RESUMO
Chronic intestinal inflammation is associated with strong alterations of the microbial composition of the gut. Probiotic treatments and microbiota-targeting approaches have been considered to reduce the inflammation, improve both gut barrier function as well as overall gastrointestinal health. Here, a murine model of experimental colitis was used to assess the beneficial health effects of Bacillus subtilis SF106 and Bacillus clausii (recently renamed Shouchella clausii) SF174, two spore-forming strains previously characterised in vitro as potential probiotics. Experimental colitis was induced in BALB/c mice by the oral administration of dextran sodium sulphate (DSS) and groups of animals treated with spores of either strain. Spores of both strains reduced the DSS-induced inflammation with spores of B. clausii SF174 more effective than B. subtilis SF106. Spores of both strains remodelled the mouse gut microbiota favouring the presence of beneficial microbes such as members of the Bacteroidetes and Akkermansia genera.
Assuntos
Bacillus clausii , Bacillus subtilis , Colite , Sulfato de Dextrana , Modelos Animais de Doenças , Microbioma Gastrointestinal , Camundongos Endogâmicos BALB C , Probióticos , Esporos Bacterianos , Animais , Probióticos/administração & dosagem , Colite/microbiologia , Colite/induzido quimicamente , Colite/terapia , Camundongos , Sulfato de Dextrana/toxicidade , Inflamação/microbiologia , Bacteroidetes , Akkermansia , FemininoRESUMO
A well-established rat model of diet-induced metabolic syndrome was used to evaluate the effects of the oral administration of spores or cells of HU16, a carotenoid-producing strain of Bacillus indicus. Symptoms of metabolic syndrome were induced in 90-days old, male Sprague-Dawley rats maintained for eight weeks on a high-fat diet, as previously reported. Parallel groups of animals under the same diet regimen also received a daily dose of 1×1010 cells or spores of B. indicus HU16. Cells of strain HU16 were able to reduce symptoms of metabolic syndrome, plasma markers of inflammation and oxidative markers in plasma and liver to levels similar to those observed in rats under a standard diet. HU16 cells did not affect obesity markers or the accumulation of triglycerides in the liver of treated animals. Denaturing gradient gel electrophoresis analysis showed that the oral administration of HU16 cells did not significantly affect the gut microbiota of high fat-fed rats, suggesting that the observed beneficial effects are not due to a reshaping of the gut microbiota but rather to metabolites produced by HU16 cells.
Assuntos
Antioxidantes/metabolismo , Bacillus/crescimento & desenvolvimento , Bacillus/metabolismo , Carotenoides/metabolismo , Dieta Hiperlipídica/efeitos adversos , Síndrome Metabólica/terapia , Probióticos/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Inflamação/patologia , Masculino , Síndrome Metabólica/patologia , Estresse Oxidativo , Plasma/química , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.
Assuntos
Bacillus megaterium/fisiologia , Células Epiteliais/microbiologia , Bacillus megaterium/classificação , Bacillus megaterium/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/uso terapêutico , Células CACO-2 , Ciclo Celular , Linhagem Celular Tumoral , Meios de Cultura , Células Epiteliais/citologia , Proteínas de Choque Térmico HSP27/metabolismo , Células HT29 , Humanos , Percepção de Quorum , Proteínas Repressoras/metabolismo , Proteínas Repressoras/uso terapêutico , Transdução de Sinais , Estresse FisiológicoRESUMO
AIMS: Litopenaeus vannamei do not have an adaptive immune response system. White spot syndrome virus (WSSV) is the most severe pathogen in shrimps. Bacillus subtilis spores carrying heterologous antigens on its surface have been evaluated as a vaccine inducing specific systemic responses on vertebrates. Orally administrated Vp28 vaccines have been investigated in crustaceans. Vp26 is also an important constituent of WSSV structure but little is known about its oral vaccination capacity in L. vannamei. METHODS AND RESULTS: In this study, for first time, L. vannamei WSSV protection was carried out using B. subtilis recombinant spores (RS), displaying CotC::Vp26 fusion protein (FP) on its surface. RS-expressing FP were coated on shrimp food pellets and used to feed L. vannamei. Results have shown that orally administered B. subtilis RS protected 100% L. vannamei against WSSV infection. CONCLUSIONS: Bacillus subtilis spores orally administrated expressing CotC::Vp26 fusion protein on its surface demonstrated the great capacity of Vp26 to induce immune protection, equally or even greater than Vp28 in L. vannamei. SIGNIFICANCE AND IMPACT OF THE STUDY: The biotechnological process developed represents an easy to produce, practical to handle, environmentally stable, human-safe and economically feasible opportunity to apply a new Vp26 vaccine in a massively way in shrimp farms.
