RESUMO
BACKGROUND: Management of blood cholesterol is a major focus of efforts to prevent cardiovascular diseases. The objective of this study was to investigate how the gut microbiota affects host cholesterol homeostasis at the organism scale. RESULTS: We depleted the intestinal microbiota of hypercholesterolemic female Apoe-/- mice using broad-spectrum antibiotics. Measurement of plasma cholesterol levels as well as cholesterol synthesis and fluxes by complementary approaches showed that the intestinal microbiota strongly regulates plasma cholesterol level, hepatic cholesterol synthesis, and enterohepatic circulation. Moreover, transplant of the microbiota from humans harboring elevated plasma cholesterol levels to recipient mice induced a phenotype of high plasma cholesterol levels in association with a low hepatic cholesterol synthesis and high intestinal absorption pattern. Recipient mice phenotypes correlated with several specific bacterial phylotypes affiliated to Betaproteobacteria, Alistipes, Bacteroides, and Barnesiella taxa. CONCLUSIONS: These results indicate that the intestinal microbiota determines the circulating cholesterol level and may thus represent a novel therapeutic target in the management of dyslipidemia and cardiovascular diseases.
Assuntos
Colesterol/metabolismo , Dislipidemias/metabolismo , Microbioma Gastrointestinal/fisiologia , Homeostase , Intestinos/microbiologia , Animais , Transplante de Microbiota Fecal , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: In eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied. RESULTS: The two genes encoding for putative serpins from the human gut bacterium Eubacterium sireaum, display low sequence identities. These genes were overexpressed and the encoded proteins, named Siropins, were purified. Activity studies demonstrated that both purified proteins inhibited serine proteases but surprisingly they preferentially inhibited two human serine proteases (Human Neutrophil Elastase and Proteinase3). The biochemical characterization of these Siropins revealed that Siropin 1 was the most active and stable at low pH values while Siropin 2 was more thermoactive and thermostable. Kinetic analysis allowed the determination of the stoichiometry of inhibition (SI) which was around 1 and of the association rate constants of 7.7 × 104 for the Human Neutrophil Elastase and 2.6 × 105 for the Proteinase3. Moreover, both Siropins displayed the ability to inhibit proteases usually present in fecal waters. Altogether our data indicate the high efficiency of Siropins and their probable involvement in the control of the overall intestine protease activity. CONCLUSIONS: Here we report the purification and the biochemical characterization of two novel serpins originated from Eubacterium sireaum, a human gastro-intestinal tract commensal bacteria. These proteins that we called Siropins, efficiently inhibited two human proteases reported to be associated with inflammatory bowel diseases. The determination of the biochemical properties of these enzymes revealed different temperature and pH behaviours that may reflect adaptation of this human commensal bacterium to different ecological environments. To the best of our knowledge, it is the first bacterial serpins showing an attractive inhibition of fecal proteases recovered from a mice group with chemically induced inflammation. Altogether our data highlight the interesting potential of Siropins, and serpins from the human gut microbiota in general, to be used as new alternative to face inflammatory diseases.
Assuntos
Doenças Inflamatórias Intestinais/tratamento farmacológico , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Serpinas/farmacologia , Animais , Eubacterium/química , Eubacterium/metabolismo , Microbioma Gastrointestinal , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Camundongos , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/metabolismo , Serpinas/isolamento & purificação , Serpinas/metabolismoRESUMO
BACKGROUND: The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. RESULTS: The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. CONCLUSIONS: We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the L-arabinose isomerase has the ability to produce D-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo , Hipoglicemiantes/uso terapêutico , Animais , Hipoglicemiantes/administração & dosagem , CamundongosRESUMO
BACKGROUND: Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. RESULTS: Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. CONCLUSIONS: The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.
