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Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N-terminal domain [NTD] and lysozyme-like domain [LD]) and a C-terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD-deficient D29 (D29∆NTD) by deleting the NTD-coding region from D29 genome using CRISPY-BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD-mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD-deficient LysA produced by D29∆NTD, despite having catalytically-active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment.
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Pared Celular , Endopeptidasas , Micobacteriófagos , Mycobacterium tuberculosis , Peptidoglicano , Micobacteriófagos/genética , Micobacteriófagos/metabolismo , Endopeptidasas/metabolismo , Endopeptidasas/genética , Pared Celular/metabolismo , Peptidoglicano/metabolismo , Mycobacterium tuberculosis/virología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Dominios Proteicos , Virión/metabolismo , Bacteriólisis , Mycobacterium smegmatis/virología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismoRESUMEN
The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.
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Cisteína , Glicopéptidos , Inositol , Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Factor sigma/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Formaldehído/metabolismo , beta-Lactamasas/metabolismo , Formiatos/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the development of phage therapy. Hence, it is both essential and important to understand the mechanism of phage resistance in bacteria. First identified in Mycobacterium smegmatis, the gene mpr, upon overexpression, confers resistance against D29 mycobacteriophage. Presently, the mechanism behind phage resistance by mpr is poorly understood. Here we show that Mpr is a membrane-bound DNA exonuclease, which digests DNA in a non-specific manner independent of the sequence, and shares no sequence or structural similarity with any known nuclease. Exonuclease activity of mpr provides resistance against phage infection, but the role of mpr may very well go beyond just phage resistance. Our experiments show that mpr plays a crucial role in the appearance of mutant colonies (phage resistant strains). However, the molecular mechanism behind the emergence of these mutant/resistant colonies is yet to be understood. Nevertheless, it appears that mpr is involved in the survival and evolution of M. smegmatis against phage. A similar mechanism may be present in other organisms, which requires further exploration.
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Bacteriófagos , Bacteriófagos/genética , Mycobacterium smegmatis/genéticaRESUMEN
Fisetin (Fis), a natural flavonoid with anticancer effects, suffers from delivery constraints. Fisetin-nanostructured lipid carriers (NLCs) were developed for better efficacy against metastatic melanoma, employing the design of experiment (DoE) approach. The optimized NLCs depict a particle diameter of 135.0 ± 5.5 nm, a polydispersity index (PDI) of 0.176 ± 0.035, and an entrapment efficiency of 78.16 ± 1.58%. The formulation was stable over a period of 60 days and demonstrated sustained release of the drug (74.79 ± 3.75%) over 96 h. Fis-NLCs depicted at least â¼3.2 times lower IC50 value and â¼1.8 times higher drug uptake at 48 h in A-375 and B16F10 cells compared to that of Fis. It also inhibited the mobility of melanoma cells and induced cell cycle arrest at the G1/S phase. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot results show enhanced expression of Nrf2/NQO1 genes and an apoptotic effect by the upregulation of BAX mRNA expression. The protein levels of BAX and p53 were â¼2-fold higher compared with that of pure Fis. In-vivo studies demonstrated 5.9- and 10.7-fold higher inhibition in melanoma-associated metastasis in the lungs and liver, respectively. The outcomes from this study demonstrated Fis-NLCs as an effective tool against melanoma.
