RESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH3-THF), which is required for the de novo biosynthesis of methionine in bacteria. Here, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein is essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis and found that these mutants were more sensitive to the antifolates para-aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research is the first to identify an MTHFR in M. tuberculosis and reveals that the activity of this enzyme is associated with susceptibility to antifolates. These findings have particular value for antitubercular drug design for the treatment of drug-resistant TB.
Assuntos
Ácido Aminossalicílico , Mycobacterium tuberculosis , Ácido Aminossalicílico/metabolismo , Ácido Aminossalicílico/farmacologia , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) manipulates multiple host defence pathways to survive and persist in host cells. Understanding Mtb-host cell interaction is crucial to develop an efficient means to control the disease. Here, we applied the Mtb proteome chip, through separately interacting with H37Ra and H37Rv stimulated macrophage lysates, screened 283 Mtb differential proteins. Through primary screening, we focused on fatty acylCoA synthetase FadD13. Mtb FadD13 is a potential drug target, but its role in infection remains unclear. Deletion of FadD13 in Mtb reduced the production of proinflammatory cytokines IL-1ß, IL-18, and IL-6. Bimolecular fluorescence complementation and colocalization showed that the binding partner of FadD13 in macrophage was eEF1A1 (a translation elongation factor). Knockdown eEF1A1 expression in macrophage abrogated the promotion of proinflammatory cytokines induced by FadD13. In addition, ΔfadD13 mutant decreased the expression of the NF-κB signalling pathway related proteins p50 and p65, so did the eEF1A1 knockdown macrophage infected with H37Rv. Meanwhile, we found that deletion of FadD13 reduced Mtb survival in macrophages during Mtb infection, and purified FadD13 proteins induced broken of macrophage membrane. Taken together, FadD13 is crucial for Mtb proliferation in macrophages, and it plays a key role in the production of proinflammatory cytokines during Mtb infection.
Assuntos
Coenzima A Ligases/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Inflamação/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Proteínas de Bactérias/genética , Parede Celular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteoma/genética , Proteômica , Transdução de SinaisRESUMO
Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Proteômica/métodos , Proteínas Ribossômicas/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Grupo dos Citocromos b/química , Ferritinas/química , Células HEK293 , Humanos , Imunidade Inata , Macrófagos/citologia , Macrófagos/metabolismo , Espectrometria de Massas , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , NF-kappa B/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Proteínas Ribossômicas/química , Células THP-1RESUMO
Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.
Assuntos
Mycobacterium tuberculosis/metabolismo , Proteoma/análise , Proteínas de Bactérias/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.
Assuntos
Ácido 4-Aminobenzoico/metabolismo , Ácido Aminossalicílico/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Fator sigma/genética , Sulfametoxazol/farmacologia , Ácido Fólico/metabolismo , Deleção de Genes , Liases/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimentoRESUMO
Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.
Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Pirofosfatases/genética , Salmonella enterica/efeitos dos fármacos , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Testes de Sensibilidade Microbiana , Neopterina/análogos & derivados , Neopterina/farmacologia , Pterinas/farmacologia , Pirofosfatases/antagonistas & inibidores , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.
