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1.
PLoS One ; 19(6): e0304876, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38848336

RESUMO

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.


Assuntos
Proteína de Transporte de Acila , Proteínas de Bactérias , Mycobacterium tuberculosis , Animais , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Camundongos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/genética , Macrófagos/microbiologia , Macrófagos/metabolismo , Virulência , Regulação Bacteriana da Expressão Gênica , Tuberculose/microbiologia , Lipídeos/química
2.
FASEB J ; 38(11): e23724, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38837712

RESUMO

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.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Cristalografia por Raios X , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Modelos Moleculares , Sequência de Aminoácidos
3.
Nat Commun ; 15(1): 4161, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755122

RESUMO

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.


Assuntos
Proteínas de Bactérias , Biotina , Mycobacterium tuberculosis , Biotina/metabolismo , Biotina/análogos & derivados , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sulfurtransferases/metabolismo , Sulfurtransferases/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/enzimologia , Escherichia coli/metabolismo , Escherichia coli/genética
4.
Nat Commun ; 15(1): 4065, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38744895

RESUMO

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.


Assuntos
Antibacterianos , Proteínas de Bactérias , Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteólise , Proteólise/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Aprendizado de Máquina
5.
Molecules ; 29(9)2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38731549

RESUMO

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.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Biossíntese de Proteínas , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Antituberculosos/farmacologia , Antituberculosos/química , Ribossomos/metabolismo , Modelos Moleculares , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Tuberculose/metabolismo , Conformação Proteica
6.
Nat Commun ; 15(1): 4216, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760394

RESUMO

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.


Assuntos
Alanina , Peptídeos Antimicrobianos , Macrófagos , Mycobacterium tuberculosis , NF-kappa B , Tuberculose , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/metabolismo , Animais , Camundongos , NF-kappa B/metabolismo , Humanos , Macrófagos/microbiologia , Macrófagos/metabolismo , Macrófagos/imunologia , Alanina/metabolismo , Peptídeos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/genética , Tuberculose/microbiologia , Tuberculose/imunologia , Alanina Desidrogenase/metabolismo , Alanina Desidrogenase/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transdução de Sinais , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Feminino
7.
PLoS Pathog ; 20(5): e1012214, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38722857

RESUMO

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.


Assuntos
Proteínas de Bactérias , Caderinas , Células Epiteliais , Mycobacterium tuberculosis , Serina Proteases , Caderinas/metabolismo , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/metabolismo , Animais , Humanos , Camundongos , Serina Proteases/metabolismo , Serina Proteases/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Células A549 , Tuberculose/microbiologia , Tuberculose/metabolismo , Feminino
8.
Sci Rep ; 14(1): 10904, 2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740859

RESUMO

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.


Assuntos
Antituberculosos , Desenho de Fármacos , Mycobacterium tuberculosis , Proteômica , Tuberculose , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Antituberculosos/farmacologia , Humanos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Proteômica/métodos , Genoma Bacteriano , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Filogenia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Genômica/métodos
9.
Elife ; 132024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38739431

RESUMO

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.


Assuntos
Proteínas de Bactérias , AMP Cíclico , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , Estresse Fisiológico , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , AMP Cíclico/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Viabilidade Microbiana , Macrófagos/microbiologia , Macrófagos/metabolismo
10.
J Inorg Biochem ; 257: 112576, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38761578

RESUMO

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1.0 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1 s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1 s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Oxigênio , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Oxigênio/metabolismo , Oxigênio/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Adaptação Fisiológica , Protamina Quinase/metabolismo , Protamina Quinase/química , Cinética , Proteínas Quinases/metabolismo , Proteínas Quinases/química
11.
Cell Mol Life Sci ; 81(1): 203, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698289

RESUMO

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.


Assuntos
Macrófagos , Mycobacterium tuberculosis , Transaminases , Camundongos , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Animais , Células RAW 264.7 , Virulência , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Transaminases/metabolismo , Transaminases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/enzimologia , Citocinas/metabolismo , Receptor 4 Toll-Like/metabolismo , Humanos , Imunidade Inata , Interações Hospedeiro-Patógeno/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia
12.
Nat Microbiol ; 9(6): 1607-1618, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38740932

RESUMO

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.


Assuntos
Mycobacterium tuberculosis , Propionatos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Propionatos/farmacologia , Propionatos/metabolismo , Virulência , Lipídeos/química , Ésteres do Colesterol/metabolismo , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Ácidos Graxos/metabolismo , Vitamina B 12/farmacologia , Vitamina B 12/metabolismo , Humanos , Mutação , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Colesterol/metabolismo , Acil Coenzima A
13.
Biochem Biophys Res Commun ; 717: 150040, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38718566

RESUMO

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.


Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Peso Molecular , Sequência de Aminoácidos , Conformação Proteica , Humanos
14.
Elife ; 122024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38805257

RESUMO

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.


Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Macrófagos , Mycobacterium tuberculosis , Fagossomos , Anticorpos de Domínio Único , Humanos , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Anticorpos de Domínio Único/metabolismo
15.
Microbiology (Reading) ; 170(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38717801

RESUMO

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.


Assuntos
Adaptação Fisiológica , Mycobacterium tuberculosis , Tuberculose , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/fisiologia , Concentração de Íons de Hidrogênio , Animais , Humanos , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico , Macrófagos/microbiologia , Virulência , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Antituberculosos/farmacologia
16.
Nat Commun ; 15(1): 3088, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600064

RESUMO

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.


Assuntos
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano/genética , Óperon/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Regulação Bacteriana da Expressão Gênica
17.
Biochem Biophys Res Commun ; 710: 149898, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38598903

RESUMO

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.


Assuntos
Antitoxinas , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Antitoxinas/química , Modelos Moleculares , Fatores de Transcrição/metabolismo , DNA/metabolismo , Proteínas de Bactérias/metabolismo
18.
Cell Chem Biol ; 31(4): 627-629, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38640900

RESUMO

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.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Tuberculose/tratamento farmacológico , Virulência
19.
mSphere ; 9(4): e0006124, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38564709

RESUMO

Mycobacterium tuberculosis (Mtb), 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 melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm 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 whiB3, a universal stress sensor. The absence of melH 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 melH 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 melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH'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 melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.


Assuntos
Proteínas de Bactérias , Cisteína , Metabolismo Energético , Glicopeptídeos , Homeostase , Mycobacterium tuberculosis , Oxirredução , Estresse Oxidativo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antituberculosos/farmacologia , Ergotioneína/metabolismo , Inositol/metabolismo , Mycobacterium marinum/efeitos dos fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Deleção de Genes
20.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 55(2): 330-336, 2024 Mar 20.
Artigo em Chinês | MEDLINE | ID: mdl-38645872

RESUMO

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.


Assuntos
Proteínas de Bactérias , Biologia Computacional , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Biologia Computacional/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Clonagem Molecular
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