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1.
FASEB J ; 38(11): e23724, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38837712

ABSTRACT

Mycobacterium tuberculosis, the pathogen of the deadly disease tuberculosis, depends on the redox cofactor mycofactocin (MFT) to adapt to and survive under hypoxic conditions. MftR is a TetR family transcription regulator that binds upstream of the MFT gene cluster and controls MFT synthesis. To elucidate the structural basis underlying MftR regulation, we determined the crystal structure of Mycobacterium tuberculosis MftR (TB-MftR). The structure revealed an interconnected hydrogen bond network in the α1-α2-α3 helices of helix-turn-helix (HTH) DNA-binding domain that is essential for nucleic acid interactions. The ligand-binding domain contains a hydrophobic cavity enclosing long-chain fatty acyl-CoAs like the key regulatory ligand oleoyl-CoA. Despite variations in ligand-binding modes, comparative analyses suggest regulatory mechanisms are largely conserved across TetR family acyl-CoA sensors. By elucidating the intricate structural mechanisms governing DNA and ligand binding by TB-MftR, our study enhances understanding of the regulatory roles of this transcription factor under hypoxic conditions, providing insights that could inform future research into Mycobacterium tuberculosis pathogenesis.


Subject(s)
Bacterial Proteins , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Crystallography, X-Ray , Transcription Factors/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Models, Molecular , Amino Acid Sequence
2.
Microbiology (Reading) ; 170(5)2024 May.
Article in English | MEDLINE | ID: mdl-38717801

ABSTRACT

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.


Subject(s)
Adaptation, Physiological , Mycobacterium tuberculosis , Tuberculosis , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/physiology , Hydrogen-Ion Concentration , Animals , Humans , Tuberculosis/microbiology , Tuberculosis/drug therapy , Macrophages/microbiology , Virulence , Gene Expression Regulation, Bacterial , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Antitubercular Agents/pharmacology
3.
Molecules ; 29(9)2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38731549

ABSTRACT

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.


Subject(s)
Bacterial Proteins , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Protein Biosynthesis , Peptide Elongation Factors/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Ribosomes/metabolism , Models, Molecular , Tuberculosis/drug therapy , Tuberculosis/microbiology , Tuberculosis/metabolism , Protein Conformation
4.
Nat Commun ; 15(1): 4216, 2024 May 17.
Article in English | MEDLINE | ID: mdl-38760394

ABSTRACT

Antimicrobial peptides (AMPs), ancient scavengers of bacteria, are very poorly induced in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis), but the underlying mechanism remains unknown. Here, we report that L-alanine interacts with PRSS1 and unfreezes the inhibitory effect of PRSS1 on the activation of NF-κB pathway to induce the expression of AMPs, but mycobacterial alanine dehydrogenase (Ald) Rv2780 hydrolyzes L-alanine and reduces the level of L-alanine in macrophages, thereby suppressing the expression of AMPs to facilitate survival of mycobacteria. Mechanistically, PRSS1 associates with TAK1 and disruptes the formation of TAK1/TAB1 complex to inhibit TAK1-mediated activation of NF-κB pathway, but interaction of L-alanine with PRSS1, disables PRSS1-mediated impairment on TAK1/TAB1 complex formation, thereby triggering the activation of NF-κB pathway to induce expression of AMPs. Moreover, deletion of antimicrobial peptide gene ß-defensin 4 (Defb4) impairs the virulence by Rv2780 during infection in mice. Both L-alanine and the Rv2780 inhibitor, GWP-042, exhibits excellent inhibitory activity against M. tuberculosis infection in vivo. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses its own alanine dehydrogenase to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.


Subject(s)
Alanine , Antimicrobial Peptides , Macrophages , Mycobacterium tuberculosis , NF-kappa B , Tuberculosis , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/metabolism , Animals , Mice , NF-kappa B/metabolism , Humans , Macrophages/microbiology , Macrophages/metabolism , Macrophages/immunology , Alanine/metabolism , Antimicrobial Peptides/metabolism , Antimicrobial Peptides/genetics , Tuberculosis/microbiology , Tuberculosis/immunology , Alanine Dehydrogenase/metabolism , Alanine Dehydrogenase/genetics , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Kinase Kinases/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Signal Transduction , Mice, Inbred C57BL , RAW 264.7 Cells , Female
5.
Nat Commun ; 15(1): 4065, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38744895

ABSTRACT

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteolysis , Proteolysis/drug effects , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Anti-Bacterial Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Humans , Microbial Sensitivity Tests , Machine Learning
6.
Nat Commun ; 15(1): 4161, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38755122

ABSTRACT

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.


