RESUMO
During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.
Assuntos
Proteínas de Bactérias , Proteína Mitocondrial Trifuncional , Mycobacterium tuberculosis , Humanos , Metabolismo dos Lipídeos , Filogenia , Ubiquitinas , Proteína Mitocondrial Trifuncional/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Sturgeons are chondrostean fish of high economic value and critically endangered due to anthropogenic activities, which has led to sturgeon aquaculture development. Russian sturgeon (Acipenser gueldenstaedtii), the second most important species reared for caviar, is successfully farmed in subtropical countries, including Uruguay. However, during the Uruguayan summer, sturgeons face intolerable warmer temperatures that weaken their defences and favour infections by opportunistic pathogens, increasing fish mortality and farm economic losses. Since innate immunity is paramount in fish, for which the liver plays a key role, we used deep RNA sequencing to analyse differentially expressed genes in the liver of Russian sturgeons exposed to chronic heat stress and challenged with Aeromonas hydrophila. We assembled 149.615 unigenes in the Russian sturgeon liver transcriptome and found that metabolism and immune defence pathways are among the top five biological processes taking place in the liver. Chronic heat stress provoked profound effects on liver biological functions, up-regulating genes related to protein folding, heat shock response and lipid and protein metabolism to meet energy demands for coping with heat stress. Besides, long-term exposure to heat stress led to cell damage triggering liver inflammation and diminishing liver ability to mount an innate response to A. hydrophila challenge. Accordingly, the reprogramming of liver metabolism over an extended period had detrimental effects on fish health, resulting in weight loss and mortality, with the latter increasing after A. hydrophila challenge. To our knowledge, this is the first transcriptomic study describing how chronic heat-stressed sturgeons respond to a bacterial challenge, suggesting that liver metabolism alterations have a negative impact on the innate anti-bacterial response.
Assuntos
Infecções Bacterianas , Peixes , Adaptação Psicológica , Animais , Peixes/metabolismo , Resposta ao Choque Térmico , LipídeosRESUMO
Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation of natural habitat and overfishing for meat and caviar production. Consequently, sturgeon aquaculture has extensively developed lately, being Russian sturgeon (Acipenser gueldenstaedtii) the second most important species reared for caviar production. However, Russian sturgeon aquaculture in subtropical countries, such as Uruguay, confronts difficulties because fish have to endure excessive summertime warm temperatures, which weaken their innate defences facilitating opportunistic infections. To address this problem, we look for identifying putative acute phase proteins (APPs), which might be robust serum biomarkers of both infection and chronic thermal stress, applied to monitoring Russian sturgeon health status in farms. We focused on the C-Reactive Protein/Serum Amyloid P (CRP/SAP) pentraxin since the pentraxin family includes well-known APPs, better characterised in mammals than fish. We identified A.gueldenstaedtii CRP/SAP (AgCRP/SAP), as a member of the universal CRP/SAP pentraxin sub-family, and studied AgCRP/SAP involvement in sturgeon response to bacterial challenge and chronic thermal stress, in comparison with A. gueldenstaedtii Serum Amyloid A (AgSAA), a previously described positive APP. Results showed that AgCRP/SAP is a constitutive serum component that remained constant upon Aeromonas hydrophila challenge and chronic thermal stress. Contrastingly, serum AgSAA was subjected to regulation by bacterial and thermal stress challenges, showing a 50-fold increase and 3-fold decline in serum levels, respectively. Overall, results highlight the potential value of AgSAA, but not of AgCRP/SAP, as a biomarker of bacterial infection and the need to continue searching for robust chronic thermal stress biomarkers in sturgeons.