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Melanoma , Nanoestructuras , Humanos , Portadores de Fármacos , Proteína X Asociada a bcl-2 , Melanoma/tratamiento farmacológico , Lípidos , Tamaño de la PartículaRESUMEN
BACKGROUND: Nrf2 regulates oxidative stress, which is essential for cellular function. Fundamental initiation of Nrf2 in many malignancies increases prosurvival genes & endorses tumour cell propagation via metabolic reprogramming, suppression of tumour programmed cell death, & increased cancer stem cell self-renewal potential. More specifically, Nrf2 has been associated with cancer cell chemoresistance, radioresistance & inflammation-induced carcinogenesis. METHODS AND RESULTS: Many Nrf2 inhibitors have been revealed for tumour treatment and targeting Nrf2 could be an effective cancer therapeutic method. Before spreading, cancer cells adapt to their surroundings. Cancer cells usually have mutations in tumor suppressor genes. In a variety of malignancies, somatic mutations & other anomalies in the Nrf2 genes, as well as renowned cancer suppressor genes including TP53, CDKN2A, PTEN & PIK3CA, have been found. In tumour cells, somatic mutations in the Nrf2 genes, as well as additional mechanisms that affect Nrf2 binding, and produce aberrant Nrf2 activation. Uncontrolled Nrf2 causes tumour cells to become resistant to antineoplastic drugs & reactive oxygen species (ROS), as well as guiding them toward metabolic reprogramming. CONCLUSIONS: As a result, Nrf2 has been studied as potential malignancy treatment target. We covered the pathways, mechanisms, and dual characteristics of Nrf2 in malignancy in this article. We also discussed how Nrf2 inhibitors are targeted against cancer in this review.
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Antineoplásicos , Neoplasias , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Antineoplásicos/farmacología , Carcinogénesis , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genéticaRESUMEN
Even though chemotherapy stands as a standard option in the therapy of TNBC, problems associated with it such as anemia, bone marrow suppression, immune suppression, toxic effects on healthy cells, and multi-drug resistance (MDR) can compromise their effects. Nanoparticles gained paramount importance in overcoming the limitations of conventional chemotherapy. Among the various options, nanotechnology has appeared as a promising path in preclinical and clinical studies for early diagnosis of primary tumors and metastases and destroying tumor cells. PLGA has been extensively studied amongst various materials used for the preparation of nanocarriers for anticancer drug delivery and adjuvant therapy because of their capability of higher encapsulation, easy surface functionalization, increased stability, protection of drugs from degradation versatility, biocompatibility, and biodegradability. Furthermore, this review also provides an overview of PLGA-based nanoparticles including hybrid nanoparticles such as the inorganic PLGA nanoparticles, lipid-coated PLGA nanoparticles, cell membrane-coated PLGA nanoparticles, hydrogels, exosomes, and nanofibers. The effects of all these systems in various in vitro and in vivo models of TNBC were explained thus pointing PLGA-based NPs as a strategy for the management of TNBC.
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Nanopartículas , Neoplasias de la Mama Triple Negativas , Humanos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ácido Láctico , Ácido Poliglicólico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Portadores de Fármacos , Línea Celular TumoralRESUMEN
Development of viable therapeutics to effectively combat tier I pneumopathogens such as Yersinia pestis requires a thorough understanding of proteins vital for pathogenicity. The host invasion protein Ail, although indispensable for Yersinia pathogenesis, has evaded detailed characterization, as it is an outer membrane protein with intrinsically low stability and high aggregation propensity. Here, we identify molecular elements of the metastable Ail structure that considerably alter protein-lipid and intraprotein thermodynamics. In addition, we find that four residues Q50, L88, L92, and A94 contribute additively to the lowered stability of Ail, and their conserved substitution is sufficient to re-engineer Ail to Out14, a thermodynamically hyperstable low-aggregation variant with a functional scaffold. Interestingly, Ail also shows two (parallel) folding pathways, which has not yet been reported for ß-barrel membrane proteins. Additionally, we identify the molecular mechanism of enhanced thermodynamic stability of Out14. We show that this enhanced stability of Out14 is due to a favorable change in the nonpolar accessible surface, and the accumulation of a kinetically accelerated off-pathway folding intermediate, which is absent in wild-type Ail. Such engineered hyperstable Ail ß-barrels can be harnessed for targeted drug screening and developing medical countermeasures against Yersiniae. Application of similar strategies will help design effective translational therapeutics to combat biopathogens.