Assuntos
Bacillus subtilis/genética , Penaeidae/virologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Vírus da Síndrome da Mancha Branca 1/imunologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Penaeidae/imunologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/imunologia , Esporos Bacterianos/genética , Vacinação/veterinária , Proteínas do Envelope Viral/genética , Vacinas Virais/genéticaRESUMO
Coeliac disease (CD) is a very common food-sensitive enteropathy, which is triggered by gluten ingestion and is mediated by CD4(+) T cells. In addition, alterations in the intestinal microbiota that is normally involved in the homeostasis of GALT (gut-associated lymphoid tissue) seem to play a role in CD. In accordance with these findings, we previously reported that Lactobacillus casei can induce a strong enhancement of the T cell-mediated response to gliadin without inducing enteropathy. In this study, we analysed the effects of L. casei administration in a mouse model of gliadin-induced villous damage that was recently developed and involves the inhibition of cyclo-oxygenase (COX) activities in gliadin-sensitized HLA-DQ8 transgenic mice. To address the issue, we assessed the weight loss, the intestinal cytokine pattern, the density of CD25(+) cells and morphometry of the gut mucosa. We confirmed that COX inhibition in sensitized mice caused villus blunting, dysregulated expression of tumour necrosis factor (TNF)-α and reduced gliadin-specific IL-2 production. Notably, the administration of probiotic strain induced a complete recovery of villus blunting. This finding was associated with a delay in weight decrease and a recovery of basal TNF-α levels, whereas the numbers of CD25(+) cells and the levels of IL-2 remained unchanged. In conclusion, our data suggest that the administration of L. casei can be effective in rescuing the normal mucosal architecture and GALT homeostasis in a mouse model of gliadin-induced enteropathy.
Assuntos
Doença Celíaca , Gliadina/imunologia , Antígenos HLA-DQ , Lacticaseibacillus casei/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Citocinas/análise , Gliadina/metabolismo , Glutens , Fatores Imunológicos , Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2 , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Redução de PesoRESUMO
AIMS: To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation. MATERIALS AND RESULTS: Sampling of heat-resistant bacterial counts from soil, sea water and the human gastrointestinal tract. Phylogenetic profiling using analysis of 16S rRNA sequences to define species. Pigment profiling using high-performance liquid chromatography-photo diode array analysis. CONCLUSIONS: The most commonly found pigments were yellow, orange and pink. Isolates were nearly always members of the Bacillus genus and in most cases were related with known species such as Bacillus marisflavi, Bacillus indicus, Bacillus firmus, Bacillus altitudinis and Bacillus safensis. Three types of carotenoids were found with absorption maxima at 455, 467 and 492 nm, corresponding to the visible colours yellow, orange and pink, respectively. Although the presence of other carotenoids cannot be ruled out, these three predominant carotenoids appear to account for the pigments obtained in most pigmented bacilli, and our analysis reveals the existence of a C30 biosynthetic pathway. Interestingly, we report the presence of a water-soluble pigment that may also be a carotenoid. The function of carotenoids is photoprotection, and carotenoid-containing spores exhibited significantly higher levels of resistance to UV radiation than non-carotenoid-containing Bacillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that pigmented bacilli are ubiquitous and contain new carotenoid biosynthetic pathways that may have industrial importance.
Assuntos
Bacillus/química , Carotenoides/química , Bacillus/classificação , Bacillus/genética , Bacillus/isolamento & purificação , Trato Gastrointestinal/microbiologia , Humanos , Filogenia , Pigmentação , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Microbiologia do Solo , Esporos Bacterianos/química , Esporos Bacterianos/classificação , Esporos Bacterianos/genética , Esporos Bacterianos/isolamento & purificação , Microbiologia da ÁguaRESUMO
In the present paper a factor analysis is presented for the enzymatic transesterification of waste oil for biodiesel production. The experimental data on batch reactor evidence two key variables: enzyme loading and mixing conditions. These variables were subjected to a factor analysis and their combined effect on the reaction performance was determined. Response surface methodology (RSM) was used based on a linear first order model (steepest ascent method) and on a second order one in proximity of the optimal solution. The result was a model able to predict reaction performance within the range of mixing rates and enzyme amount considered for model formulation and outside of it, as shown in the final validation. Best performances were obtained at high stirring and high enzyme loading.
Assuntos
Fontes de Energia Bioelétrica , Óleos de Plantas/química , Resíduos/análise , Esterificação , Modelos Químicos , Reprodutibilidade dos Testes , Propriedades de Superfície , Fatores de TempoRESUMO
AIMS: To isolate and characterize spore-former bacteria able to colonize the human gastrointestinal tract (GIT). METHODS AND RESULTS: A total of 25 spore-formers was isolated from faeces and ileal biopsies of healthy human volunteers and identified at the species level. Physiological analysis was performed to evaluate the ability of the various isolates to form biofilms, to swarm, to produce surfactants and molecules that have antimicrobial activity against selected pathogens. To assess the potential probiotic activity of the isolates, we tested the resistance of cells and spores to simulated gastric conditions, the ability to grow and sporulate in anaerobic conditions and the presence of toxin-encoding genes in their genome. CONCLUSIONS: Spore-formers belonging to various bacterial species have been isolated from the gut of healthy human volunteers. These strains appear to be well adapted to the intestinal environment and we propose them as potential probiotic strains for human use and as oral vaccine vehicles. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge this is the first detailed characterization of spore-forming Bacilli from the human GIT. Our data suggest that the isolated species do not transit, but rather colonize this specific habitat and propose them as probiotic strains for human use.
