This study purposes to examine extracellular proteases of the anaerobic rumen bacterium, Bacteroides amylophilus H-18. An enzyme was isolated and purified from 29 litres of 23 hr cell-free culture supernatant using DEAE Sephadex A-50, Sephadex G-200 and isoelectrofocusing techniques. Although proteolytic activity in the supernatant had a peak of activity at pH 6.7, there was activity at pH values from 4.5 to 11.5. Therefore, an attempt was made to purify the pH 6.7 activity and to follow the activity at other pH values as an index of purity. It was found that separation of the activities at different pH values was not achieved, even though the enzyme was purified 1265 times. Gel filtration of this purified material revealed the presence of two proteases, one of 60,000 and the other of 30,000 molecular weight. Since these enzymes were otherwise identical, they could have represented the monomeric and dimeric forms of a single protein. If the protease of 30,000 molecular weight was separated and resubjected to gel filtration, protease activity of molecular weight 60,000 reappeared. Ultracentrifugation of the 30,000 molecular weight protease demonstrated only one component. Therefore, if the two forms were in equilibrium, it appeared that the dimer was the more stable form of the enzyme. The purified protease did not contain cysteine, so that any tertiary structure in the enzyme could not involve disulfide bridges. All attempts to dissociate the dimeric into the monomeric form were unsuccessful. Examination of the inhibition of Nα benzoyl-L-arginine methyl ester esterase and protease activities with Nα tosyl-L-chloromethane revealed a complete inhibition of esterase activity at pH 8.0 but only a 30% inhibition of protease activity at the same pH, suggesting that more than one enzyme was responsible for the proteolytic activity exhibited by the purified enzyme. Because it was not possible to achieve separation of proteolytic activities at different pH values after a 1265 times purification, it must be assumed that if there are actually different proteases present they must have very similar structures. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Anxiety disorders, including generalized anxiety disorder (GAD), are prevalent mental health conditions with a complex etiology. The gut microbiota and diet have emerged as important factors in modulating anxiety symptoms. This thesis aimed to investigate the relationship between gut microbiota composition and anxiety symptoms in the context of GAD. This involved an in silico analysis of a cohort of GAD patients and healthy controls, coupled with experiments using a microbiota-humanized mouse model. A comprehensive in silico analysis was conducted using basic statistics and machine learning techniques on the human cohort. The analysis explored the associations between anxiety symptoms and various factors, including demographics, dietary intake, gastrointestinal symptoms, inflammatory markers, stool metabolites, microbiota composition, and PICRUSt2 microbiota predicted function. GAD patients exhibited higher gut microbiota abundance of Bacteroides, which was positively associated to carbohydrate degradation pathways. Machine learning analysis identified abdominal pain as a key indicator for distinguishing GAD donors. Moreover, anxiety symptoms were negatively correlated with inulin intake in GAD patients, altogether suggesting an association between a carbohydrate degradation imbalance in the GAD microbiota, abdominal pain, and anxiety symptoms. To investigate further, germ-free mice were colonized with stool samples from healthy controls and GAD patients, and they were fed either a low or high-fiber (inulin) diet. Assessments included behavioral tests, microbiota analysis, colonic gene expression, and mucus degradation. Bacteroides abundance positively correlated with anxiety-like behavior, mucus degradation, and expression of colonic genes related to immune activation, pain, and intestinal permeability, further supporting the results observed in the donor cohort. Improvements in metabolic parameters were also observed in mice fed high-fiber diet. Furthermore, we found that inulin dosage played a crucial role in mediating the observed immune activation and anxiety-like behavior, with excessive inulin supplementation showing a detrimental effect. This study provides insights into the complex relationship between dietary fiber, gut microbiota composition, and anxiety symptoms in mice and humans. Further studies are needed to determine optimal dosages of inulin supplementation as a potential therapeutic approach for managing anxiety symptoms. / Dissertation / Doctor of Philosophy (Medical Science) / Generalized anxiety disorder (GAD) is a common anxiety disorder that has complex causes. There's growing evidence that gut bacteria, known as microbiota, along with diet, can impact anxiety symptoms. In our study, we explored this connection by examining a group of GAD patients and healthy individuals, and by conducting experiments using a mouse model. We found that GAD patients have a microbiota with higher levels of Bacteroides and higher ability to feed from carbohydrates compared to healthy controls. We believe this is associated to abdominal pain and higher anxiety symptoms. To explore this in more depth, we introduced the gut microbiota from GAD patients and healthy controls into mice with no previous microbiota. Because we also found that GAD patients who ate foods with more specific fiber (inulin) content had less anxiety symptoms, we fed them a low or high-inulin diet and assessed anxiety-like behavior. We found that Bacteroides levels were associated with high anxiety-like behavior and gene expression in the colon associated to inflammation and pain in mice, further supporting the results found in the humans. Although more research is still needed, this study helps us better understand how the interaction between dietary fiber and gut bacteria can affect anxiety.

