RESUMO
Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.
Assuntos
Azospirillum brasilense , Microalgas , Microalgas/genética , Biocombustíveis , Transcriptoma , Ácidos Indolacéticos/metabolismo , Perfilação da Expressão Gênica , Adaptação Fisiológica/genética , Riboflavina/genética , Riboflavina/metabolismoRESUMO
Autonomous control of gene expression through engineered quorum-sensing processes is broadly applicable to biosynthetic pathways, including simultaneous control of different genes. It is also a powerful tool for balancing growth and production. We had previously engineered a modular autoinduction device for the control of gene expression in B. subtilis. Now, we expand its functionality to repress gene expression autonomously. The engineered R8 promoter responds to AHL accumulation in the culture medium. In a riboflavin-producing strain, the AHL-Lux complex exerts 5-fold repression on the R8-driven expression of the flavokinase/FAD synthetase gene ribC, resulting in a higher titer of the vitamin. We engineered a strain able to autonomously induce and repress different genes simultaneously, demonstrating the potential of the device for use in metabolic engineering.
Assuntos
Bacillus subtilis , Riboflavina , Bacillus subtilis/metabolismo , Riboflavina/metabolismo , Regiões Promotoras Genéticas , Vias Biossintéticas , Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia MetabólicaRESUMO
AIMS: To evaluate a mixture of selected lactic acid bacteria (LAB) (a riboflavin-producer, a folate-producer and an immunomodulatory strain) as co-adjuvant for 5-fluorouracil (5-FU) chemotherapy in cell culture and using a 4T1 cell animal model of breast cancer. METHODS AND RESULTS: The viability of Caco-2 cells exposed to 5-FU and/or LAB was analysed. Mice bearing breast tumour were treated with 5-FU and/or LAB. Tumour growth was measured. Intestinal mucositis (IM) was evaluated in small intestine; haematological parameters and plasma cytokines were determined. The bacterial mixture did not negatively affect the cytotoxic activity of 5-FU on Caco-2 cells. The LAB mixture attenuated the IM and prevented blood cell decreases associated with 5-FU treatment. Mice that received 5-FU and LAB mixture decreased tumour growth and showed modulation of systemic cytokines modified by both tumour growth and 5-FU treatment. The LAB mixture by itself delayed tumour growth. CONCLUSIONS: The mixture of selected LAB was able to reduce the side-effects associated with chemotherapy without affecting its primary anti-tumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This bacterial mixture could prevent the interruption of conventional oncologic therapies by reducing undesirable side-effects. In addition, this blend would provide essential nutrients (vitamins) to oncology patients.
Assuntos
Adjuvantes Imunológicos , Neoplasias da Mama/terapia , Fluoruracila/uso terapêutico , Lactobacillales/imunologia , Lactobacillales/metabolismo , Animais , Antineoplásicos/uso terapêutico , Células CACO-2 , Linhagem Celular , Sobrevivência Celular , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Ácido Fólico/metabolismo , Humanos , Imunomodulação , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosite/microbiologia , Mucosite/patologia , Riboflavina/metabolismo , VitaminasRESUMO
Reactive oxygen species (ROS) have been described in their double physiological function, helping in the maintenance of health as well as contributing to oxidative stress. Diabetes mellitus is a chronical disease nearly related to oxidative stress, whose treatment (in type II variant) consists in the administration of antidiabetic compounds (Andb) such as Gliclazide (Gli) and Glipizide (Glip). In this context, as Andb may be exposed to high ROS concentrations in diabetic patients, we have studied the potential ROS-mediated degradation of Gli and Glip through photosensitized processes, in the presence of Riboflavin (Rf) vitamin. We found that singlet oxygen (O2 (1 ∆g )) participated in the Rf-sensitized photodegradation of both Andb, and also superoxide radical anion in the case of Gli. Two principal products derived from O2 (1 ∆g )-mediated degradation of Gli were identified and their chemical structures characterized, through HPLC mass spectrometry. O2 (1 ∆g )-mediated degradation products and their toxicity was assayed on Vero cell line. These studies demonstrated that neither Gli nor its photoproducts caused cytotoxic effect under the experimental conditions assayed. Our results show strong evidences of ROS-mediated Andb degradation, which may involve the reduction or loss of their therapeutic action, as well as potential cytotoxicity derived from their oxidation products.
