RESUMEN
Pseudomonas spp. collected from areas where human regularly comes into contact with were tested for their susceptibility to antibiotics. Twenty-nine samples were collected and screened for Pseudomonas spp. Of the nine isolated strains Pseudomonas spp. six were resistant to antibiotics. A few were used for an antimicrobial study on the interaction with silver and zinc oxide nanoparticles individually and as a mixture. A mixture of silver and zinc oxide nanoparticles showed synergy against resistant Pseudomonas spp.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Nanopartículas del Metal/química , Pseudomonas/efectos de los fármacos , Antibacterianos/administración & dosificación , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Plata/administración & dosificación , Plata/química , Plata/farmacología , Óxido de Zinc/administración & dosificación , Óxido de Zinc/química , Óxido de Zinc/farmacologíaRESUMEN
The World Health Organization acknowledges tuberculosis as a global threat. Tuberculosis infection is one of the top 10 causes of death worldwide. Nanotechnology and microbiology researchers are looking for new and safe nano drugs for eliminating Mycobacterium tuberculosis, the causative agent of tuberculosis. In this study, AgZnO nano-crystals (AgZnONCs) is synthesized via the decomposition of the precursor of oxalate method. Characterization of AgZnONCs were evaluated. Next, various concentrations of AgZnONCs, as well AgZnONCs+Rifampicin, were prepared. The MTT assay was employed to study the viability of human macrophage cell lines (THP-1) exposed to AgZnONCs. The bactericidal effects of AgZnONCs and AgZnONCs+Rifampicin were studied by Minimum Bactericidal Concentration (MBC) test. Subsequently, THP-1 were infected by H37 Rv strain of M. tuberculosis (H37 RvMtb). Also, bactericidal effects of AgZnONCs and AgZnONCs+Rifampicin were compared with ex-vivo conditions. The MBC of AgZnONCs and AgZnONCs+Rifampicin were ratios of 1:4 and 1:32 respectively (p-value <0.05). Also, more than 50% and 80% of THP-1 were alive in ratios of 1:4 and 1:32 in the presence of AgZnONCs, respectively. All phagocytic H37 RvMtb were killed in the presence of AgZnONCs+Rifampicin (p-value <0.05), while AgZnONCs were not able to kill all the H37 RvMtb (p-value >0.05). This study showed that, AgZnONCs+Rifampicin has the most anti-tubercular behavior with respect to the macrophages.
Asunto(s)
Antituberculosos/toxicidad , Macrófagos/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Nanopartículas/química , Rifampin/toxicidad , Plata/toxicidad , Óxido de Zinc/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , FagocitosisRESUMEN
The purpose of this research project was to infection of human macrophages (THP-1) cell lines by H37Rv strain of Mycobacterium tuberculosis (H37RvMTB) and find out the ratio/dilution of mixture silver (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) whose ability to eliminate phagocytized bacteria compared to rifampicin. The colloidal Ag NPs and ZnO NPs were synthesized and their characteristics were evaluated. The THP-1 cell lines were infected with different concentration of H37RvMTB. Next, the infected cells were treated with different ratios/dilutions of Ag NPs, ZnO NPs and rifampicin. The THP-1 were lysed and were cultured in Lowenstein-Jensen agar medium, for eight weeks. The TEM and AFM images of NPs and H37RvMTB were supplied. It is observed that Ag NPs, 2Ag:8ZnO and 8Ag:2ZnO did not have any anti-tubercular effects on phagocytized H37RvMTB. Conversely, ZnO NPs somehow eliminated 18.7 × 104 CFU ml-1 of H37RvMTB in concentration of â¼ 0.468 ppm. To compare with 40 ppm of rifampicin, â¼ 0.663 ppm of 5Ag:5ZnO had the ability to kill of H37RvMTB, too. Based on previous research, ZnO NPs had strong anti-tubercular impact against H37RvMTB to in-vitro condition, but it was toxic in concentration of â¼ 0.468 ppm to both of THP-1 and normal lung (MRC-5) cell lines. It also seems that 5Ag:5ZnO is justified because in concentration of â¼ 0.663 ppm of 5Ag:5ZnO, phagocytized H37RvMTB into the THP-1 had died without any toxicity effects against THP-1 and also MRC-5 cell lines. It is obvious that the mixture of colloidal silver and zinc oxide NPs with ratio of 5Ag:5ZnO would be trustworthy options as anti-tubercular nano-drugs in future researches.