No description available.

Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250 / Ph. D.

The effect of various growth conditions on the type of superoxide dismutase (SGD) formed anaerobically in three Bacteriodes species was studied. B. fragilis, B. distasonis, and B. thetaiotaomicron were grown in ironâ restricted media with or without manganese supplementation. Iron availability was decreased by treatment of the media with chelex-100, a metal-chelating resin, and addition of desferrioxamine mesylate (desferal, Ciba-Geigy), an iron chelator. Mn-containing (MnSOD) and Fe-containing superoxide dismutase (EeSOD) activities in cell extracts were differentiated by inhibition with azide and inactivation by H₂0₂. The amount of Mn-containing superoxide dismutase was estimated by the fraction of azide- and H₂0₂, -resistant activity. Cells grown in untreated media contained approximately 90% FeSOD and 10% MnSOD. Cells grown in Fe-restricted media supplemented with graded amounts of manganese synthesized a progressively larger fraction of MnSOD. Hemin, added to the Fe-restricted media, did not serve as an iron source for FeSOD formation. Superoxide dismutase specific activities varied (3-6 U/mg) in each extract but not as a function of manganese concentration. / Master of Science

Thirty Bacteroides strains from the human colon were tested for ability to degrade cyclodextrins (CD). Twenty four strains were able to degrade CD. Cyclodextrinase in two of these strains B. ovatus 3524 and B. distasonis Cl8-7 has been studied. Organisms were grown on a minimal medium containing CD (0.5%), and cyclodextrinase activity was assayed by measuring the increase in reducing sugar (as glucose) when CD was incubated at 37℃ for 4 h with crude enzyme preparations. Cyclodextrinase activity was predominantly cell bound and induced in both organisms by growth on CD. Analysis via high performance liquid chromatography showed that products of CD hydrolysis by the crude enzyme preparations from the 2 strains were sharply different. B. ovatus 3524 cyclodextrinase yielded glucose only, while the B. distasonis Cl8-7 enzyme catalyzed production of a series of maltooligomers. Cyclodextrinase of both strains was stable at 4℃ for at least 48 h. B. distasonis Cl8-7 cyclodextrinase showed greater than 75% retention of activity at temperatures up to 55℃ after 48 h, whereas the B. ovatus 3524 enzyme was labile above 25℃. Optimum activity and stability of cyclodextrinase from both strains occured at pH 7.0. Salt precipitation and chromatographic methods were utilized in an attempt to purify the enzyme(s) in crude cyclodextrinase. No enzymes were purified to homogeneity, but a 15- to 17-fold increase in specific cyclodextrinase activity was obtained via hydrophobic interaction chromatography. Also, the products obtained by the action of cyclodextrinase from B. ovatus 3524 were markedly altered during purification, suggesting that the crude cyclodextrinase contains a mixture of enzymes. / M.S.

The serological and chemical properties of antigens extracted from strains of Bacteroides fragilis and related species belonging to several different DNA homology groups were investigated. Antisera prepared against formalin-treated whole cells suspensions of representative strains were tested against cell suspensions, cell wall preparations, and extracts of homologous and heterologous strains using agglutination, immunodiffusion, and hemagglutination techniques. Serological results indicated that the species were antigenically distinct, although minor cross reactions were observed across species boundaries. However, the serological properties did not appear to distinguish genetic heterogeneity at levels down to approximately 65% homology. Homology groups, including the two B. fragilis subgroups, were relatively homogeneous, although the presence of serotypes within each homology group was suggested. Immunodiffusion tests demonstrated, however, that the "homogeneity" was not always represented by a single "common" antigen but rather implied a mosaic antigen composition for each strain. A minimum of ten and six different antigenic factors were demonstrated on B. fragilis 2393 and 2553, respectively. Similar mosaics were observed with members of the other species. Hemagglutination patterns using crude antigen extracts were also consistent with the antigenic mosaic. Many of the strains were found to be capsulated. Preliminary studies demonstrated a similar sugar composition in the capsular material to that in lipopolysaccharide extracted from the same strain with aqueous phenol. Studies also suggested that the capsule influenced the serological properties of the cell. The chemical make-up of all of the Bacteroides LPS was found to be similar to that of LPS from facultative organisms, although KDO and heptose were not detected in any of the Bacteroides preparations. Electron microscopy of Bacteroides LPS demonstrated a trilaminar structure characteristic of LPS from other gram negative organisms. However, gel-filtration experiments suggested that the Bacteroides LPS may be of a different architecture, particularly regarding the core region. When hydrolyzed by weak acid, Bacteroides LPS behaved differently to LPS from facultative bacteria yielding large amounts of high molecular weight polymers and very little core-type material. B. distasonis and B. thetaiotaomicron displayed lower levels of high molecular weight material, as compared with the other strains, and may represent semi-rough strains. A comparison of the sugar patterns of the antigen extracts from one member of each homology group implied distinct differences among the strains. However, the sugar patterns of two B. fragilis strains which are genetically more related to each other than to the other species, were very similar. The constituents identified in the Bacteroides LPS were glucosamine, galactosamine, glucose, galactose, mannose, fucose, and rhamnose. An unidentified aniline pthalate reacting compound with similar mobility to colitose in chromatography studies was observed in the B. thetaiotaomicron 5482 LPS. It was not thought justified to assign definite chemotypes to individual homology groups due to the possible contamination of LPS preparation with non-LPS material, e.g. capsules or a glycan polymer. / Ph. D.