Assuntos
Infecções Bacterianas/veterinária , Peixes , Resposta ao Choque Térmico , Imunidade Inata , Proteínas de Fase Aguda , Animais , Infecções Bacterianas/imunologia , Proteína C-Reativa , Peixes/imunologia , Peixes/microbiologia , Sistema Imunitário , Proteína Amiloide A Sérica , Componente Amiloide P SéricoRESUMO
The immune system of sturgeons, one of the most ancient and economically valuable fish worldwide, is poorly understood. The lack of molecular tools and data about infection biomarkers hinders the possibility to monitor sturgeon health during farming and detect infection outbreaks. To tackle this issue, we mined publicly available transcriptomic datasets and identified putative positive acute-phase proteins (APPs) of Russian sturgeons that could be induced by a bacterial infection and monitored using non-invasive methods. Teleost literature compelled us to focus on five promising candidates: hepcidin, a warm acclimation associated hemopexin, intelectin, serum amyloid A protein (SAA) and serotransferrin. Among them, SAA was the most upregulated protein at the mRNA level in the liver of sturgeons challenged with heat-inactivated or live Aeromonas hydrophila. To assess whether this upregulation yielded increasing SAA levels in circulation, we developed an in-house ELISA to quantify SAA levels in sturgeon serum. Circulating SAA rose upon bacterial challenge and positively correlated with hepatic saa expression. This is the first time serum SAA has been quantified in an Actinopterygii fish. Since APPs vary across different fish species, our work sheds light on sturgeon acute-phase response, revealing that SAA is a positive APP with potential value as infection biomarker.
Assuntos
Proteínas de Fase Aguda/genética , Aeromonas hydrophila , Peixes/genética , Peixes/microbiologia , Interações Hospedeiro-Patógeno/genética , Proteína Amiloide A Sérica/genética , Proteínas de Fase Aguda/química , Reação de Fase Aguda , Sequência de Aminoácidos , Animais , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Peixes/imunologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Modelos Moleculares , Conformação Proteica , Proteína Amiloide A Sérica/química , Relação Estrutura-Atividade , TranscriptomaRESUMO
Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence.
RESUMO
Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past ten years. However, during the Uruguayan summer fish endure high water temperatures and increased bacterial infections that threaten aquaculture. Our understanding of sturgeon's immune system and its interplay with environmental factors like temperature is almost unknown. This study analysed the way in which seasonal variations affect enzymatic blood components of Russian sturgeon's innate defences, including the serum alternative complement pathway (ACP), ceruloplasmin (Cp) and lysozyme activities. Results showed that summertime conditions in the farm altered these defences in different ways, inducing a significant decrease in ACP and Cp, and an increase in lysozyme. In addition, serum levels of total protein and cortisol decreased in summer, suggesting a chronic stress response was induced in parallel. Subsequently, we analysed whether the increase in water river temperature during summer could account for the observed results. To that end, we acclimated juvenile sturgeons to mild (18 °C) or warm (24 °C) temperatures for 37 days. Like in summer, sturgeons exposed to 24 °C showed lower levels of serum ACP, Cp and total proteins, together with a progressive decrease in body weight and increased fish mortality. Administration of an immunostimulant containing Se and Zn slightly reverted the temperature-induced effects on sturgeon's defences. Altogether, our study provides novel data on various physiological parameters of the Russian sturgeon and highlights the impact warm temperature has on stress and innate immunity in this chondrostean fish.
Assuntos
Aquicultura/normas , Peixes/imunologia , Temperatura Alta , Imunidade Inata , Estresse Fisiológico , Animais , Ceruloplasmina/metabolismo , Via Alternativa do Complemento , Feminino , Muramidase/metabolismo , Distribuição Aleatória , Estações do Ano , UruguaiRESUMO
Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.
Assuntos
Vírus do Orf/enzimologia , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Cristalografia por Raios X , Dissulfetos/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Especificidade por SubstratoRESUMO
The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Dicroísmo Circular , Humanos , Macrófagos/imunologia , Espectrometria de Massas , Mutação , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes pi stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents.
Assuntos
Antituberculosos/química , Chalconas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Chalconas/síntese química , Chalconas/química , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97%) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 degrees C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.