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Proteínas de la Membrana Bacteriana Externa/química , Factores de Virulencia/química , Yersinia pestis/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cinética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica en Lámina beta , Pliegue de Proteína , Estabilidad Proteica , Alineación de Secuencia , Termodinámica , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming non-specific holes in the host cell membrane at a specific time post-infection. Thus, Holins are also termed as "Protein clocks". Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among Holins, has not yet been examined in detail. Here, we characterize the molecular properties of mycobacteriophage D29 Holin C-terminal region, and investigate the significance of the charged residues and coiled coil (CC) domain present therein. We show that the CC domain is indispensable for Holin-mediated efficient bacterial cell lysis. We further demonstrate that out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders Holin non-toxic. Moreover, the basic residues present between the 59th and the 79th amino acids are the most crucial for Holin-mediated toxicity. We also constructed an engineered Holin, HolHC, by duplicating the C-terminal region. The HolHC protein shows higher toxicity in both Escherichia coli and Mycobacterium smegmatis, and causes rapid killing of both bacteria upon expression, as compared to the wild-type. A similar oligomerization property of HolHC as the wild-type Holin allows us to propose that the C-terminal region of D29 Holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage Holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including M. tuberculosis ImportanceHolins are bacteriophage-encoded small membrane perforators that play an important role in determining the timing of host cell lysis towards the end of the phage infection cycle. Holin's ability to precisely time the hole formation in the cell membrane ensuing cell lysis is both interesting and intriguing. Here, we examined the molecular properties of the mycobacteriophage D29 Holin C-terminal region that harbours several polar charged residues and a coiled-coil domain. Our data allowed us to engineer Holin with an ability to rapidly kill bacteria and show higher toxicity than the wild-type protein. Due to their ability to kill host bacteria by membrane disruption, it becomes important to explore the molecular properties of Holins that allow them to function in a timely and efficient manner. Understanding these details can help us modulate Holin activity and engineer bacteriophages with superior lytic properties to kill pathogenic bacteria, curtail infections, and combat antimicrobial resistance.
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NQO1 is an enzyme present in humans which is encoded by NQO1 gene. It is a protective antioxidant agent, versatile cytoprotective agent and regulates the oxidative stresses of chromatin binding proteins for DNA damage in cancer cells. The oxidization of cellular pyridine nucleotides causes structural alterations to NQO1 and changes in its capacity to binding of proteins. A strategy based on NQO1 to have protective effect against cancer was developed by organic components to enhance NQO1 expression. The quinone derivative compounds like mitomycin C, RH1, E09 (Apaziquone) and ß-lapachone causes cell death by NQO1 reduction of two electrons. It was also known to be overexpressed in various tumor cells of breast, lung, cervix, pancreas and colon when it was compared with normal cells in humans. The mechanism of NQO1 by the reduction of FAD by NADPH to form FADH2 is by two ways to inhibit cancer cell development such as suppression of carcinogenic metabolic activation and prevention of carcinogen formation. The NQO1 exhibit suppression of chemical-mediated carcinogenesis by various properties of NQO1 which includes, detoxification of quinone scavenger of superoxide anion radical, antioxidant enzyme, protein stabilizer. This review outlines the NQO1 structure, mechanism of action to inhibit the cancer cell, functions of NQO1 against oxidative stress, drugs acting on NQO1 pathways, clinical significance.
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NAD(P)H Deshidrogenasa (Quinona) , Neoplasias , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Mama/metabolismo , Femenino , Humanos , NAD , NAD(P)H Deshidrogenasa (Quinona)/genética , Neoplasias/tratamiento farmacológicoRESUMEN
Samples of ambient aerosols were collected at an urban site of eastern central India from monsoon to summer 2016-17 for the characterization of biogenic secondary organic aerosols (BSOA). The BSOA tracers derived from isoprene, α/ß-pinene and ß-caryophyllene in size-distributed aerosols were studied. Concentrations of total SOAI (Isoprene secondary organic aerosols) were found more abundant than α/ß-pinene in summer, while contradictory trends were found in the winter season, where SOAM (monoterpene derived SOA) and SOAS (sesquiterpenes derived SOA) were dominated. Size-distribution study revealed that most of the BSOA were formed in the aerosol phase and dominated in fine mode, except cis-pinonic acid. They were formed in the gaseous phase and partitioned onto the aerosol phase. The alkaline nature of mineral dust particles that triggered the adsorption of gaseous species onto pre-existing particles could be the reason for bimodal size distribution with major coarse mode peak and miner fine mode peak. Temporal variations suggest that the BSOA must be derived from terrestrial vegetation and biomass burning. The isoprene SOC (secondary organic carbon) contributed 0.91%, 1.38%, 0.88% and 1.04% to OC during winter, summer, post-monsoon and monsoon season, respectively. The isoprene SOC in fine mode was found to be higher than the coarse mode.