O objetivo deste trabalho foi analisar comparativamente alguns microrganismos que compõe a microbiota intestinal endógena de crianças eutróficas (30), com sobrepeso (24) e obesas (30) entre 3 a 11 anos, a partir de amostras fecais. Foi realizado o isolamento de espécies de Bacteroides, Parabacteroides e Clostridium; a identificação de B. fragilis e C. perfringens enterotoxigênicos; e a detecção quantitativa por PCR (SybrGreen) de B. fragilis, B.vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales e Clostridium (cluster I). As espécies C. perfringens e B. vulgatus foram as mais isoladas; nenhum isolado B. fragilis foi enterotoxigênico; todos C. perfringens foram classificados como tipo A e destes 8,7% e 12,2% possuiam os genes tpeL e netB, respectivamente. C. perfringens, C. difficile e Bifidobacterium spp. estavam em maior quantidade em crianças eutróficas, enquanto obesos e com sobrepeso apresentaram maior número de Lactobacillus spp. e Bacteroidales. / The aim of this study was to evaluate some microorganism from endogenous intestinal microbiota of normal weight (30), overweight (24) and obese (30) children between 3 and 11 years, from fecal samples. It was performed the isolation of species of Bacteroides, Parabacteroides and Clostridium; the identification of B. fragilis and C. perfringens enterotoxigenic; and the quantitative detection by PCR (SybrGreen) B. fragilis, B. vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales and Clostridium (cluster I). The species C. perfringens and B. vulgatus were the most isolated; no isolated B. fragilis was enterotoxigenic; all C. perfringens were classified as type A and these 8.7% and 12.2% harbored tpeL and netB genes, respectively. C. perfringens, C. difficile and Bifidobacterium spp. were in greater quantity in normal weight children while obese and overweight showed a higher number of Lactobacillus spp. and Bacteroidales.