Assuntos
Gliclazida/química , Glipizida/química , Hipoglicemiantes/química , Fármacos Fotossensibilizantes/química , Riboflavina/química , Oxigênio Singlete/química , Superóxidos/química , Animais , Biotransformação/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/tratamento farmacológico , Gliclazida/metabolismo , Gliclazida/farmacologia , Glipizida/metabolismo , Glipizida/farmacologia , Humanos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Cinética , Luz , Oxirredução , Fotólise , Fármacos Fotossensibilizantes/metabolismo , Riboflavina/metabolismo , Oxigênio Singlete/metabolismo , Soluções , Espectrometria de Fluorescência , Superóxidos/metabolismo , Células VeroRESUMO
Vibrio cholerae, a pandemic diarrheagenic bacterium, is able to synthesize the essential vitamin riboflavin through the riboflavin biosynthetic pathway (RBP) and also to internalize it through the RibN importer. In bacteria, the way riboflavin biosynthesis and uptake functions correlate is unclear. To gain insights into the role of the riboflavin provision pathways in the physiology of V. cholerae, we analyzed the transcriptomics response to extracellular riboflavin and to deletions of ribD (RBP-deficient strain) or ribN. Many riboflavin-responsive genes were previously reported to belong to the iron regulon, including various iron uptake genes. Real time PCR analysis confirmed this effect and further documented that reciprocally, iron regulates RBP and ribN genes in a riboflavin-dependent way. A subset of genes were responding to both ribD and ribN deletions. However, in the subset of genes specifically affected in the ∆ribD strain, the functional terms protein folding and oxidation reduction process were enriched, as determined by a Gene Ontology analysis. In the gene subset specifically affected in the ∆ribN strain, the cytochrome complex assembly functional term was enriched. Results suggest that iron and riboflavin interrelate to regulate its respective provision genes and that both common and specific effects of biosynthesized and internalized riboflavin exist.
Assuntos
Perfilação da Expressão Gênica , Ferro/metabolismo , Riboflavina/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Transporte Biológico , MutaçãoRESUMO
The Brazilian Healthy Eating Index-Revised (BHEI-R) can be used to determine overall dietary patterns. We assessed the BHEI-R scores in children and adolescents, aged from 9 to 13 years old, and associated its component scores with biomarkers of health and dietary exposure. Three 24-h recalls were used to generate BHEI-R. Biomarkers were analyzed in plasma and red blood cells. Correlation tests, agreement, and covariance analyses were used to associate BHEI-R components with biomarkers. Data from 167 subjects were used. The strongest correlations were between fruits, vegetables and legumes with omega-6 and omega-3 fatty acids, and ß-carotene intakes. Milk and dairy correlated with plasma retinol and pyridoxine. All components rich in vegetable and animal protein sources correlated with plasma creatine. Total BHEI-R scores were positively associated with intakes of omega-6, omega-3, fiber and vitamin C, and inversely associated with energy and saturated fat intakes of individuals. Plasma ß-carotene and riboflavin biomarkers were positively associated with total BHEI-R. An inadequate food consumption pattern was captured by both biomarkers of health and dietary exposure. BHEI-R was validated for the above dietary components and can be associated with metabolomics and nutritional epidemiological data in future pediatric studies.