Assuntos
Evolução Molecular , Genes Bacterianos , Lactobacillus delbrueckii/genética , Aminoácidos/biossíntese , Proteínas de Bactérias/genética , Metabolismo dos Carboidratos , Fermentação , Transferência Genética Horizontal , Genoma Bacteriano , Tipagem de Sequências Multilocus , Proteoma/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70-75 °C and pH 6.5-7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca(2+) ion and exhibited high catalytic efficiency of 8.3 s(-1) µM(-1) towards phytic acid.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Bacillus/enzimologia , Bacillus/genética , Ácido Fítico/metabolismo , 6-Fitase/química , Sequência de Aminoácidos , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Clonagem Molecular , Coenzimas/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , TemperaturaRESUMO
The gene encoding the ß-galactosidase from the dairy Lactococcus lactis IL1403 strain was cloned, sequenced and overexpressed in Escherichia coli. The purified enzyme has a tetrameric arrangement composed of four identical 120 kDa subunits. Biochemical characterization showed that it is optimally active within a wide range of temperatures from 15 to 55 °C and of pH from 6.0 to 7.5. For its maximal activity this enzyme requires only 0.8 mM Fe(2+) and 1.6 mM Mg(2+). Purified protein displayed a high catalytic efficiency of 102 s(-1) mM(-1) for lactose. The enzyme stability was increased by immobilization mainly at low pH (from 4.0 to 5.5) and high temperatures (55 and 60 °C). The bioconversion of lactose using the L. lactis ß-galactosidase allows the production of lactose with a high bioconversion rate (98 %) within a wide range of pH and temperature.
Assuntos
Lactococcus lactis/enzimologia , Lactose/metabolismo , beta-Galactosidase/metabolismo , Clonagem Molecular , Ativadores de Enzimas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Hidrólise , Lactococcus lactis/genética , Peso Molecular , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/isolamento & purificaçãoRESUMO
Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/farmacologia , Hidrolases/metabolismo , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Hidrolases/genética , Lactococcus lactis/genética , Estresse Oxidativo/genética , Proteômica , Transdução de Sinais/genéticaRESUMO
BACKGROUND: L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. RESULTS: The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. CONCLUSIONS: Here we reported the purification and the biochemical characterization of the novel Shewanella sp. ANA-3 L-arabinose isomerase. Determination of the biochemical properties demonstrated that this enzyme was highly active at low temperatures. The generated T268K mutant displays an increase of the enzyme stability essentially at low pH. These features seem to be very attractive for the bioconversion of D-galactose into D-tagatose at low temperature which is very interesting from industrial point of view.
Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Shewanella/enzimologia , Ácidos/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Aldose-Cetose Isomerases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Temperatura Baixa , Estabilidade Enzimática , Hexoses/metabolismo , Cinética , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Shewanella/química , Shewanella/genética , Especificidade por SubstratoRESUMO
The role of the gut microbiota in health and disease is well recognized and the microbiota dysbiosis observed in many chronic diseases became a new therapeutic target. The challenge is to get a better insight into the functionality of commensal bacteria and to use this knowledge to select live biotherapeutics as new preventive or therapeutic products. In this study, we set up a screening approach to evaluate the functional capacities of a set of 21 strains isolated from the gut microbiota of neonates and adults. For this purpose, we selected key biological processes involved in the microbiome-host symbiosis and known to impact the host physiology i.e., the production of short-chain fatty acids and the ability to strengthen an epithelial barrier (Caco-2), to induce the release of the anti-inflammatory IL-10 cytokine after co-culture with human immune cells (PBMC) or to increase GLP-1 production from STC-1 endocrine cell line. This strategy highlighted fifteen strains exhibiting beneficial activities among which seven strains combined several of them. Interestingly, this work revealed for the first time a high prevalence of potential health-promoting functions among intestinal commensal strains and identified several appealing novel candidates for the management of chronic diseases, notably obesity and inflammatory bowel diseases.
RESUMO
Excess chronic contact between microbial motifs and intestinal immune cells is known to trigger a low-grade inflammation involved in many pathologies such as obesity and diabetes. The important skewing of intestinal adaptive immunity in the context of diet-induced obesity (DIO) is well described, but how dendritic cells (DCs) participate in these changes is still poorly documented. To address this question, we challenged transgenic mice with enhanced DC life span and immunogenicity (DChBcl-2 mice) with a high-fat diet. Those mice display resistance to DIO and metabolic alterations. The DIO-resistant phenotype is associated with healthier parameters of intestinal barrier function and lower intestinal inflammation. DChBcl-2 DIO-resistant mice demonstrate a particular increase in tolerogenic DC numbers and function, which is associated with strong intestinal IgA, T helper 17, and regulatory T-cell immune responses. Microbiota composition and function analyses reveal that the DChBcl-2 mice microbiota is characterized by lower immunogenicity and an enhanced butyrate production. Cohousing experiments and fecal microbial transplantations are sufficient to transfer the DIO resistance status to wild-type mice, demonstrating that maintenance of DCs' tolerogenic ability sustains a microbiota able to drive DIO resistance. The tolerogenic function of DCs is revealed as a new potent target in metabolic disease management.