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Nanopartículas del Metal/química , Mycobacterium tuberculosis/efectos de los fármacos , Fagosomas/efectos de los fármacos , Plata/farmacología , Células THP-1/microbiología , Óxido de Zinc/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Línea Celular/efectos de los fármacos , Humanos , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/ultraestructura , Mycobacterium tuberculosis/patogenicidad , Fagocitosis , Fagosomas/microbiología , Fagosomas/ultraestructura , Plata/química , Células THP-1/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Óxido de Zinc/químicaRESUMEN
Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.
Asunto(s)
Interacciones Huésped-Patógeno , Lipopolisacáridos/análisis , Manosa/análisis , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patología , Animales , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Modelos Animales de Enfermedad , Cobayas , Macrófagos/microbiología , Ratones , Viabilidad Microbiana , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Pulmonar/microbiología , Factores de Virulencia/análisisRESUMEN
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.
Asunto(s)
Proteína Transportadora de Acilo , Proteínas Bacterianas , Mycobacterium tuberculosis , Animales , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Ratones , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteína Transportadora de Acilo/metabolismo , Proteína Transportadora de Acilo/genética , Macrófagos/microbiología , Macrófagos/metabolismo , Virulencia , Regulación Bacteriana de la Expresión Génica , Tuberculosis/microbiología , Lípidos/químicaRESUMEN
Mycobacterium tuberculosis thrives in oxidative environments such as the macrophage. To survive, the bacterium must sense and adapt to the oxidative conditions. Several antioxidant defenses including a thick cell wall, millimolar concentrations of small molecule thiols, and protective enzymes are known to help the bacterium withstand the oxidative stress. However, oxidation-sensing regulators that control these defenses have remained elusive. In this study, we report a new oxidation-sensing regulator, Rv1049 or MosR (M. tuberculosis oxidation-sensing regulator). MosR is a transcriptional repressor of the MarR family, which, similarly to Bacillus subtilis OhrR and Staphylococcus aureus MgrA, dissociates from DNA in the presence of oxidants, enabling transcription. MosR senses oxidation through a pair of cysteines near the N terminus (Cys-10 and Cys-12) that upon oxidation forms a disulfide bond. Disulfide formation rearranges a network of hydrogen bonds, which leads to a large conformational change of the protein and dissociation from DNA. MosR has been shown previously to play an important role in survival of the bacterium in the macrophage. In this study, we show that the main role of MosR is to up-regulate expression of rv1050 (a putative exported oxidoreductase that has not yet been characterized) in response to oxidants and propose that it is through this role that MosR contributes to the bacterium survival in the macrophage.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Mycobacterium tuberculosis/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , Oxidantes/farmacología , Oxidación-Reducción , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the ã-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.
Asunto(s)
Francisella tularensis , Fructosa-Bifosfatasa , Fructosa-Bifosfatasa/metabolismo , Francisella tularensis/metabolismo , Catálisis , Dominio Catalítico , Fructosa/metabolismo , Cristalografía por Rayos XRESUMEN
Tuberculosis (TB) is a major global cause of mortality, primarily stemming from latent tuberculosis infection (LTBI). Failure to fully treat LTBI can result in drug-resistant forms of TB. Therefore, it is essential to develop novel drugs with unique mechanisms of action to combat TB effectively. One crucial metabolic pathway in Mycobacterium tuberculosis (Mtb), which contributes to TB infection and persistence, is gluconeogenesis. Within this pathway, the enzyme fructose bisphosphatase (FBPase) plays a significant role and is considered a promising target for drug development. By targeting MtbFBPaseII, a specific class of FBPase, researchers have employed molecular dynamics simulations to identify regions capable of binding new drugs, thereby inhibiting the enzyme's activity and potentially paving the way for the development of effective treatments.Communicated by Ramaswamy H. Sarma.