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Contaminantes Atmosféricos , Material Particulado , Aerosoles/análisis , Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente , India , Material Particulado/análisis , Estaciones del AñoRESUMEN
Adrenal oncocytomas are rare neoplasms that are usually benign and nonfunctional, and often detected incidentally. Very few cases have been reported of functioning adrenal oncocytomas. We report a rare case of adrenocortical oncocytoma in a 29-year-old female presenting with hirsutism and irregular menstrual history. The tumor was functional and was successfully managed by laparoscopic adrenalectomy. Detailed radiological, histological, and immunohistochemical workup was done to come to a definitive diagnosis of adrenal oncocytoma.
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The author regrets that one of the author's name was incorrectly presented in the published version of this article. The third author's name original read as "Tajwar Singh Negi" this should have been "Tajwer Singh Negi".
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The objectives of this prospective case-control study were to determine liver stiffness (LSM) by transient elastography (TE) in children with newly diagnosed chronic liver disease (CLD) and to find out normal values in healthy Indian children. Two groups (A: 50 CLD who underwent liver biopsy and B: 50 healthy) aged 5-18 years were recruited prospectively. Liver biopsies were scored as per Metavir scoring and compared with TE. The median age of 100 recruited children was 13.6 years. In group B, normal LSM was 4.9 (2.5-7.3) kPa with significantly higher LSM in adolescent males (5.6 (4.1-7.3) kPa) as compared with females (4.3 (3.7-4.9) kPa), p = 0.001. In group A, TE was excellent in discriminating significant fibrosis (≥ F2) (P = 0.001) at a cut-off value of 10.6 kPa with area under receiver operating characteristic curve of 0.96. Metavir fibrosis stage (ß = 0.611; R2 = 0.586) and age (ß = 0.230; R2 = 0.586) were independent variables associated with higher LSM in stepwise multiple logistic regression analysis.Conclusions: TE is an excellent non-invasive tool to assess significant liver fibrosis and can be used as an alternative to liver biopsy. Normative value of TE in adolescent males is higher than in females.What is Known:⢠Transient elastography is a good non-invasive test for liver fibrosis assessment.⢠Normal liver stiffness depends on race, gender, and age.What is New:⢠This is the first study from India to show the normative data of transient elastography in healthy Indian children.⢠We have documented that liver stiffness measurement by fibroscan in treatment naïve chronic liver disease has excellent correlation in significant fibrosis, severe fibrosis, and cirrhosis.
