RESUMO
The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.
Assuntos
Proteínas de Bactérias/metabolismo , Fatores Corda/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Decanoatos/metabolismo , Escherichia coli , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/ultraestrutura , Trealose/metabolismoRESUMO
Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.
Assuntos
Proteínas de Bactérias/ultraestrutura , Mycobacterium smegmatis/ultraestrutura , Peroxidases/ultraestrutura , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/patogenicidade , Organelas/metabolismo , Organelas/fisiologia , Peroxidases/metabolismoRESUMO
An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection. It is mainly useful for the investigation of various microorganisms in the sort of Mycobacteria in cell culture laboratories. Some Mycobacterium species groups that is normal, unsafe ailments, likely to Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis. At present, various laboratories are clean and culture this type of species to make an opinion that fascinating route of harmful Mycobacteria. This publication provides aggregate data on cell shape, genome studies, ecology, pathology and utilization of M. smegmatis.
Assuntos
Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Lipossomos/metabolismo , Modelos Biológicos , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/ultraestruturaRESUMO
The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.
Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/química , Transporte Proteico/genética , Sistemas de Secreção Tipo VII/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Cromossomos/química , Cromossomos/genética , Epitopos/química , Epitopos/genética , Mycobacterium smegmatis/ultraestrutura , Óperon/genética , Sistemas de Secreção Tipo VII/genética , Sistemas de Secreção Tipo VII/ultraestruturaRESUMO
Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems1. A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines2, and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.
Assuntos
Microscopia Crioeletrônica , Mycobacterium smegmatis/química , Sistemas de Secreção Tipo VII/química , Sistemas de Secreção Tipo VII/ultraestrutura , Actinobacteria/química , Actinobacteria/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/ultraestrutura , Domínios Proteicos , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Relação Estrutura-Atividade , Thermomonospora , Sistemas de Secreção Tipo VII/isolamento & purificaçãoRESUMO
Mycobacterium tuberculosis, a causative pathogen of tuberculosis (TB), still threatens human health worldwide. To find a novel drug to eradicate this pathogen, we tested taurine-5- bromosalicylaldehyde Schiff base (TBSSB) as an innovative anti-mycobacterial drug using Mycobacterium smegmatis as a surrogate model for M. tuberculosis. We investigated the antimicrobial activity of TBSSB against M. smegmatis by plotting growth curves, examined the effect of TBSSB on biofilm formation, observed morphological changes by scanning electron microscopy and transmission electron microscopy, and detected differentially expressed proteins using two-dimensional gel electrophoresis coupled with mass spectrometry. TBSSB inhibited mycobacterial growth and biofilm formation, altered cell ultrastructure and intracellular content, and inhibited cell division. Furthermore, M. smegmatis adapted itself to TBSSB inhibition by regulating the metabolic pathways and enzymatic activities of the identified proteins. NDMA-dependent methanol dehydrogenase, NAD(P)H nitroreductase, and amidohydrolase AmiB1 appear to be pivotal factors to regulate the M. smegmatis survival under TBSSB. Our dataset reinforced the idea that Schiff base-taurine compounds have the potential to be developed as novel anti-mycobacterial drugs.
Assuntos
Aldeídos/farmacologia , Antibacterianos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Proteômica , Bases de Schiff/farmacologia , Taurina/análogos & derivados , Proteínas de Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis , Taurina/farmacologiaRESUMO
Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracycline-responsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.
Assuntos
Carboxipeptidases/genética , Genes Bacterianos , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Carboxipeptidases/metabolismo , Biologia Computacional , Deleção de Genes , Família Multigênica , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Óperon/genética , Peptidoglicano/metabolismo , Regiões Promotoras Genéticas/genética , Homologia de Sequência do Ácido NucleicoRESUMO
D-Alanyl-D-alanine ligase A (DdlA) catalyses the dimerization of two D-alanines yielding D-alanyl-D-alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti-DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N-Nitrosodimethylamine (NDMA)-dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.