Assuntos
Lisina/metabolismo , Metaboloma , Mycobacterium tuberculosis/metabolismo , Succinatos/metabolismo , Acetato-CoA Ligase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Western Blotting , Carbono/farmacologia , Sequência Conservada , Humanos , Immunoblotting , Imunoprecipitação , Metaboloma/efeitos dos fármacos , Metabolômica , Simulação de Dinâmica Molecular , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , NAD/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
NÉ-lysine acetylation is one of the most abundant post-translational modifications in eukaryote and prokaryote. Protein acetylome of Escherichia coli has been screened using mass spectrometry (MS) technology, and many acetylated proteins have been identified, including the pyridoxine 5'-phosphate oxidase (EcPNPOx), but the biological roles played by lysine acetylation in EcPNPOx still remain unknown. In this study, EcPNPOx was firstly overexpressed and purified, and two acetylated lysine residues were identified by the subsequent liquid chromatography-tandem mass spectrometry analysis. Site-directed mutagenesis analysis demonstrated that acetylated lysine residues play important roles in the enzymatic activity and enzymatic properties of the protein. EcPNPOx could be non-enzymatically acetylated by acetyl-phosphate and deacetylated by CobB in vitro. Furthermore, enzymatic activities of acetylated and deacetylated EcPNPOx were compared in vitro, and results showed that acetylation led to a decrease of its enzymatic activity, which could be rescued by CobB deacetylation. Taken together, our data suggest that CobB modulates the enzymatic activity of EcPNPOx in vitro.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Piridoxaminafosfato Oxidase/metabolismo , Acetilação , Sequência de Aminoácidos , Western Blotting , Cromatografia Líquida , Ensaios Enzimáticos/métodos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Concentração de Íons de Hidrogênio , Lisina/genética , Mutagênese Sítio-Dirigida , Piridoxaminafosfato Oxidase/genética , Homologia de Sequência de Aminoácidos , Sirtuínas/metabolismo , Espectrometria de Massas em Tandem , TemperaturaRESUMO
Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) is able to acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) techniques. The antituberculosis activity of the acetylated PAS is significantly reduced. As a result, overexpression of TBNAT in M. tuberculosis results in PAS resistance, as determined by MIC tests and drug exposure experiments. Taken together, our results suggest that TBNAT from M. tuberculosis is able to inactivate PAS by acetylating the compound.
Assuntos
Ácido Aminossalicílico/metabolismo , Antituberculosos/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Acetilação , Ácido Aminossalicílico/química , Ácido Aminossalicílico/isolamento & purificação , Ácido Aminossalicílico/farmacologia , Antituberculosos/química , Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Arilamina N-Acetiltransferase/genética , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Expressão Gênica , Inativação Metabólica , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Acetilação , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico/genética , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Gluconeogênese/genética , Glicólise/genética , Histona Desacetilases do Grupo III/deficiência , Histona Desacetilases do Grupo III/genética , Anotação de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mapeamento de Interação de Proteínas , Proteoma/genéticaRESUMO
Imaging of protein-protein and RNA-protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the 'tissue optical window' for imaging of protein-protein and RNA-protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3'long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA-protein and protein-protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.
Assuntos
Fluorometria/métodos , Mapeamento de Interação de Proteínas/métodos , RNA Mensageiro/análise , Proteínas de Ligação a RNA/análise , Animais , Linhagem Celular , HIV/genética , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análise , RNA Viral/análiseRESUMO
Lysine acetylation is one of the most abundant post-translational modifications. However, physiological roles of this modification in bacteria are largely unknown. Previous protein acetylome analysis showed that Escherichia coli adenosylmethionine synthase (MAT) undergoes acetylation in vivo, but the biological functions of this modification still need to be uncovered. In this study, MAT of E. coli was over-expressed and purified. Subsequent mass spectrometry analysis showed that 12 lysine residues of the protein were acetylated. Site-directed mutagenesis analysis was performed and the results showed that acetylated lysine residues play important roles in the enzymatic activity of MAT. Next, deacetylation assay was performed by using CobB as the deacetylase, and the results showed that CobB could deacetylate MAT in vitro In addition, the enzymatic activities of acetylated and deacetylated MAT were compared in vitro, and results showed that acetylation led to a decrease in its enzymatic activity, which could be reversed by CobB deacetylation. Altogether, our data suggest that CobB modulates the enzymatic activity of E. coli MAT in vitro.
Assuntos
Escherichia coli/enzimologia , Lisina/metabolismo , Metionina Adenosiltransferase/metabolismo , Acetilação , Metionina Adenosiltransferase/isolamento & purificaçãoRESUMO
DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro, implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro. Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Fluoroquinolonas/farmacologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Proteínas de Bactérias/fisiologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Proteínas Monoméricas de Ligação ao GTP/fisiologiaRESUMO
The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.