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Diagnóstico por Imagen de Elasticidad/normas , Cirrosis Hepática/diagnóstico , Adolescente , Estudios de Casos y Controles , Niño , Diagnóstico por Imagen de Elasticidad/métodos , Femenino , Humanos , Cirrosis Hepática/fisiopatología , Masculino , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Approximately 40% of compounds in clinical drug development suffer from solubility and bioavailability challenges. Evidence from literature demonstrates the growing interest to utilize flavonoids as potential compounds owing to their widespread therapeutic utility in various ailments. Nobiletin (NOB), one such dietary polymethoxylated flavonoid found in citrus fruits, has multiple pharmacological effects such as antioxidant, anti-microbial, anti-cancer, and anti-inflammatory. It is useful in cancer, inflammatory bowel diseases, atherosclerosis, obesity, and Alzheimer's disease. Although preclinical studies demonstrate the therapeutic utility of NOB, it suffers from serious biopharmaceutical limitations such as low aqueous solubility (below 1 µg/ml), poor permeability across biological barriers, and low bioavailability. To overcome these biopharmaceutical challenges associated with NOB, the use of advanced formulations and nanotechnology-based strategies appears to be a promising approach to potentiate its therapeutic action. Multiple reviews cover the various therapeutic benefits of NOB in various diseases; however, there is an absence of a comprehensive review that focuses on the formulation development strategies of NOB. The purpose of this review is to provide a concise perspective on NOB as a candidate molecule for formulation development. The manuscript covers various aspects related to NOB, such as its chemistry, physicochemical properties, and pharmacological effects. This is also a thorough review of various formulation development strategies with advances made in the past years to improve the solubility, bioavailability, and therapeutic efficacy of NOB. The review also contains information related to toxicity and patents involving NOB and its formulation.
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Antioxidantes/química , Composición de Medicamentos , Flavonas/química , Nanotecnología , Animales , Antioxidantes/farmacocinética , Disponibilidad Biológica , Flavonas/farmacocinética , Humanos , SolubilidadRESUMEN
Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatisM. smegmatis harbors two identical copies of atpD, which codes for the ß subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the ß-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections.IMPORTANCEM. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase ß subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.
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Eliminación de Gen , ATPasas de Translocación de Protón Mitocondriales/genética , Mycobacterium smegmatis/fisiología , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Diarilquinolinas/farmacología , Metabolismo Energético/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Operón , Fosforilación Oxidativa/efectos de los fármacos , FenotipoRESUMEN
Mycobacterium smegmatis possesses (N,N-dimethyl-4-nitrosoaniline)-dependent (NDMA) methanol dehydrogenase (Mno) to establish methylotrophy by utilizing methanol as the source of both carbon and energy. In this study, we show that Mno forms decamer and has NADPH as the bound cofactor. Interestingly, Mno uses NDMA and not NADP+ as an electron acceptor in in vitro reactions. We further show that the operon mftAD required for the biosynthesis of mycofactocin, a ribosomally-synthesized electron carrier, is indispensable for the growth of M. smegmatis on methanol. Our data obtained from 2,6-Dichlorophenolindophenol reduction assays also suggest that Mno uses mycofactocin as an in vivo electron acceptor for the oxidation of methanol to formaldehyde. We thus provide here biochemical evidence for mycofactocin as an electron carrier in mycobacterial physiology.
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Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/metabolismo , Metanol/metabolismo , Mycobacterium smegmatis/metabolismo , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/crecimiento & desarrollo , NADP/metabolismo , Compuestos Nitrosos/metabolismoRESUMEN
Mycobacteriophages are viruses that infect and kill mycobacteria. The peptidoglycan hydrolase, lysin A (LysA), coded by one of the most potent mycobacteriophages, D29, carries two catalytic domains at its N-terminus and a cell wall-binding domain at its C-terminus. Here, we have explored the importance of the centrally located lysozyme-like catalytic domain (LD) of LysA in phage physiology. We had previously identified an R198A substitution that causes inactivation of the LD when it is present alone on a polypeptide. Here, we show that upon incorporation of the same mutation (i.e. R350A) in full-length LysA, the protein demonstrates substantially reduced activity in vitro, even in the presence of the N-terminal catalytic domain, and has less efficient mycobacterial cell lysis ability when it is expressed in Mycobacterium smegmatis. These data suggest that an active LD is required for the full-length protein to function optimally. Moreover, a mutant D29 phage harbouring this substitution (D29R350A) in its LysA protein shows significantly delayed host M. smegmatis lysis. However, the mutant phage demonstrates an increase in burst size and plaque diameter. Taken together, our data show the importance of an intact LD region in D29 LysA PG hydrolase, and indicate an evolutionary advantage over other phages that lack such a domain in their endolysins.