Assuntos
Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Peptídeo Sintases/deficiência , Proteoma/análise , Eletroforese em Gel Bidimensional , Técnicas de Silenciamento de Genes , Espectrometria de Massas , Microscopia Eletroquímica de Varredura , Microscopia Eletrônica de Transmissão , Mycobacterium smegmatis/química , Mycobacterium smegmatis/ultraestrutura , Peptídeo Sintases/metabolismo , VirulênciaRESUMO
The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two-gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon's function. Interestingly, influx of the fluorescent dyes BCECF-AM and calcein-AM in de-energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana Transportadoras/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Óperon , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Deleção de Genes , Lactococcus lactis , Lipopolissacarídeos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Mutação , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Permeabilidade , Estrutura Terciária de Proteína , Rifampina/farmacologia , VirulênciaAssuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestrutura , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Estrutura Secundária de ProteínaRESUMO
Trehalose analogues bearing fluorescent and click chemistry tags have been developed as probes of bacterial trehalose metabolism, but these tools have limitations with respect to in vivo imaging applications. Here, we report the radiosynthesis of the 18F-modified trehalose analogue 2-deoxy-2-[18F]fluoro-d-trehalose ([18F]-2-FDTre), which in principle can be used in conjunction with positron emission tomography (PET) imaging to allow in vivo imaging of trehalose metabolism in various contexts. A chemoenzymatic method employing the thermophilic TreT enzyme from Thermoproteus tenax was used to rapidly (15-20â¯min), efficiently (70% radiochemical yield; ≥ 95% radiochemical purity), and reproducibly convert the commercially available radiotracer 2-deoxy-2-[18F]fluoro-d-glucose ([18F]-2-FDG) into the target radioprobe [18F]-2-FDTre in a single step; both manual and automated syntheses were performed with similar results. Cellular uptake experiments showed that radiosynthetic [18F]-2-FDTre was metabolized by Mycobacterium smegmatis but not by various mammalian cell lines, pointing to the potential future use of this radioprobe for selective PET imaging of infections caused by trehalose-metabolizing bacterial pathogens such as M. tuberculosis.
Assuntos
Radioisótopos de Flúor/química , Mycobacterium smegmatis/ultraestrutura , Trealose/análogos & derivados , Trealose/análise , Linhagem Celular , Química Click , Células HT29 , Humanos , Estrutura Molecular , Mycobacterium smegmatis/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Thermoproteus/enzimologia , Trealose/química , Trealose/metabolismoRESUMO
Ribosomes are the dynamic protein synthesis machineries of the cell. They may exist in different functional states in the cell. Therefore, it is essential to have structural information on these different functional states of ribosomes to understand their mechanism of action. Here, we present single particle cryo-EM reconstructions of the Mycobacterium smegmatis 70S ribosomes in the hibernating state (with HPF), trans-translating state (with tmRNA), and the P/P state (with P-tRNA) resolved to 4.1, 12.5, and 3.4 Å, respectively. A comparison of the P/P state with the hibernating state provides possible functional insights about the Mycobacteria-specific helix H54a rRNA segment. Interestingly, densities for all the four OB domains of bS1 protein is visible in the hibernating 70S ribosome displaying the molecular details of bS1-70S interactions. Our structural data shows a Mycobacteria-specific H54a-bS1 interaction which seems to prevent subunit dissociation and degradation during hibernation without the formation of 100S dimer. This indicates a new role of bS1 protein in 70S protection during hibernation in Mycobacteria in addition to its conserved function during translation initiation.
Assuntos
Mycobacterium smegmatis/ultraestrutura , RNA Bacteriano/química , RNA Ribossômico/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Modelos Moleculares , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Time-resolved atomic force microscopy (AFM) offers countless new modes by which to study bacterial cell physiology on relevant time scales, from mere milliseconds to hours and days on end. In addition, time-lapse AFM acts as a complementary tool to optical fluorescence microscopy (OFM), for which the combination offers a correlative link between the physical manifestation of bacterial phenotypes and molecular mechanisms obeying those principles. Herein we describe the essential materials and methods necessary for conducting time-resolved AFM and dual AFM/OFM experiments on bacteria.
Assuntos
Membrana Celular/ultraestrutura , Microscopia de Força Atômica/métodos , Mycobacterium smegmatis/ultraestrutura , Imagem com Lapso de Tempo/métodos , Ciclo Celular , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Dimetilpolisiloxanos/química , Microscopia de Fluorescência , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Polilisina/farmacologiaRESUMO
The ribosome carries out the synthesis of proteins in every living cell. It consequently represents a frontline target in anti-microbial therapy. Tuberculosis ranks among the leading causes of death worldwide, due in large part to the combination of difficult-to-treat latency and antibiotic resistance. Here, we present the 3.3-Å cryo-EM structure of the 70S ribosome of Mycobacterium smegmatis, a close relative to the human pathogen Mycobacterium tuberculosis. The structure reveals two additional ribosomal proteins and localizes them to the vicinity of drug-target sites in both the catalytic center and the decoding site of the ribosome. Furthermore, we visualized actinobacterium-specific rRNA and protein expansions that extensively remodel the ribosomal surface with implications for polysome organization. Our results provide a foundation for understanding the idiosyncrasies of mycobacterial translation and reveal atomic details of the structure that will facilitate the design of anti-tubercular therapeutics.
