Lymph Node
PubMed: 36048328
DOI: 10.1007/s12275-022-2284-8 -
Microbiology Spectrum Aug 2022Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality...
Unveiling the Biosynthetic Pathway for Short Mycolic Acids in Nontuberculous Mycobacteria: Mycobacterium smegmatis MSMEG_4301 and Its Ortholog Mycobacterium abscessus MAB_1915 Are Essential for the Synthesis of α'-Mycolic Acids.
Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.
Topics: Biosynthetic Pathways; Fatty Acids; Mycobacterium; Mycobacterium abscessus; Mycobacterium smegmatis; Mycolic Acids; Nontuberculous Mycobacteria
PubMed: 35862962
DOI: 10.1128/spectrum.01288-22 -
Nature Communications Mar 2024The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive...
The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. The phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM) are cell envelope components critical for host-pathogen interactions, but their physiological functions in mycobacteria remained elusive. In this work, using biosynthetic mutants of these lipoglycans, we examine their roles in maintaining cell envelope integrity in Mycobacterium smegmatis and Mycobacterium tuberculosis. We find that mutants defective in producing mature LAM fail to maintain rod cell shape specifically at the new pole and para-septal regions whereas a mutant that produces a larger LAM becomes multi-septated. Therefore, LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell wall integrity and septal placement.
Topics: Lipopolysaccharides; Mycobacterium smegmatis; Cell Wall; Mycobacterium tuberculosis
PubMed: 38467648
DOI: 10.1038/s41467-024-46565-5 -
Microbial Pathogenesis Jan 2023Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is...
Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is critically linked to biofilm formation by NTM which also includes opportunistic pathogens such as Mycobacterium abscessus. Although GPLs have been investigated in many earlier studies, the biosynthesis of GPLs using exogenous fatty acids in M. smegmatis subjected to stresses encountered by mycobacteria during infection of the human body has not been studied. Therefore, we subjected M. smegmatis to different combinations of the three stresses of hypoxia, acidic pH and nutrient starvation and report here that the metabolic incorporation of radiolabeled long-chain fatty acids into alkali-stable GPLs was significantly increased under these stress conditions. Endogenously synthesized fatty acids were not preferred for GPL biosynthesis by M. smegmatis subjected to the triple stress combination. Our observations indicate that GPLs may play important roles in cell surface modifications associated with the non-replicating state of M. smegmatis. Our experimental model reported here would be useful in the further study of GPL biosynthesis from exogenous fatty acid sources in M. smegmatis subjected to hypoxia, nutrient starvation and acidic stress conditions and help in the screening of candidate drugs that target this biochemical pathway in pathogenic NTM.
Topics: Humans; Mycobacterium smegmatis; Fatty Acids; Glycopeptides; Mycobacterium; Nontuberculous Mycobacteria
PubMed: 36502992
DOI: 10.1016/j.micpath.2022.105943 -
MSphere Oct 2023Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal...
Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine survival in the presence of the chelator sodium citrate. We observed that instead of restricting growth, concentrated sodium citrate killed . RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, , , and we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen . Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.
Topics: Mycobacterium smegmatis; Sodium Citrate; Osmotic Pressure; Mycobacterium tuberculosis; Homeostasis; Cations; Chelating Agents
PubMed: 37681985
DOI: 10.1128/msphere.00358-23 -
Molecular Informatics Mar 2022Tuberculosis has been the serious disease threatening human health and public safety due to the emergence of MDR and XDR-TB. Mycobacterium tuberculosis peptide...
Tuberculosis has been the serious disease threatening human health and public safety due to the emergence of MDR and XDR-TB. Mycobacterium tuberculosis peptide deformylase (MtPDF) is a valuable target for antituberculotics. In order to discover new potential inhibitor candidates of MtPDF as leads for antituberculotics, Discovery Studio (DS) 2019 was used to perform molecular docking for virtual screening in silico with the bioactive compound library-I (L1700) against MtPDF. Six compounds with high docking scores and favourable ligand-protein interactions by LibDock and CDOCKER were selected for the evaluation of the inhibition potencies against MtPDF and Mycobacterium smegmatis. GST-6×His tagged MtPDF was recombinant expressed and purified firstly by Glutathione Sepharose 4B, and secondly by Ni Sepharose 6 FF after the cleavage of human rhinovirus 3C protease. These compounds showed IC values from 0.5 μmol/L to 112 μmol/L against MtPDF, among which CUDC-101 bearing hydroxamic acid exhibited IC of 0.5 μmol/L on MtPDF and MIC against Mycobacterium smegmatis of 32 μg/mL, and Ixazomib Citrate with IC of 63 μmol/L and MIC of 16 μg/mL. CUDC-101 and Ixazomib Citrate are promising as the potential leads for antituberculotics.