Assuntos
Fenômenos Fisiológicos da Nutrição do Adolescente , Fenômenos Fisiológicos da Nutrição Infantil , Dieta Saudável , Avaliação Nutricional , Cooperação do Paciente , Adolescente , Fenômenos Fisiológicos da Nutrição do Adolescente/etnologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Brasil , Criança , Fenômenos Fisiológicos da Nutrição Infantil/etnologia , Dieta Saudável/etnologia , Eritrócitos/metabolismo , Fabaceae/química , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/administração & dosagem , Ácidos Graxos Ômega-6/sangue , Ácidos Graxos Ômega-6/metabolismo , Frutas/química , Humanos , Estudos Longitudinais , Valor Nutritivo , Cooperação do Paciente/etnologia , Riboflavina/administração & dosagem , Riboflavina/sangue , Riboflavina/metabolismo , Sementes/química , Autorrelato , Verduras/química , beta Caroteno/administração & dosagem , beta Caroteno/sangue , beta Caroteno/metabolismoRESUMO
AIM: To assess the anti-inflammatory effect associated with individual probiotic suspensions of riboflavin-producing lactic acid bacteria (LAB) in a colitis murine model. METHODS AND RESULTS: Mice intrarectally inoculated with trinitrobenzene sulfonic acid (TNBS) were orally administered with individual suspensions of riboflavin-producing strains: Lactobacillus (Lact.) plantarum CRL2130, Lact. paracasei CRL76, Lact. bulgaricus CRL871 and Streptococcus thermophilus CRL803; and a nonriboflavin-producing strain or commercial riboflavin. The extent of colonic damage and inflammation and microbial translocation to liver were evaluated. iNOs enzyme was analysed in the intestinal tissues and cytokine concentrations in the intestinal fluids. Animals given either one of the four riboflavin-producing strains showed lower macroscopic and histologic damage scores, lower microbial translocation to liver, significant decreases of iNOs+ cells in their large intestines and decreased proinflammatory cytokines, compared with mice without treatment. The administration of pure riboflavin showed similar benefits. Lact. paracasei CRL76 accompanied its anti-inflammatory effect with increased IL-10 levels demonstrating other beneficial properties in addition to the vitamin production. CONCLUSION: Administration of riboflavin-producing strains prevented the intestinal damage induced by TNBS in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: Riboflavin-producing phenotype in LAB represents a potent tool to select them for preventing/treating IBD.
Assuntos
Colite/tratamento farmacológico , Lactobacillus/metabolismo , Probióticos/administração & dosagem , Riboflavina/metabolismo , Streptococcus thermophilus/metabolismo , Animais , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Fezes/microbiologia , Feminino , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/metabolismo , Riboflavina/metabolismo , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Crithidia fasciculata/genética , Crithidia fasciculata/metabolismo , Humanos , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Estágios do Ciclo de Vida , Modelos Lineares , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Proteínas de Protozoários/genética , Ratos , Riboflavina/análogos & derivados , Trypanosoma cruzi/metabolismoRESUMO
Riboflavin derivatives are essential cofactors for a myriad of flavoproteins. In bacteria, flavins importance extends beyond their role as intracellular protein cofactors, as secreted flavins are a key metabolite in a variety of physiological processes. Bacteria obtain riboflavin through the endogenous riboflavin biosynthetic pathway (RBP) or by the use of importer proteins. Bacteria frequently encode multiple paralogs of the RBP enzymes and as for other micronutrient supply pathways, biosynthesis and uptake functions largely coexist. It is proposed that bacteria shut down biosynthesis and would rather uptake riboflavin when the vitamin is environmentally available. Recently, the overlap of riboflavin provisioning elements has gained attention and the functions of duplicated paralogs of RBP enzymes started to be addressed. Results point towards the existence of a modular structure in the bacterial riboflavin supply pathways. Such structure uses subsets of RBP genes to supply riboflavin for specific functions. Given the importance of riboflavin in intra and extracellular bacterial physiology, this complex array of riboflavin provision pathways may have developed to contend with the various riboflavin requirements. In riboflavin-prototrophic bacteria, riboflavin transporters could represent a module for riboflavin provision for particular, yet unidentified processes, rather than substituting for the RBP as usually assumed.
Assuntos
Bactérias/genética , Bactérias/metabolismo , Redes e Vias Metabólicas , Riboflavina/metabolismoRESUMO
Inflammatory bowel diseases (IBD) are idiopathic diseases of the gastrointestinal tract characterised by recurrent inflammation that require lifelong treatments. It has been shown that certain strains of lactic acid bacteria (LAB) can produce specific health-promoting compounds in foods or in the gastrointestinal tract that can in turn prevent and/or treat IBD. This study was designed to evaluate the possible therapeutic potential of soymilk fermented by the riboflavin-producing strain Lactobacillus plantarum CRL 2130 in a trinitrobenzene sulfonic induced colitis mouse model. Mice that received soymilk fermented by L. plantarum CRL 2130 showed a decrease in weight loss, lower damage scores in their large intestines, lower microbial translocation to liver and decreased cytokines levels in their intestinal fluids compared to animals that received unfermented soymilk or soymilk fermented by a non-riboflavin-producing L. plantarum strain. This is the first report that demonstrates that a riboflavin-producing LAB was able to prevent experimental colitis in a murine model.