Assuntos
Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Potyvirus/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/metabolismo , Endopeptidases/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glutaral/química , Histidina/metabolismo , Cinética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Sefarose/química , Proteínas Virais/químicaRESUMO
Fork-head associated (FHA) domains are widely found in bacteria, but their cellular functions remain unclear. Here, we focus on Mycobacterium tuberculosis GarA, an FHA-containing protein conserved in actinomycetes that is phosphorylated by different Ser/Thr protein kinases. Using various physicochemical approaches, we show that phosphorylation significantly stabilizes GarA, and that its FHA domain interacts strongly with the phosphorylated N-terminal extension. Altogether, our results indicate that phosphorylation triggers an intra-molecular protein closure, blocking the phosphothreonine-binding site and switching off the regulatory properties of GarA. The model can explain the reported functions of this mycobacterial protein as regulator of glycogen degradation and glutamate metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/química , Fatores de Transcrição Forkhead/química , Ácido Glutâmico/metabolismo , Glicogênio/metabolismo , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
In the search for lead compounds for new drugs for tuberculosis, the activity of 38 synthetic chalcones were assayed for their potential inhibitory action towards a protein tyrosine phosphatase from Mycobacterium tuberculosis--PtpA. The compounds were obtained by aldolic condensation between aldehydes and acetophenones, under basic conditions. Five compounds presented moderate or good activity. The structure-activity analysis reveals that the predominant factor for the activity is the molecule planarity/hydrophobicity and the nature of the substituents.
Assuntos
Chalconas/síntese química , Chalconas/farmacologia , Mycobacterium tuberculosis/enzimologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas de Bactérias , Chalconas/química , Técnicas de Química Combinatória , Farmacorresistência Bacteriana/efeitos dos fármacos , Estrutura Molecular , Proteínas Tirosina Fosfatases/genética , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológicoRESUMO
Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antituberculosos/uso terapêutico , Cristalografia por Raios X , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Tuberculose/tratamento farmacológico , VirulênciaRESUMO
Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the alpha isoform of human PP2C (PP2Calpha), which folds into an alpha/beta domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn(2+)-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.
Assuntos
Proteínas de Bactérias/química , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Fosfoproteínas Fosfatases/química , Monoéster Fosfórico Hidrolases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Manganês/química , Monoéster Fosfórico Hidrolases/química , Conformação ProteicaRESUMO
The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.
Assuntos
Mycobacterium tuberculosis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/enzimologia , Primers do DNA , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Culturable cells and non-culturable cells of fecal coliforms, obtained by irradiation at 312 nm were submitted to the combined stress conditions of salinity and starvation. After 14 days, beta-galactosidase activity of UV-irradiated cells was at least twice the value of non-irradiated cells. UV-irradiated cells thus contribute more than non-irradiated cells to the enzyme assay after incubation in saline water. This finding is essential for the interpretation of quantitative investigations into the environment using enzymatic methods.
Assuntos
Contagem de Colônia Microbiana/métodos , Monitoramento Ambiental/métodos , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Água do Mar/microbiologia , beta-Galactosidase/metabolismo , Relação Dose-Resposta à Radiação , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/efeitos da radiação , Ativação Enzimática , Escherichia coli/isolamento & purificação , Escherichia coli/efeitos da radiação , Glucose/metabolismo , Estatística como Assunto , Raios Ultravioleta , Poluentes da Água/análise , beta-Galactosidase/análiseRESUMO
Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a consequence, the interpretation of the state of 'nonculturable' cells is often ambiguous. Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost 'culturability' but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden-Meyerhof and pentose-phosphate pathways. However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities. Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/efeitos da radiação , Raios Ultravioleta , Aerobiose , Técnicas Bacteriológicas/métodos , Relação Dose-Resposta à Radiação , Escherichia coli/citologia , Proteínas de Escherichia coli/biossíntese , Citometria de Fluxo , Glucose/metabolismo , Espectroscopia de Ressonância Magnética , Metionina/metabolismo , Microscopia de Fluorescência , Via de Pentose FosfatoRESUMO
beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.