Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

Horizontal gene transfer (HGT) is one of the man driving forces of evolution in prokaryotes, and can also promote within-strain variation of bacterial species. The genomes of three previously sequenced Bacteroides fragilis strains, NCTC9343, 638R and YCH46 displayed evidence of extensive HGT, demonstrated by the presence of 28 divergent capsular polysaccharide-associated biosynthesis loci. The genomes of a further four B. fragilis strains, LS66, GNAB92, RD48 and BE1 were sequenced and analysed. Genomic comparisons of BE1 and GNAB92 with NCTC9343, 638R and YCH46 identified ten new divergent polysaccharide biosynthesis loci. There is consequently, the potential to express 38 different polysaccharides amongst these five strains. Such a high level of variation in capsular polysaccharides, in so few strains has not been previously observed. HGT has occurred in B. fragilis despite the presence of diverse Restriction-Modification systems. The genome sequences of NCTC9343 and 638R contained a gene, ubb, the product of which, BfUbb, has 63% identity to human ubiquitin. The closest DNA sequence homology is to a migratory grasshopper entomopox virus, suggesting acquisition of this gene was via inter-kingdom HGT. The ubb gene was also identified in the newly sequenced genomes of B. fragilis strains LS66 and RD48. BfUbb had a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts by Western blot analysis. The inability to detect BfUbb in periplasmic extracts isolated from a B. fragilis strain containing an ubb signal sequence deletion construct, supported the periplasmic location of the processed form of the protein and the requirement for the signal peptide for transport from the cytoplasm. BfUbb was also detected in concentrated supernatants containing outer membrane vesicles, suggesting a mechanism by which the protein may be delivered to the host. This is the first example of ubiquitin being produced by a prokaryote. Transduction by bacteriophages is one mechanism by which horizontal gene transfer can occur and can also be a useful tool for genetic manipulation. Fifteen potentially new B. fragilis-specific bacteriophages were isolated from filtered sewage and characterised by phage titres and restriction endonuclease cleavage profiles. Of the fifteen, seven phages appeared to be different to the previously identified phage ФED01. None of the bacteriophages were capable of transduction. B. fragilis is a predominant member of the gastrointestinal microbiota. To survive within this specific niche, bacteria must successfully compete with other organisms for nutrients and space, and withstand attacks from bacteriophages. HGT may aid in the survival of B. fragilis as a commensal.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/11). Includes bibliographical references.

Thesis (doctoral)--Vrije Universiteit te Amsterdam, 1986. / Summary in Dutch. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

REIS, A. C. M. Fatores de virulência e produção de biofilme de Bacteroides fragilis isolados da microbiota intestinal de cães. 2013. 87 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-07-27T12:53:54Z No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-07-27T15:17:11Z (GMT) No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) / Made available in DSpace on 2017-07-27T15:17:12Z (GMT). No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) Previous issue date: 2013-01-08 / Bacteroides fragilis is part of the fecal microbiota of animals, yet it is the most pathogenic species of the genus Bacteroides. The transformation these microrganisms commensals in pathogens agents is mainly due to its virulence factors. This microorganism has been isolated from several infections in dogs. Considering the pathogenic potential of B. fragilis and its importance in veterinary medicine, this study was designed to evaluate the virulence factors and biofilm formation of Bacteroides fragilis isolated from fecal samples from dogs. The samples used in this study were collected at the Veterinary Clinic of the State University of Ceará and processed at the Laboratory of Bacteriology of the Federal University of Ceará. Were phenotypically analyzed the virulence factors (production of hemolysins and hemagglutinins, hydrophobicity, the presence of the enzyme β-lactamase and capsule) and biofilm production of 13 strains of B. fragilis. To determine the pattern of sensitivity to clindamycin, metronidazole, chloramphenicol and penicillin was used in agar dilution technique. In vitro assays for detection of biofilms were performed by the methods Congo Red agar and Accession in microplates. All strains of B. fragilis showed capsule and produced no haemolysis. Regarding the production of hemagglutinin, 38% of the strains showed hemagglutination when used canine blood and 15% in human blood (A+). Of the strains studied, 7% had a cell surface hydrophobic. In