Assuntos
Células Dendríticas/metabolismo , Microbioma Gastrointestinal/fisiologia , Inflamação/metabolismo , Doenças Metabólicas/metabolismo , Obesidade/metabolismo , Animais , Células Dendríticas/patologia , Dieta Hiperlipídica , Inflamação/patologia , Masculino , Doenças Metabólicas/patologia , Camundongos , Camundongos Transgênicos , Obesidade/patologiaRESUMO
Alterations in the gut microbiota composition and diversity seem to play a role in the development of chronic diseases, including inflammatory bowel disease (IBD), leading to gut barrier disruption and induction of proinflammatory immune responses. This opens the door for the use of novel health-promoting bacteria. We selected five Parabacteroides distasonis strains isolated from human adult and neonates gut microbiota. We evaluated in vitro their immunomodulation capacities and their ability to reinforce the gut barrier and characterized in vivo their protective effects in an acute murine model of colitis. The in vitro beneficial activities were highly strain dependent: two strains exhibited a potent anti-inflammatory potential and restored the gut barrier while a third strain reinstated the epithelial barrier. While their survival to in vitro gastric conditions was variable, the levels of P. distasonis DNA were higher in the stools of bacteria-treated animals. The strains that were positively scored in vitro displayed a strong ability to rescue mice from colitis. We further showed that two strains primed dendritic cells to induce regulatory T lymphocytes from naïve CD4+ T cells. This study provides better insights on the functionality of commensal bacteria and crucial clues to design live biotherapeutics able to target inflammatory chronic diseases such as IBD.
Assuntos
Bacteroidetes/genética , Bacteroidetes/imunologia , Colite/induzido quimicamente , Colite/microbiologia , Microbioma Gastrointestinal/imunologia , Ácido Trinitrobenzenossulfônico/efeitos adversos , Adulto , Animais , Bacteroidetes/isolamento & purificação , Células CACO-2 , Colite/imunologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Humanos , Recém-Nascido , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T Reguladores/imunologiaRESUMO
Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H(2)O(2) production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior.
Assuntos
Ferro/metabolismo , Lactobacillus delbrueckii/crescimento & desenvolvimento , Leite/microbiologia , Nitrogênio/metabolismo , Purinas/metabolismo , Streptococcus thermophilus/química , Streptococcus thermophilus/genética , Animais , Proteínas de Bactérias/análise , Técnicas de Cocultura , Perfilação da Expressão Gênica , Proteoma/análise , Streptococcus thermophilus/crescimento & desenvolvimentoRESUMO
Lactic acid bacteria (LAB) gradually acidify their environment through the conversion of pyruvate to lactate, an essential process to regenerate NAD(+) used during glycolysis. A clear demonstration of acidification can be found in yogurt, the product of milk fermentation by the LAB Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus, where the pH falls to 4.2. Acid adaptation therefore plays an important role in the physiology of LAB. Here we present the results of a proteomic approach to reveal cellular changes associated with acid adaptation in L. bulgaricus. These results were complemented with transcription data for selected genes to show three major effects: (i) induction of the chaperones GroES, GroEL, HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP, and ClpL, and the repression of ClpC; (ii) induction of genes involved in the biosynthesis of fatty acids (fabH, accC, fabI); (iii) repression of genes involved in the mevalonate pathway of isoprenoid synthesis (mvaC, mvaS). Together with changes in the expression of other genes from the local metabolic network, these results for the first time show a coherent picture of changes in gene expression expected to result in a rerouting of pyruvate metabolism to favor fatty acid biosynthesis, and thereby affect membrane fluidity.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Lactobacillus/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Bactérias/genética , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Meios de Cultura/farmacologia , Eletroforese em Gel Bidimensional , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Concentração de Íons de Hidrogênio , Lactobacillus/efeitos dos fármacos , Lactobacillus/genética , Modelos Biológicos , Reação em Cadeia da PolimeraseRESUMO
Streptococcus thermophilus is a thermophilic lactic acid bacterium widely used as starter in the manufacture of dairy products in particular in yoghurt manufacture in combination with Lactobacillus delbrueckii ssp. bulgaricus. However, in spite of its massive use, the physiological state of S. thermophilus in milk has hardly been investigated. We established the first map of the cytosolic proteome of S. thermophilus LMG18311 grown in milk. It comprises 203 identified proteins corresponding to 32% of theoretical proteome. In addition, using proteomic and transcriptomic approaches, we analyzed the physiology of LMG18311 during its late stage of growth in milk (between 2h30 and 5h30). It revealed the up-regulation of (i) peptides and AA transporters and of specific AA biosynthetic pathways notably for sulfur AA and (ii) genes and proteins involved in the metabolism of various sugars. These two effects were also observed in LMG18311 grown in milk in coculture with L. bulgaricus although the effect on sugar metabolism was less pronounced. It suggests that the stimulatory effect of Lactobacillus on the Streptococcus growth is more complex than AA or peptides supply.