Topics: Amidohydrolases; Antitubercular Agents; Citrates; Humans; Molecular Docking Simulation; Mycobacterium tuberculosis
PubMed: 34708566
DOI: 10.1002/minf.202100002 -
Microbiology Spectrum Oct 2021tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a...
tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and -chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis . Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.
Topics: Chloramines; Chlorine; DNA Replication; DnaB Helicases; Gene Expression Regulation, Bacterial; Hypochlorous Acid; Inteins; Mycobacterium leprae; Mycobacterium smegmatis; Oxidants; Oxidation-Reduction; Protein Splicing; Reactive Oxygen Species; Taurine
PubMed: 34549994
DOI: 10.1128/Spectrum.00301-21 -
Frontiers in Microbiology 2021is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to...
is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of in the period of infection. However, the mechanism by which utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. is fast-growing and generally used to study its pathogenic counterpart, . In this work, we found that the phosphate sensing regulator RegX3 of is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of and related genes () in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that RegX3 also binds to the operon , the RegX3-mediated regulation might be general in and other mycobacteria for nutrient sensing and environmental adaptation.
PubMed: 33603724
DOI: 10.3389/fmicb.2021.619387 -
Applied and Environmental Microbiology Feb 2024The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in...
The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, . We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with . Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate operon expression. In addition, sensitivity to formaldehyde in -knockout could be alleviated by overexpression of . Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in , the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from , plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in . Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for eradication.
Topics: Humans; Mycobacterium smegmatis; Sigma Factor; Mycobacterium tuberculosis; Formaldehyde; beta-Lactamases; Tuberculosis; Formates; Bacterial Proteins; Cysteine; Glycopeptides; Inositol
PubMed: 38259108
DOI: 10.1128/aem.02039-23 -
Journal of Theoretical Biology Jan 2022Mycobacterium tuberculosis can exist within a host for lengthy periods, tolerating even antibiotic challenge. This non-heritable, antibiotic tolerant "persister" state,...
Mycobacterium tuberculosis can exist within a host for lengthy periods, tolerating even antibiotic challenge. This non-heritable, antibiotic tolerant "persister" state, is thought to underlie latent Tuberculosis (TB) infection and a deeper understanding thereof could inform treatment strategies. In addition to experimental studies, mathematical and computational modelling approaches are widely employed to study persistence from both an in vivo and in vitro perspective. However, specialized models (partial differential equations, agent-based, multiscale, etc.) rely on several difficult to determine parameters. In this study, a dynamic mathematical model was developed to predict the response of Mycobacterium smegmatis (a model organism for M. tuberculosis) grown in batch culture and subjected to a range of in vitro environmental stresses. Lag phase dynamics, pH variations and internal nitrogen storage were mechanistically modelled. Experimental results were used to train model parameters using global optimization, with extensive subsequent model validation to ensure extensibility to more complex modelling frameworks. This included an identifiability analysis which indicated that seven of the thirteen model parameters were uniquely identifiable. Non-identifiable parameters were critically evaluated. Model predictions compared to validation data (based on experimental results not used during training) were accurate with less than 16% maximum absolute percentage error, indicating that the model is accurate even when extrapolating to new experimental conditions. The bulk growth model can be extended to spatially heterogeneous simulations such as an agent-based model to simulate in vitro granuloma models or, eventually, in vivo conditions, where distributed environmental conditions are difficult to measure.
Topics: Hydrogen-Ion Concentration; Models, Theoretical; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nutrients
PubMed: 34582827
DOI: 10.1016/j.jtbi.2021.110921