O objetivo deste trabalho foi analisar comparativamente alguns microrganismos que compõe a microbiota intestinal endógena de crianças eutróficas (30), com sobrepeso (24) e obesas (30) entre 3 a 11 anos, a partir de amostras fecais. Foi realizado o isolamento de espécies de Bacteroides, Parabacteroides e Clostridium; a identificação de B. fragilis e C. perfringens enterotoxigênicos; e a detecção quantitativa por PCR (SybrGreen) de B. fragilis, B.vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales e Clostridium (cluster I). As espécies C. perfringens e B. vulgatus foram as mais isoladas; nenhum isolado B. fragilis foi enterotoxigênico; todos C. perfringens foram classificados como tipo A e destes 8,7% e 12,2% possuiam os genes tpeL e netB, respectivamente. C. perfringens, C. difficile e Bifidobacterium spp. estavam em maior quantidade em crianças eutróficas, enquanto obesos e com sobrepeso apresentaram maior número de Lactobacillus spp. e Bacteroidales. / The aim of this study was to evaluate some microorganism from endogenous intestinal microbiota of normal weight (30), overweight (24) and obese (30) children between 3 and 11 years, from fecal samples. It was performed the isolation of species of Bacteroides, Parabacteroides and Clostridium; the identification of B. fragilis and C. perfringens enterotoxigenic; and the quantitative detection by PCR (SybrGreen) B. fragilis, B. vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales and Clostridium (cluster I). The species C. perfringens and B. vulgatus were the most isolated; no isolated B. fragilis was enterotoxigenic; all C. perfringens were classified as type A and these 8.7% and 12.2% harbored tpeL and netB genes, respectively. C. perfringens, C. difficile and Bifidobacterium spp. were in greater quantity in normal weight children while obese and overweight showed a higher number of Lactobacillus spp. and Bacteroidales.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T17:57:41Z No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:18:16Z (GMT) No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) / Made available in DSpace on 2017-08-07T19:18:16Z (GMT). No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) Previous issue date: 2010-05-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Bacteroides fragilis é um anaeróbio Gram negativo membro da microbiota indígena do trato gastrintestinal, e é comumente isolado de infecções clínicas. À medida que as drogas antimicrobianas são introduzidas no ambiente, os microrganismos respondem, tornando-se resistentes, podendo gerar alterações na fisiologia celular, e que podem interferir na relação bactéria-hospedeiro. Nosso objetivo foi investigar alterações em B . fragilis após exposição a concentrações subinibitórias de ampicilina (AMP), ampicilina/sulbactam (AMS), clindamicina (CLI) e cloranfenicol (CLO); e investigar aspectos da relação droga-bactéria-hospedeiro, após desafio experimental em camundongos. Foram obtidas 4 linhagens bacterianas: BfAMP, BfAMS, BFCLI e BfCLO. Alterações na morfologia, padrões de crescimento, habilidades bioquímicas e fisiológicas, perfis de expressão protéica e semelhança genética foram avaliadas comparativamente entre as linhagens obtidas e a linhagem Bf parental ATCC 25285. Infecção experimental foi realizada em camundongos convencionais, desafiados intraperitonealmente. Histologia de baço e fígado, além de dosagem das citocinas TNF-α, IL-6, IL-10, IL-17 e IFN-γ no intestino, foram realizadas. Ampicilina e ampicilina/sulbactam induziram alterações morfológicas significativas nas células bacterianas na primeira exposição. Foi verificada diminuição da sensibilidade aos antimicrobianos em todas as linhagens selecionadas. Além disso, a exposição aos antimicrobianos foi capaz de induzir alterações nos padrões bioquímico-fisiológicos bacterianos, se comparado com a amostra parental. Em relação à capacidade de formação de biofilme, observou-se alteração em todas as linhagens, quando comparadas à parental, sendo que as linhagens selecionadas pela exposição aos antimicrobianos tiveram capacidade de aderência diminuída. A identidade genética das linhagens bacterianas obtidas foi confirmada e não foram detectados fragmentos de DNA polimórfico. Variações nos perfis de citocinas foram verificadas nos animais desafiados com a linhagem BfAMS. Alterações histopatológicas também foram observadas no baço e fígado dos animais desafiados com as linhagens de B . fragilis selecionadas por exposição aos antimicrobianos. Nossos resultados permitem sugerir fenômenos relacionados à regulação da expressão gênica como uma resposta momentânea ou evolutiva dos microrganismos. Estes resultados podem ainda sugerir que essas drogas tem diferentes interações com a célula bacteriana, que podem ser prejudiciais para a interação bactéria-hospedeiro. Assim, nossos resultados chamam a atenção para o risco de terapia antimicrobiana inadequada, mesmo com baixas concentrações de fármacos, que poderiam alterar os níveis de expressão gênica de uma bactéria e modular sua patogenicidade. / Bacteroides fragilis is an anaerobic Gram negative rod member of resident gut microbiota, isolated from clinical infections due in part to its potent virulence factors. As antimicrobial drugs are introduced into the environment, microorganisms respond by becoming resistant and may cause changes in cell physiology. These changes can interfere with the bacterium-host relationship. The aim of this study was to investigate changes in B . fragilis after exposure to ampicillin (AMP), ampicillin/sulbactam (AMS), clindamycin (CLI) and chloramphenicol (CLO); and to investigate changes in host-bacteria relationships after experimental challenge in mice. Four bacterial strains were obtained: BfAMP, BfAMS, BFCLI e BfCLO. Morphological changes, growth patterns, biochemical and physiological abilities, protein expression profile and genetic relationships were comparatively evaluated among the drug-selected strains and Bf parent ATCC 25285. Experimental infection was performed intraperitoneally in conventional mice. Liver and spleen histology, besides cytokines determinations were performed in the intestines. Ampicillin and ampicillin/sulbactam induced the highest significant morphological alterations in the bacterial cells after the initial drug exposure. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria strains. Furthermore, drug-exposure was able to induce changes in biochemical pathways and bacterial physiology, if compared with the Bf parent strain. Regarding the ability of biofilm formation, alterations in adherence properties were observed for all drug-selected bacteria, being this characteristic down regulated. The genetic identities were confirmed for the selected strains and no DNA polymorphisms were detected. The cytokine patterns in the intestines from mice challenged with drug-selected bacteria were highly altered, mainly considering the BfAMS strain, as well as the histological aspect concerning the liver and the spleen excised from the experimentally challenged animals. Our results may suggest phenomena related to the gene expression regulation, as a microbial momentary or evolutionary response to the selective pressure of the antimicrobial drugs. These results may also suggest that these drugs have different interactions with the bacterial cell which can be harmful to the host-bacteria relationships. In this way, the data ask for attention regarding the risks of inappropriate chemotherapy, even with low concentrations of the antimicrobial drugs, which might the bacteria genetic expression levels and modulate their pathogenicity