Assuntos
Aminoácidos Sulfúricos/metabolismo , Proteínas de Bactérias , Fermentação/fisiologia , Leite/metabolismo , Streptococcus thermophilus/fisiologia , Animais , Técnicas de Cocultura , Genes Bacterianos/fisiologia , Lactobacillus delbrueckii/crescimento & desenvolvimento , Nitrogênio/metabolismo , Proteoma/metabolismo , Streptococcus thermophilus/crescimento & desenvolvimento , Regulação para CimaRESUMO
Phytases catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol phosphates, myo-inositol (having important role in metabolism and signal transduction pathways), and inorganic phosphate. These enzymes have been widely used in animal feed in order to improve phosphorus nutrition and to decrease pollution in animal waste. Compared to previously described phytases, the phytase (PhyL) from Bacillus licheniformis ATCC 14580 has attractive biochemical properties which can increase the profitability of several biotechnological procedures (animal nutrition, humain health etc). Due to its amino acid sequence with critical substitutions, the PhyL could be a model to enhance other phytases features, in terms of thermal stability and high activity. Otherwise, an engineered PhyL, with low pH optimum, will represent a challenge within the class of ß- propeller phytases.
Assuntos
6-Fitase/química , Bacillus/enzimologia , Proteínas de Bactérias/química , Engenharia de Proteínas , 6-Fitase/genética , 6-Fitase/metabolismo , Bacillus/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Peso Molecular , TemperaturaRESUMO
Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain.
RESUMO
"Candidatus Arthromitus" sp. strain SFB-mouse-NL (SFB, segmented filamentous bacteria) is a commensal bacterium necessary for inducing the postnatal maturation of homeostatic innate and adaptive immune responses in the mouse gut. Here, we report the genome sequence of this bacterium, which sets it apart from earlier sequenced mouse SFB isolates.
RESUMO
The Serine Protease Inhibitors (Serpins) have been a focus of research by biomedical industries due to their critical role in human health. The use of serpin in the treatment of many diseases was widely investigated through the identification of new genes encoding these proteins in all kingdoms of life. The characterization of these genes revealed that they encoded proteins having low sequence homologies. Future developments are focusing not only on the protease inhibition activity, but also on the other effects due to the interactions of serpins with other components such as hormone transport. Here we give a concise overview of the most recent patents that have been reported in this field of research.
Assuntos
Serpinas/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Antivirais/química , Antivirais/uso terapêutico , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Fibrinólise , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Neoplasias/diagnóstico , Patentes como Assunto , Serpinas/química , Viroses/tratamento farmacológicoRESUMO
Silver nanoparticles capped with nine different sulphonated calix[n]arenes were tested for their anti-bacterial effects against B. subtilis and E. coli at an apparent concentration of 100 nM in calix[n]arene. The results show the para-sulphonato-calix[n]arenes are active against Gram positive bacteria and the derivatives having sulphonate groups at both para and alkyl terminal positions are active against Gram negative bacteria. The calix[6]arene derivative with only O-alkyl sulphonate groups shows bactericidal activity.
Assuntos
Antibacterianos/síntese química , Calixarenos/síntese química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/química , Antibacterianos/química , Antibacterianos/farmacologia , Calixarenos/química , Calixarenos/farmacologia , Estrutura MolecularRESUMO
Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli-B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.