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Endopeptidasas/genética , Micobacteriófagos , Mycobacterium smegmatis/virología , N-Acetil Muramoil-L-Alanina Amidasa/genética , Dominio Catalítico/genética , Pared Celular/metabolismo , Endopeptidasas/química , Endopeptidasas/metabolismo , Mutación , Micobacteriófagos/genética , Micobacteriófagos/crecimiento & desarrollo , Micobacteriófagos/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Mycobacterium smegmatis and several other mycobacteria are able to utilize methanol as the sole source of carbon and energy. We recently showed that N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol dehydrogenase (Mno) is essential for the growth of M. smegmatis on methanol. Although Mno from this bacterium shares high homology with other known methanol dehydrogenases, methanol metabolism in M. smegmatis differs significantly from that of other described methylotrophs. In this study, we dissect the regulatory mechanism involved in the methylotrophic metabolism in M. smegmatis We identify a two-component system (TCS), mnoSR, that is involved in the regulation of mno expression. We show that the MnoSR TCS is comprised of a sensor kinase (MnoS) and a response regulator (MnoR). Our results demonstrate that MnoS undergoes autophosphorylation and is able to transfer its phosphate to MnoR by means of phosphotransferase activity. Furthermore, MnoR shows specific binding to the putative mno promoter region in vitro, thus suggesting its role in the regulation of mno expression. Additionally, we find that the MnoSR system is involved in the regulation of MSMEG_6239, which codes for a putative 1,3-propanediol dehydrogenase. We further show that M. smegmatis lacking mnoSR is unable to utilize methanol and 1,3-propanediol as the sole carbon source, which confirms the role of MnoSR in the regulation of alcohol metabolism. Our data, thus, suggest that the regulation of mno expression in M. smegmatis provides new insight into the regulation of methanol metabolism, which furthers our understanding of methylotrophy in mycobacteria.IMPORTANCE Methylotrophic metabolism has gained huge attention considering its broad application in ecology, agriculture, industries, and human health. The genus Mycobacterium comprises both pathogenic and nonpathogenic species. Several members of this genus are known to utilize methanol as the sole carbon source for growth. Although various pathways underlying methanol utilization have been established, the regulation of methylotrophic metabolism is not well studied. In the present work, we explore the regulation of methanol metabolism in M. smegmatis and discover a dedicated two-component system (TCS), MnoSR, that is involved in its regulation. We show that the loss of MnoSR renders the bacterium incapable of utilizing methanol and 1,3-propanediol as the sole carbon sources. Additionally, we establish that MnoS acts as the common sensor for the alcohols in M. smegmatis.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Metanol/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas Quinasas/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/genética , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Proteínas Quinasas/genéticaRESUMEN
OBJECTIVE: To control the rate of growth of the cancer cell is the objective of this paper. In cancer, the rate of the growth of the cancer cell is indefinite. This paper proposes a method to transform into definite rate of growth of the cancer cell from indefinite. This indefiniteness lies with the set of unknown elements. This paper finds these unknown elements by matrix method.
Asunto(s)
Proliferación Celular , Neoplasias , HumanosRESUMEN
The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as Mycobacterium tuberculosis, have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C1) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including M. tuberculosis and M. smegmatis, are capable of utilizing alternative C1 compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in M. smegmatis We present N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of M. smegmatis on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities in vitro Further, M. smegmatis is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde in vivo Finally, we show that M. smegmatis devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO2 in M. tuberculosis, does not grow on methanol, suggesting that the final step of methanol utilization requires CO2 fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species.IMPORTANCE Methylotrophy, the ability to utilize single-carbon (C1) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including Mycobacterium tuberculosis, are capable of utilizing C1 compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in Mycobacterium smegmatis With several genetic knockouts, we have dissected the entire methanol metabolism pathway in M. smegmatis We show that while methanol dissimilation in M. smegmatis differs from that in other mycobacterial species, the concluding step of CO2 fixation is similar to that in M. tuberculosis It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.