RESUMEN
Aromatic oleo-gum-resin secreted from B. sacra, reputed as frankincense, is widely used in traditional medicine to treat Alzheimer's disease, gastric disorders, hepatic disorders, etc. Frankincense is also used in the cosmetic, perfume, and beverage and food industries. Frankincense is a rich resource for bioactive compounds, especially boswellic acids and derivatives. Although several reports have described frankincense's constituents and pharmacological activities, there is no comprehensive study that covers the valuable information on this species. Therefore, the current review will focus on the phytochemistry, traditional uses, and pharmacological activities of B. sacra.
RESUMEN
Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7â Å and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 131.3, c = 143.2â Å. The structure has been solved by molecular replacement and is currently undergoing refinement.
Asunto(s)
Fructosadifosfatos/química , Mycobacterium tuberculosis/enzimología , Cristalización , Cristalografía por Rayos X , Fructosadifosfatos/aislamiento & purificaciónRESUMEN
BACKGROUND: Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. RESULTS: Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. CONCLUSIONS: We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.
Asunto(s)
Inositol/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Monoéster Fosfórico Hidrolasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Extractos Celulares/química , Biología Computacional , Expresión Génica , Lipopolisacáridos/metabolismo , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Alineación de SecuenciaRESUMEN
Purpose: Paclitaxel (PTX) has transpired as a significant agent in the treatment of breast cancer. Meanwhile, polylactic glycolic acid (PLGA) nanoparticles (NPs) are able to increase the anticancer effect of the PTX in the blood. Methods: Nano-precipitation was used to prepare the PLGA-PTX-VitD3 co-delivery NPs. Drug loading, encapsulation efficiency, in vitro release profile, cell viability, migration, apoptosis, and bcl2 expression of NPs were evaluated. Results: The average size of co-delivery NPs was 231 ± 46 nm. Observed was a controlled release of the PTX and vitamin D3 from co-delivery NPs between 0.5 and 240 hours. MTT showed the ability of 8 µg.mL-1 of co-delivery NPs to kill 50 % of the MCF-7; likewise, the co-delivery NPs prevented MCF-7 migration. The co-delivery NPs led 46.35 % MCF-7 to enter primary apoptosis. 60.8% of MCF-7 in the control group were able to enter the G (1) phase of the cell cycle. The co-delivery NPs increased expression of bax. In addition to its higher toxicity against MCF-7 than that of PTX, co-delivery NPs were able to release drugs continuously for a long period, which indeed increased the efficiency of the drugs. Conclusion: The effect of co-delivery NPs on MCF-7 cell viability was different from that in other drugs. In fact, the co-deliver NPs were able to release drugs continuously for a long time, this could induce primary apoptosis in the MCF-7 and decrease the metastasis and toxicity of drugs.
RESUMEN
The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4â Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02â Å, ß = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.
Asunto(s)
Francisella tularensis/enzimología , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/enzimología , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/aislamiento & purificación , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Conformación Proteica , Estructura Cuaternaria de Proteína , Synechocystis/enzimologíaRESUMEN
The true identity of the protein found in the crystals reported by Bondoc et al. [(2019), Acta Cryst. F75, 646-651] is given.
RESUMEN
Deletion of genes in a pathogen is commonly associated with a reduction in its ability to cause disease. However, some rare cases have been described in the literature whereby deletion of a gene results in an increase in virulence. Recently, there have been several reports of hypervirulence resulting from gene deletion in Mycobacterium tuberculosis. Here, we explore this phenomenon in the context of the interaction between the pathogen and the host response.