Subject(s)
Bacterial Proteins , Biotin , Mycobacterium tuberculosis , Biotin/metabolism , Biotin/analogs & derivatives , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Sulfurtransferases/metabolism , Sulfurtransferases/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/enzymology , Escherichia coli/metabolism , Escherichia coli/genetics
7.
Sci Rep ; 14(1): 10904, 2024 05 13.
Article in English | MEDLINE | ID: mdl-38740859

ABSTRACT

Tuberculosis (TB), caused by Mycobacterium tuberculosis, ranks among the top causes of global human mortality, as reported by the World Health Organization's 2022 TB report. The prevalence of M. tuberculosis strains that are multiple and extensive-drug resistant represents a significant barrier to TB eradication. Fortunately, having many completely sequenced M. tuberculosis genomes available has made it possible to investigate the species pangenome, conduct a pan-phylogenetic investigation, and find potential new drug targets. The 442 complete genome dataset was used to estimate the pangenome of M. tuberculosis. This study involved phylogenomic classification and in-depth analyses. Sequential filters were applied to the conserved core genome containing 2754 proteins. These filters assessed non-human homology, virulence, essentiality, physiochemical properties, and pathway analysis. Through these intensive filtering approaches, promising broad-spectrum therapeutic targets were identified. These targets were docked with FDA-approved compounds readily available on the ZINC database. Selected highly ranked ligands with inhibitory potential include dihydroergotamine and abiraterone acetate. The effectiveness of the ligands has been supported by molecular dynamics simulation of the ligand-protein complexes, instilling optimism that the identified lead compounds may serve as a robust basis for the development of safe and efficient drugs for TB treatment, subject to further lead optimization and subsequent experimental validation.


Subject(s)
Antitubercular Agents , Drug Design , Mycobacterium tuberculosis , Proteomics , Tuberculosis , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Antitubercular Agents/pharmacology , Humans , Tuberculosis/drug therapy , Tuberculosis/microbiology , Proteomics/methods , Genome, Bacterial , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Phylogeny , Molecular Docking Simulation , Molecular Dynamics Simulation , Genomics/methods
8.
Cell Mol Life Sci ; 81(1): 203, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38698289

ABSTRACT

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.


Subject(s)
Macrophages , Mycobacterium tuberculosis , Transaminases , Mice , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/metabolism , Animals , RAW 264.7 Cells , Virulence , Macrophages/microbiology , Macrophages/immunology , Macrophages/metabolism , Transaminases/metabolism , Transaminases/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Mycobacterium smegmatis/pathogenicity , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/enzymology , Cytokines/metabolism , Toll-Like Receptor 4/metabolism , Humans , Immunity, Innate , Host-Pathogen Interactions/immunology , Tuberculosis/immunology , Tuberculosis/microbiology
9.
PLoS Pathog ; 20(5): e1012214, 2024 May.
Article in English | MEDLINE | ID: mdl-38722857

ABSTRACT

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.


Subject(s)
Bacterial Proteins , Cadherins , Epithelial Cells , Mycobacterium tuberculosis , Serine Proteases , Cadherins/metabolism , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/metabolism , Animals , Humans , Mice , Serine Proteases/metabolism , Serine Proteases/genetics , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , A549 Cells , Tuberculosis/microbiology , Tuberculosis/metabolism , Female
10.
Biochem Biophys Res Commun ; 717: 150040, 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38718566

ABSTRACT

Mtb12, a small protein secreted by Mycobacterium tuberculosis, is known to elicit immune responses in individuals infected with the pathogen. It serves as an antigen recognized by the host's immune system. Due to its immunogenic properties and pivotal role in tuberculosis (TB) pathogenesis, Mtb12 is considered a promising candidate for TB diagnosis and vaccine development. However, the structural and functional properties of Mtb12 are largely unexplored, representing a significant gap in our understanding of M. tuberculosis biology. In this study, we present the first structure of Mtb12, which features a unique tertiary configuration consisting of four beta strands and four alpha helices. Structural analysis reveals that Mtb12 has a surface adorned with a negatively charged pocket adjacent to a central cavity. The features of these structural elements and their potential effects on the function of Mtb12 warrant further exploration. These findings offer valuable insights for vaccine design and the development of diagnostic tools.


Subject(s)
Antigens, Bacterial , Bacterial Proteins , Mycobacterium tuberculosis , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Models, Molecular , Molecular Weight , Amino Acid Sequence , Protein Conformation , Humans
11.
Nat Microbiol ; 9(6): 1607-1618, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38740932

ABSTRACT

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.


Subject(s)
Mycobacterium tuberculosis , Propionates , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Propionates/pharmacology , Propionates/metabolism , Virulence , Lipids/chemistry , Cholesterol Esters/metabolism , Tuberculosis/microbiology , Tuberculosis/prevention & control , Fatty Acids/metabolism , Vitamin B 12/pharmacology , Vitamin B 12/metabolism , Humans , Mutation , Virulence Factors/metabolism , Virulence Factors/genetics , Cholesterol/metabolism , Acyl Coenzyme A
12.
Elife ; 122024 May 28.
Article in English | MEDLINE | ID: mdl-38805257

ABSTRACT

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.


Subject(s)
Antigens, Bacterial , Bacterial Proteins , Macrophages , Mycobacterium tuberculosis , Phagosomes , Single-Domain Antibodies , Antigens, Bacterial/metabolism , Antigens, Bacterial/immunology , Bacterial Proteins/metabolism , Phagosomes/metabolism , Hydrogen-Ion Concentration , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Single-Domain Antibodies/metabolism , Humans , Molecular Dynamics Simulation , Animals
13.
Elife ; 132024 May 13.
Article in English | MEDLINE | ID: mdl-38739431

ABSTRACT

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.


Subject(s)
Bacterial Proteins , Cyclic AMP , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis , Stress, Physiological , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/physiology , Cyclic AMP/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Microbial Viability , Macrophages/microbiology , Macrophages/metabolism
14.
Nat Commun ; 15(1): 3088, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38600064

ABSTRACT

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.


Subject(s)
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genome, Bacterial/genetics , Operon/genetics , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Gene Expression Regulation, Bacterial
15.
Biochem Biophys Res Commun ; 710: 149898, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38598903

ABSTRACT

Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.


Subject(s)
Antitoxins , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolism , Antitoxins/chemistry , Models, Molecular , Transcription Factors/metabolism , DNA/metabolism , Bacterial Proteins/metabolism
16.
Cell Chem Biol ; 31(4): 627-629, 2024 Apr 18.
Article in English | MEDLINE | ID: mdl-38640900

ABSTRACT

In this issue of Cell Chemical Biology, Gries et al.1 employ an innovative screening approach to identify anti-tuberculosis compounds with dual modes of action: anti-virulence against the type VII secretion system ESX-1 and enhanced ethionamide efficacy. These compounds hold promise for developing multi-target tuberculosis drugs with potential clinical applications.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Mycobacterium tuberculosis/metabolism , Bacterial Proteins/metabolism , Tuberculosis/drug therapy , Virulence
17.
Biochem Biophys Res Commun ; 711: 149920, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38615574