Assuntos
Mycobacterium smegmatis/química , Subunidades Ribossômicas Maiores de Bactérias/química , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Mycobacterium smegmatis/ultraestrutura , Domínios Proteicos , RNA Ribossômico/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Bactérias/ultraestruturaRESUMO
AIMS: To construct a conditional N-acetylglucosamine-1-phosphate transferase (WecA) knockdown strain of Mycobacterium smegmatis and to investigate the biological effect of WecA on mycobacterial growth, morphology and susceptibilities against anti-tuberculosis drugs. METHODS AND RESULTS: Mycobacterium smegmatis wecA knockdown strain was constructed by using a tetracycline-inducible expression vector pMind and the expression of WecA was regulated by antisense RNA. The results of growth curves and the colony formation unit curves showed that the growth rate of WecA down-regulation strain was decreased and the amount of live bacterial cells dropped. In addition, the wecA knockdown strain exhibited dramatically morphological alterations through scanning electron microscopy observation. The susceptibility of WecA low-expression strain to anti-tuberculosis drugs was detected by using a rapid resazurin microtitre assay as well as a traditional agar dilution method. Notably, the wecA knockdown strain was more sensitive to rifampin, compared with the wecA normal-expression strain. In addition, the sensitivity of wild type Myco. smegmatis mc(2) 155 strain against rifampin was also enhanced in the presence of a low concentration of tunicamycin, a natural WecA inhibitor. CONCLUSIONS: Down-regulation of WecA enhanced the sensitivity of Myco. smegmatis against rifampin. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provided a possibility of combined application of rifampin together with tunicamycin or other WecA inhibitors, which could be a new approach for the treatment of tuberculosis.
Assuntos
Antibióticos Antituberculose/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Rifampina/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Hipersensibilidade , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/genética , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
BACKGROUND: Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. OBJECTIVE: To investigate the antimycobacterial effect of mature bovine neutrophil ß-defensin (mBNBD) 4 against Mycobacterium spp. infection for the first time. DESIGN: mBNBD4 protein was expressed in Pichia pastoris GS115. We used immunofluorescent assay to detect whether the recombinant mBNBD4 had entered the macrophages. The antimycobacterial activity of mBNBD4 was tested through colony-forming unit (cfu) assay. Morphological changes in the cell wall of M. bovis treated with mBNBD4 were observed by scanning electron microscope. RESULTS: mBNBD4 was expressed and successfully purified from P. pastoris with intact antimicrobial activity. The recombinant protein was able to enter Raw 264.7 macrophages and exhibited potent in vitro bactericidal activity against M. smegmatis and M. bovis. The cell wall of M. bovis was disrupted after interaction with mBNBD4. Exogenous addition of mBNBD4 to both Raw 264.7 and THP-1 derived macrophages reduced the intracellular survival of M. bovis and M. tuberculosis relative to control cells. CONCLUSION: Our data show that mBNBD4 plays an important role in inhibiting mycobacterial growth and in controlling intracellular survival of mycobacteria. mBNBD4 could therefore an effective antimycobacterial molecule in combination with other measures.
Assuntos
Antibacterianos/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , beta-Defensinas/farmacologia , Animais , Bovinos , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/ultraestrutura , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Células RAW 264.7 , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0µg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death.
Assuntos
Antituberculosos/farmacologia , DNA Bacteriano/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Streptomyces/química , Antituberculosos/química , Antituberculosos/isolamento & purificação , Organismos Aquáticos , Técnicas de Cultura de Células , Cosmídeos/química , Cosmídeos/metabolismo , DNA Bacteriano/química , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestrutura , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Quinolonas/química , Quinolonas/isolamento & purificação , Quinolonas/farmacologia , Streptomyces/metabolismoRESUMO
BACKGROUND: Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. RESULTS: In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 µMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. CONCLUSION: We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis.
Assuntos
Amidoidrolases/metabolismo , Mycobacterium tuberculosis/enzimologia , Peptidoglicano/metabolismo , Amidoidrolases/genética , Clonagem Molecular , Cobalto/metabolismo , Ativadores de Enzimas/metabolismo , Escherichia coli/genética , Expressão Gênica , Cinética , Microscopia Eletrônica , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.