total, 61% of the strains showed β-lactamase test positive. As regards the production of biofilm was observed by the method of strokes that 12 (92.3%) of the biofilm producers were isolated by the method and Adhesion microplates, it was found that eight (61.5%) strains were able to produce biofilm. All isolates were susceptible to metronidazole and resistant to penicillin and chloramphenicol. The rate of clindamycin resistance was 69.2%. This study showed that species B. fragilis isolated from normal canine microflora showed major virulence factors for pathogenicity, they possess the ability to form biofilms and show a high rate of resistance to clindamycin. / Bacteroides fragilis faz parte da microbiota fecal de animais, contudo é a espécie mais patogênica do gênero Bacteroides. A sua transformação de um microrganismo comensal para agente patogênico deve-se principalmente aos seus fatores de virulência. Este micro-organismo tem sido isolado de diversas infecções em cães. Considerando o potencial patogênico de B. fragilis e sua importância na medicina veterinária, o presente trabalho foi elaborado com o objetivo de avaliar os fatores de virulência e formação de biofilme de Bacteroides fragilis isolados de amostras fecais de cães. As amostras utilizadas neste estudo foram coletadas na Clinica Veterinária da Universidade Estadual do Ceará (UECE) e processadas no Laboratório de Bacteriologia da Universidade Federal do Ceará (UFC). Foram analisadas fenotipicamente os fatores de virulência (produção de hemolisinas e hemaglutininas, hidrofobicidade, presença da enzima - lactamase e cápsula) e produção de biofilme de 13 cepas da espécie B. fragilis. Para determinação do perfil de sensibilidade a clindamicina, metronidazol, cloranfenicol e penicilina foi utilizada a técnica de diluição em Agar. Os ensaios in vitro para detecção de biofilme foram realizados pelos métodos Agar Vermelho Congo (AVC) e Adesão em microplacas. Todas as cepas de B. fragilis apresentaram cápsula e não produziram hemólise. Com relação à produção de hemaglutininas, 38% das cepas são capazes de aglutinar sangue canino e 15% em sangue humano (A+). Das cepas estudadas, 7% apresentaram uma superfície celular hidrofóbica. No total, 61% das cepas produziram teste da - lactamase positivo. Quanto a produção de biofilme, foi observado pelo método do AVC que 12 (92,3%) dos isolados eram produtores de biofilme e por meio do método de Adesão em Microplacas, verificou-se que 8 (61,5%) cepas foram capazes de produzir biofilme. Todos os isolados foram sensíveis ao metronidazol e cloranfenicol e resistentes à penicilina. A taxa de resistência à clindamicina foi de 69,2%. Esse estudo mostrou que espécies de B. fragilis isolados da microbiota normal de cães apresentaram fatores de virulência importantes para sua patogenicidade, possuem a capacidade de formar biofilme e demonstram uma alta taxa de resistência a clindamicina.

Bacteroides fragilis is a member of the normal microbiota that resides in the human lower gastrointestinal tract. This bacterium is of clinical significance because it is the most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is important for fibrin-mediated abscess formation and limiting the spread of infection. B. fragilis can bind and degrade fibrinogen which may aid in its escape from abscesses into the bloodstream, thereby promoting bacteraemia. In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the extracellular matrix, found in association with fibrinogen at wound sites, has also been reported. An outer membrane protein, BF1705, expressed by B. fragilis was found to share homology with BspA from Tannerella forsythia which is known to bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC 9343 genome in the present work using a markerless gene deletion technology. Proteins derived from the outer membranes of wild-type B. fragilis were able to bind fibronectin and fibrinogen in far-western blots. Similar protein extracts from the ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of BF1705 in adhesive interactions with proteins of the host extracellular matrix. The possible involvement of BF1705 in fibrinogen degradation was ruled out because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases involved in degradation of fibrinogen, four genes encoding putative extracellular metallo- and serine proteases in the size range 45-50 kDa were deleted from the NCTC 9343 genome. All of the single and multiple mutants defective in these selected proteases were still capable of degrading fibrinogen as determined by zymography. Expression of eight B. fragilis proteases in E. coli did not lead to detectable degradation of fibrinogen. These observations suggest that these proteases alone cannot degrade fibrinogen and either that an unidentified protease is responsible for degradation or that there is redundancy in the proteases involved. Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from the environment to replenish the depleting intracellular nitrogen content. By examining the differential regulation of the B. fragilis transcriptome under nitrogen replete and depleting conditions, a potential role for BF1705 and secreted proteases in nutrient binding and assimilation were studied. Growth on conventional glucose defined medium with ammonia as the nitrogen source was compared to growth in defined medium with glutamine as nitrogen source. A reduced doubling time and diauxic growth in the medium containing glutamine indicated nitrogen limitation. Comparison of the transcriptome derived from cultures of B. fragilis grown on either ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of BF1705 in response to nitrogen limitation. This observation in conjunction with its inability to degrade fibrinogen suggests that the primary role of BF1705 might be as an adhesin and does not act directly in nutrient binding and degradation. Nevertheless, nitrogen limitation was found to induce the expression of four protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular weight of three of these proteases were identified to be within the size range of 45-55 kDa which corresponded to the lysis bands detected by fibrinogen zymography with wild-type B. fragilis protein extracts. Therefore the possible involvement of these three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation (adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B, under conditions of nitrogen limitation suggest a previously uncharacterised aspartate metabolism pathway for ammonia generation via arginine catabolism in B. fragilis. Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen deprived conditions. In addition to nitrogen assimilation, significant upregulation was observed in the expression of genes involved in regulation of oxidative stress and metronidazole resistance. The observed changes in the transcriptome will add to our understanding of the B. fragilis metabolism and potential assist with unravelling the mechanisms of infection mediated by this important opportunistic pathogen.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-07-03T18:12:55Z No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 3934181 bytes, checksum: f5f6a1d475abc70500055be8358b14c7 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-08T13:14:10Z (GMT) No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 3934181 bytes, checksum: f5f6a1d475abc70500055be8358b14c7 (MD5) / Made available in DSpace on 2017-08-08T13:14:10Z (GMT). No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 3934181 bytes, checksum: f5f6a1d475abc70500055be8358b14c7 (MD5) Previous issue date: 2015-04-10 / Bastonetes Gram negativos anaeróbicos da espécie Bacteroides fragilis são membros da microbiota residente intestinal, e frequentemente associados a infecções endógenas. Para o tratamento destas infecções, o metronidazol persiste como um dos principais fármacos disponíveis. Como outros microrganismos, B. fragils são susceptíveis a perturbações ecológicas, muitas vezes resultantes de concentrações subinibitórias de antimicrobianos, que podem que podem desencadear alterações na biologia bacteriana com reflexo no seu potencial agressor. Considerando-se que, geralmente, os estudos de interação droga-bactéria utilizam ensaios in vitro, permanecem, assim, questionamentos sobre a real extensão do fenômeno considerando-se uma situação real de infecção e exposição a antimicrobianos em concentração subinibitória. Neste sentido, foi proposto o estudo de um modelo experimental de interação droga-bactéria-hospedeiro. Ratos Wistar foram submetidos à laparotomia para inserção de um corpo de prova na cavidade peritoneal, e posterior desafio percutâneo com B. fragilis ATCC 43859. Os animais foram divididos em três grupos experimentais: grupo controle negativo (CN) (n=18) sem inoculação bacteriana, apenas com o veículo; grupo controle positivo (CP) (n=30) animais infectados com a linhagem bacteriana parental; grupo experimental (GE) (n=24) animais infectados com a linhagem bacteriana parental, tratados com concentração subinibitória de metronidazol (1µg/mL - via percutânea) por um período de oito dias em intervalos de 48 horas. Foram avaliados em dois momentos durante o processo infeccioso: Após 8 dias o tratamento com o MTZ e 8 dias após o término do tratamento (16 dias). Experimentos com cultura bacteriana in vitro foram realizados considerando-se o mesmo desenho experimental para comparação. A dinâmica da colonização microbiana nos corpos de prova foi avaliada por cultura microbiana, PCR simples, PCR quantitativo e coloração de Gram. No hospedeiro, foram avaliados parâmetros histológicos e imunológicos, pela avaliação transcricional da expressão de citocinas. Características morfo-fisiológicas, bioquímicas e moleculares de bactérias selecionadas in vivo e in vitro foram avaliadas, ainda, pela coloração de Gram, Kit API20, formação de