Assuntos
Ácido Aminossalicílico/farmacologia , Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/fisiologia , Alelos , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismoRESUMO
Bacteria like Escherichia coli can use propionate as sole carbon and energy source. All pathways for degradation of propionate start with propionyl-CoA. However, pathways of propionyl-CoA synthesis from propionate and their regulation mechanisms have not been carefully examined in E. coli. In this study, roles of the acetyl-CoA synthetase encoding gene acs and the NAD(+)-dependent protein deacetylase encoding gene cobB on propionate utilization in E. coli were investigated. Results from biochemical analysis showed that, reversible acetylation also modulates the propionyl-CoA synthetase activity of Acs. Subsequent genetic analysis revealed that, deletion of acs in E. coli results in blockage of propionate utilization, suggesting that acs is essential for propionate utilization in E. coli. Besides, deletion of cobB in E. coli also results in growth defect, but only under lower concentrations of propionate (5mM and 10mM propionate), suggesting the existence of other propionyl-CoA synthesis pathways. In combination with previous observations, our data implies that, for propionate utilization in E. coli, a primary amount of propionyl-CoA seems to be required, which is synthesized by Acs.
Assuntos
Acetato-CoA Ligase/metabolismo , Coenzima A Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Propionatos/metabolismo , Acetato-CoA Ligase/genética , Acetilação , Coenzima A Ligases/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
For living deep-tissue imaging, the optical window favorable for light penetration is in near-infrared wavelengths, which requires fluorescent proteins with emission spectra in the near-infrared region. Here, we report that a single mutant Ser28His of mNeptune with a near-infrared (≥650 nm) emission maxima of 652 nm is found to improve the brightness, photostability, and pH stability when compared with its parental protein mNeptune, while it remains as a monomer, demonstrating that there is still plenty of room to improve the performance of the existing near infrared fluorescence proteins by directed evolution.
Assuntos
Concentração de Íons de Hidrogênio , Proteínas Luminescentes/química , Mutação , Teoria Quântica , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Sequência de Bases , Cromatografia em Gel , Primers do DNA , Dados de Sequência MolecularRESUMO
As one of the factors affecting the treatment outcomes, drug tolerance in mycobacteriosis has not been paid due attention. Genome-wide association studies on 607 Mycobacterium tuberculosis clinical isolates with phenotypic drug susceptibility test data revealed that a K114N mutation on the rv2820c gene was highly enriched in capreomycin-resistant isolates (32/213, 15.02%). However, the mutation was also observed in capreomycin-sensitive isolates (10/394, 2.53%). In most cases (31/42, 73.81%), the rv2820c K114N mutation occurred in isolates with the known capreomycin resistance conferring mutation rrs A1401G. In contrast, the general frequency of the rv2820c K114N mutation was low in 7061 genomes downloaded from the National Center for Biotechnology Information database. To determine the impact of this mutation on the antimycobacterial activity of capreomycin, the intact rv2820c gene and the rv2820c K114N mutant were over-expressed in Mycobacterium smegmatis (Ms), and the results of susceptibility tests showed that the rv2820c K114N mutation did not affect the minimum inhibition concentration (MIC) of capreomycin. Subsequently, the data of time-kill assays showed that, it took only 2 h of capreomycin treatment (40 µg/ml, 5 × MIC) to kill 99.9% bacterial cells of Ms MC2155 pMV261::rv2820cH37Rv, while it took 6 h to achieve that for Ms MC2155 pMV261::rv2820cK114N. Taken together, these data suggested that the rv2820c K114N mutation is related with capreomycin tolerance, which merits further investigation.
RESUMO
Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.
Assuntos
Ácido Aminossalicílico , Antituberculosos , Proteínas de Bactérias , Farmacorresistência Bacteriana , Mutação , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Ácido Aminossalicílico/farmacologia , Humanos , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , NAD/metabolismo , Tuberculose/microbiologiaRESUMO
In a previous study, we presented the dimer structure of DNA gyrase B' domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a 'sluice-like' model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B' protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA-GyrB-DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.