ABSTRACT

Tuberculosis (TB), a deadly infectious disease induced by Mycobacterium tuberculosis (Mtb), continues to be a global public health issue that kill millions of patents every year. Despite significant efforts have been paid to identify effective TB treatments, the emergence of drug-resistant strains of the disease and the presence of comorbidities in TB patients urges us to explore the detailed mechanisms involved in TB immunity and develop more effective innovative anti-TB strategies. HIF-1α, a protein involved in regulating cellular immune responses during TB infection, has been highlighted as a promising target for the development of novel strategies for TB treatment due to its critical roles in anti-TB host immunity. This review provides a summary of current research progress on the roles of HIF-1α in TB infection, highlighting its importance in regulating the host immune response upon Mtb infection and summarizing the influences and mechanisms of HIF-1α on anti-TB immunological responses of host cells. This review also discusses the various challenges associated with developing HIF-1α as a target for anti-TB therapies, including ensuring specificity and avoiding off-target effects on normal cell function, determining the regulation and expression of HIF-1α in TB patients, and developing drugs that can inhibit HIF-1α. More deep understanding of the molecular mechanisms involved in HIF-1α signaling, its impact on TB host status, and systematic animal testing and clinical trials may benefit the optimization of HIF-1α as a novel therapeutic target for TB.


Subject(s)
Antitubercular Agents , Hypoxia-Inducible Factor 1, alpha Subunit , Mycobacterium tuberculosis , Signal Transduction , Tuberculosis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/immunology , Signal Transduction/drug effects , Tuberculosis/drug therapy , Tuberculosis/immunology , Tuberculosis/metabolism , Tuberculosis/microbiology , Animals , Antitubercular Agents/therapeutic use , Antitubercular Agents/pharmacology , Molecular Targeted Therapy/methods
18.
mBio ; 15(5): e0255223, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38567992

ABSTRACT

Since the discovery of extracellular vesicles (EVs) in mycobacterial species 15 years back, we have learned that this phenomenon is conserved in the Mycobacterium genus and has critical roles in bacterial physiology and host-pathogen interactions. Mycobacterium tuberculosis (Mtb), the tuberculosis (TB) causative agent, produces EVs both in vitro and in vivo including a diverse set of biomolecules with demonstrated immunomodulatory effects. Moreover, Mtb EVs (MEVs) have been shown to possess vaccine properties and carry biomarkers with diagnostic capacity. Although information on MEV biogenesis relative to other bacterial species is scarce, recent studies have shed light on how MEVs originate and are released to the extracellular space. In this minireview, we discuss past and new information about the vesiculogenesis phenomenon in Mtb, including biogenesis, MEV cargo, aspects in the context of host-pathogen interactions, and applications that could help to develop effective tools to tackle the disease.


Subject(s)
Extracellular Vesicles , Host-Pathogen Interactions , Mycobacterium tuberculosis , Tuberculosis , Extracellular Vesicles/metabolism , Humans , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Tuberculosis/microbiology , Tuberculosis/diagnosis , Animals , Biomarkers , Mycobacterium/genetics , Mycobacterium/metabolism
19.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 55(2): 330-336, 2024 Mar 20.
Article in Chinese | MEDLINE | ID: mdl-38645872

ABSTRACT

Objective: To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb. Methods: The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL. Results: pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of -0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured. Conclusion: We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.


Subject(s)
Bacterial Proteins , Computational Biology , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Computational Biology/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Plasmids/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Cloning, Molecular
20.
Microbiol Res ; 284: 127675, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38636239

ABSTRACT

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), mainly transmitted through droplets to infect the lungs, and seriously affecting patients' health and quality of life. Clinically, anti-TB drugs often entail side effects and lack efficacy against resistant strains. Thus, the exploration and development of novel targeted anti-TB medications are imperative. Currently, protein-protein interactions (PPIs) offer novel avenues for anti-TB drug development, and the study of targeted modulators of PPIs in M. tuberculosis has become a prominent research focus. Furthermore, a comprehensive PPI network has been constructed using computational methods and bioinformatics tools. This network allows for a more in-depth analysis of the structural biology of PPIs and furnishes essential insights for the development of targeted small-molecule modulators. Furthermore, this article provides a detailed overview of the research progress and regulatory mechanisms of PPI modulators in M. tuberculosis, the causative agent of TB. Additionally, it summarizes potential targets for anti-TB drugs and discusses the prospects of existing PPI modulators.


Subject(s)
Antitubercular Agents , Bacterial Proteins , Computational Biology , Mycobacterium tuberculosis , Protein Interaction Maps , Tuberculosis , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Antitubercular Agents/pharmacology , Humans , Tuberculosis/microbiology , Tuberculosis/drug therapy , Computational Biology/methods
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