Assuntos
Bacillus/fisiologia , Trato Gastrointestinal/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Adulto , Anaerobiose , Animais , Bacillus/patogenicidade , Biofilmes/crescimento & desenvolvimento , Eletroforese em Gel de Ágar , Enterotoxinas/biossíntese , Fezes/microbiologia , Feminino , Humanos , Ílio/microbiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Esporos Bacterianos/isolamento & purificação , Fatores de Virulência/análiseRESUMO
We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.
Assuntos
Bacillus subtilis/genética , Proteínas Recombinantes de Fusão/metabolismo , Esporos Bacterianos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Imunoglobulina G/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Toxoide Tetânico/genética , Toxoide Tetânico/imunologiaRESUMO
Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.
Assuntos
Bacillus subtilis/fisiologia , Modelos Animais , Probióticos , Esporos Bacterianos/fisiologia , Animais , Contagem de Colônia Microbiana , Fezes/microbiologia , Feminino , CamundongosRESUMO
HrcA is a negative transcriptional factor controlling the expression of the stress-specific operons dnaK and groESL in several bacteria. Although the HrcA structural gene has been identified in various organisms, studies at the protein level have been so far limited and mostly restricted to Bacillus subtilis. We have identified the HrcA protein of Streptococcus thermophilus and show here that it is a dimer with a native molecular mass of 74.5 kDa and a sequence-specific DNA-binding activity. Partially denatured and inactive S. thermophilus HrcA recovered its binding activity in the presence of the GroEL chaperone.
Assuntos
Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/genética , Streptococcus/genética , Streptococcus/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Chaperonina 60/genética , Chaperonina 60/metabolismo , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Genes Bacterianos , Proteínas de Choque Térmico HSP70/genética , Peso Molecular , Desnaturação Proteica , Renaturação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismoRESUMO
Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.
Assuntos
Bacillus , Gastroenteropatias/prevenção & controle , Gastroenteropatias/terapia , Probióticos/uso terapêutico , Administração Oral , Animais , Bacillus/efeitos dos fármacos , Bacillus/genética , Bacillus/ultraestrutura , Bacillus subtilis/genética , Sequência de Bases , Primers do DNA/genética , Resistência Microbiana a Medicamentos , Genes Bacterianos , Humanos , Microscopia Eletrônica , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Especificidade da Espécie , Esporos Bacterianos/ultraestruturaRESUMO
Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.
Assuntos
Mutagênese Insercional/métodos , Streptococcus/genética , Sequência de Bases , Southern Blotting , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Recombinante , Mutação , Plasmídeos/genéticaRESUMO
Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine.
Assuntos
Bacillus/fisiologia , Intestinos/microbiologia , Probióticos , Esporos Bacterianos/fisiologia , Administração Oral , Animais , Bacillus/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/fisiologia , Ácidos e Sais Biliares/farmacologia , Ácido Desoxicólico/farmacologia , Feminino , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Esporos Bacterianos/efeitos dos fármacos , Ácido Taurodesoxicólico/farmacologiaRESUMO
We used a PCR-based method to generate a single base pair mutation in the proB gene of Streptococcus thermophilus, which replaced an aspartic acid with a glycine residue at position 192 of the first proline biosynthetic enzyme gamma-glutamyl kinase. This was the first identified mutation in amino acid biosynthesis in S. thermophilus to our knowledge. The mutation caused an enhanced, feedback-resistant gamma-glutamyl kinase activity and conferred an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene.
Assuntos
Escherichia coli/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Mutação Puntual , Streptococcus/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Azetidinocarboxílico/farmacologia , Sequência de Bases , Escherichia coli/enzimologia , Retroalimentação , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Carboxila)/química , Reação em Cadeia da Polimerase , Prolina/análogos & derivados , Prolina/farmacologia , Streptococcus/enzimologia , Transformação BacterianaRESUMO
Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.
Assuntos
Bacillus subtilis/fisiologia , Bacillus/fisiologia , Probióticos , Antibacterianos/farmacologia , Bacillus/classificação , Bacillus/efeitos dos fármacos , Bacillus/genética , Bacillus subtilis/classificação , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Resistência Microbiana a Medicamentos , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Esporos Bacterianos/fisiologiaRESUMO
Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of sigmaH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator sigma , Fatores de Transcrição , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Deleção de Genes , Genótipo , Cinética , Modelos Biológicos , Óperon , Fenótipo , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestruturaRESUMO
We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.