biofilme experimental, tolerância ao estresse oxidativo (H2O2), e genotipagem por AP-PCR. Os resultados mostram que a infecção in vivo por Bacteroides fragilis se manteve como monoinfecção durante todo o período experimental avaliado, com viabilidade bacteriana mesmo após tratamento com concentração subinibitória de metronidazol. Análise histológica do tecido formado no corpo de prova mostrou um infiltrado inflamatório exacerbado após os 8 dias de tratamento em ambos os grupos infectados. No entanto, após 16 dias o infiltrado inflamatório diminuiu no GE. Os níveis de expressão de citocinas mostraram-se alterados quando os grupos infectados foram comparados, principalmente relacionados aos níveis de IL-10. Embora alterações no perfil bioquímico bacteriano não tenham sido observadas, morfologia aberrante, aumento na formação de biofilme e diminuição da sensibilidade ao estresse oxidativo foram observadas nas linhagens selecionadas in vitro. Polimorfismo genético não foi detectado por AP-PCR. Nossos resultados sugerem que metronidazol em baixas concentrações interfere na relação bactéria-hospedeiro, o que pode ter implicações no processo infeccioso, sem, no entanto, resultar em alterações evidentes nas células bacterianas, como observado in vitro. Percebe-se, assim, a complexidade das interações droga-bacteria-hospedeiro em situação real de infecção e suas implicações na biologia microbiana e na patogênese. / Bacteroides fragilis, a member of the resident intestinal microbiota is the most important pathogen associated to endogenous infections. Meanwhile, metronidazole persists as the main drug available for treating these diseases. As other resident microorganisms, these bacteria are exposed to ecological disturbances, often resulting from subinhibitory concentrations of antimicrobial agents that can trigger changes in microbial biology and which may affect potential aggressor. Considering that the studies of drug-bacteria interactions usually uses in vitro assays, questions about the true extension of the phenomenon in a real situation of microbial infection and exposure to antimicrobials in subinhibitory concentrations remains. Thus, for a better understanding of drug-bacteria-host relationship in real situation of infection, an in vivo model of interaction has been proposed. Wistar rats were subjected to laparotomy to insert a perforated table tenis ball into the peritoneal cavity, followed by percutaneous challenge with B. fragilis ATCC 43859. The animals were divided into three experimental groups: negative control (NC) (n = 18) animals without bacterial inoculation using the vehicle only; positive control (PC) (n = 30) animals infected with the parental bacterial strain; experimental group (EG) (n = 24) animals infected with the parental bacterial strain, treated with subinhibitory concentration of metronidazole (1µg/mL percutaneous) for a period of eight days in 48 hour intervals. To evaluate the impact of metronidazole in the bacteria-host relationship the experimental infections were evaluated two times during the infectious process; first, 8 days after the treatment with MTZ and 8 days after the end of the treatment (16 days). The dynamics of the microbial colonization into the tissue cage was evaluated by conventional PCR, real time PCR and Gram stain. In the host, histological and transcriptional analysis of cytokine expression was performed. Morpho-physiological, biochemical and molecular characteristics of bacteria selected in vivo and in vitro were evaluated by Gram stain, kit API20, experimental biofilm formation, tolerance to oxidative stress (H2O2), and genotyping by AP-PCR. The results show that in vivo infection by Bacteroides fragilis remained as monoinfection throughout the experimental period with bacterial viability even after the treatment with sub inhibitory concentration of metronidazole. Histological analysis of the tissue formed into the tissue cage showed exacerbated inflammatory infiltrate after 8 days of treatment in both infected groups. However, after 16 days the inflammatory infiltrate decreased in the EG group. Increased cytokine expression was observed when infected groups were compared, mainly related to interleukin 10. Although changes in the bacterial biochemical profile were not observed, aberrant morphology, increased biofilm formation and decreased sensitivity to oxidative stress were observed in the in vitro selected strains. Genetic polymorphism was not detected by AP-PCR. Our results suggest that low concentrations of metronidazole interfere in the bacteria-host relationship, which could have implications in the infection process even without showing obvious alterations in the bacterial cells as demonstrated in vitro. Therefore, the complexity of drug-bacteria-host interactions in real infection situations and its consequences in microbial biology and pathogenesis is clear.

Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.