Asunto(s)
Eliminación de Gen , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos , Mycobacterium tuberculosis/metabolismo , Eliminación de Secuencia , VirulenciaRESUMEN
BACKGROUND: The high chances of getting latent tuberculosis infection (LTBI) among health care workers (HCWs) will an enormous problem in low and upper-middle-income countries. METHOD: Search strategies were done through both national and international databases include SID, Barakat knowledge network system, Irandoc, Magiran, Iranian national library, web of science, Scopus, PubMed/MEDLINE, OVID, EMBASE, the Cochrane library, and Google Scholar search engine. The Persian and the English languages were used as the filter in national and international databases, respectively. Medical Subject Headings (MeSH) terms was used to controlling comprehensive vocabulary. The search terms were conducted without time limitation till January 01, 2019. RESULTS: The prevalence of LTBI in Iranian's HCWs, based on the PPD test was 27.13% [CI95%: 18.64-37.7]. The highest prevalence of LTBI in Iranian's HCWs were estimated 41.4% [CI95%: 25.4-59.5] in the north, and 33.8% [CI95%: 21.1-49.3] in the west. The lowest prevalence of LTBI was evaluated 18.2% [CI95%: 3.4-58.2] in the south of Iran. The prevalence of LTBI in Iranian's HCWs who had work-experience more than 20 years old were estimated 20.49% [CI95%: 11-34.97]. In the PPD test, the prevalence of LTBI in Iranian's HCWs who had received the Bacille Calmette-Guérin (BCG) was estimated 15% [CI95%: 3.6-47.73]. While, in the QFT, the prevalence of LTBI in Iranian's HCWs in non-vaccinated was estimated 25.71% [CI95%: 13.96-42.49]. CONCLUSIONS: This meta-analysis shows the highest prevalence of LTBI in Iranian's HCWs in the north and the west probably due to neighboring countries like Azerbaijan and Iraq, respectively. It seems that Iranian's HCWs have not received the necessary training to prevent of TB. We also found that BCG was not able to protect Iranian's HCWs from TB infections, completely.
Asunto(s)
Personal de Salud , Tuberculosis Latente/epidemiología , Adulto , Femenino , Geografía Médica , Humanos , Irán/epidemiología , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/microbiología , Masculino , Persona de Mediana Edad , Prevalencia , Vigilancia en Salud Pública , Sesgo de Publicación , Factores de Riesgo , Sensibilidad y Especificidad , Prueba de TuberculinaRESUMEN
Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9â Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Cristalización , Mycobacterium tuberculosis/química , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cristalografía por Rayos X , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6â Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.
Asunto(s)
Regulación Alostérica , Citratos/antagonistas & inhibidores , Fructosa-Bifosfatasa/química , Mycobacterium tuberculosis/enzimología , Catálisis , Dominio Catalítico , Inhibidores Enzimáticos , Proteínas de Escherichia coli , Fructosa-Bifosfatasa/genética , Cinética , Proteínas Mutantes/química , Mutación , Multimerización de Proteína , Synechocystis/químicaRESUMEN
Inositol monophosphatase (IMPase) is a crucial enzyme for the biosynthesis of phosphatidylinositol, an essential component in mycobacterial cell walls. IMPase A (ImpA) from Mycobacterium smegmatis is a bifunctional enzyme that also functions as a fructose-1,6-bisphosphatase (FBPase). To better understand the bifunctional nature of this enzyme, point mutagenesis was conducted on several key residues and their enzyme activity was tested. Our results along with active site models support the fact that ImpA is a bifunctional enzyme with residues Gly94, Thr95 hypothesized to be contributing to the FBPase activity and residues Trp220, Asp221 hypothesized to be contributing to the IMPase activity. Double mutants, W220A + D221A reduced both FBPase and IMPase activity drastically while the double mutant G94A + T95A surprisingly partially restored the IMPase activity compared to the single mutants. This study establishes the foundation toward obtaining a better understanding of the bifunctional nature of this enzyme.
RESUMEN
The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis. We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: Km 11 µM, Vmax 2.0 units/mg, kcat 1.2 s-1, kcat/Km 120 mM-1 s-1, and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme's properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis.