Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry. / Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

F1651, les pili Pap et l’antigène CS31A associé aux antigènes de surface K88 sont tout trois des membres de la famille de type P des facteurs d’adhérence jouant un rôle prépondérant lors de l’établissement d’une maladie causée par des souches Escherichia coli pathogènes, en particulier des souches d’E. coli pathogènes extra-intestinales (ExPEC, Extra-intestinal pathogenic E. coli). Leur expression est sous le contrôle d’un mécanisme de régulation transcriptionnel dépendant de l’état de méthylation de l’ADN, résultant dans l’existence de deux populations définies, l’une exprimant l’adhésine (population ON) et l’autre ne l’exprimant pas (population OFF). Malgré de fortes identités de séquences, ces trois systèmes diffèrent l’un de l’autre, principalement par le pourcentage de cellules ON rencontrées. Ainsi, quand CS31A est systématiquement orienté vers un état considéré comme OFF, F1651 présente une phase ON particulièrement élevée et Pap montre deux états OFF et ON bien distincts, selon le phénotype de départ. La protéine régulatrice sensible à la leucine (Lrp, Leucine-responsive regulatory protein) joue un rôle essentiel dans la réversibilité de ce phénomène épigénétique et il est supposé que les différences de séquences au niveau de la région régulatrice modifient la localisation à ces sites de fixation de Lrp; ce qui résulte, en final, aux différences de phase existant entre CS31A, F1651 et Pap.À l’aide de divers techniques parmi lesquelles l’utilisation de gènes rapporteurs, mutagénèses dirigées et d’analyse des interactions ADN-protéines in vitro, nous montrons dans ce présent projet que la phase OFF prédominante chez CS31A est principalement due à une faible interaction de Lrp avec la région distale de l’opéron clp, et que la présence d’un homologue du régulateur local PapI joue un rôle également clef dans la production de CS31A. Dans le cas de F1651, nous montrons dans cette étude que le taux élevé de cellules en phase ON est dû à une altération dans le maintien de Lrp sur les sites répresseurs 1-3. Ceci est dû à la présence de deux nucléotides spécifiques, situé de part et d’autre du site répresseur 1, qui défavorisent la fixation de Lrp sur ce site précis. Tout comme dans le cas de CS31A, la formation d’un complexe, activateur ou répresseur de la phase ON, dépend également de l’action de du régulatuer local FooI, qui favorise alors le déplacement de Lrp des sites répresseurs 1-3 vers les sites activateurs 4-6. / F1651, the pyelonephritis-associated pili (Pap) and the K88-related surface antigen CS31A are three members of the type P family of adhesive factors that play a key role in the establishment of disease caused by Extra-intestinal Escherichia coli (ExPEC) strains. They are all under the control of methylation-dependent transcriptional regulation that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Despite a high similarity in DNA sequence, these three adhesive systems nonetheless differ in the ratio of ON cells. While CS31A is always turned toward the OFF state, F1651 presents a particularly high level of ON cells and Pap shows two distinct OFF and ON states, depending on the starting phenotype. The leucine-responsive regulatory protein (Lrp) plays an essential role in the reversibility of this epigenetic switch and it is believed that the difference in nucleotides within the regulatory region of each operons could modify the binding of Lrp and, in turn, CS31A, Pap and F1651 phase variation. Using a variety of techniques including gene expression, site-directed mutagenesis, and in vitro protein–DNA interaction analysis, we demonstrate that the preferential OFF state observed in CS31A-positive cells is mainly due to a weak interaction of Lrp with the clp distal region and that the presence of a PapI homologue within the cell plays a key role in CS31A production. For F1651, we show in this study that the high level of ON cells found during F1651 phase variation is due to an altered stability of the DNA complex formed by Lrp at its repressor binding sites 1-3. Again, after each cell cycle, complex formation is modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp toward its activator binding sites 4-6. Furthermore, we identify two nucleotides (T490, G508) surrounding the Lrp-binding site 1 that are critical to maintaining a high OFF to ON switch rate during F1651 phase variation, as well switching Pap fimbriae toward the OFF state.

No description available.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

No description available.

A mastite subclínica em caprinos é causada principalmente pelo Staphylococcus spp., sendo os estafilococos coagulase negativa (SCN) os patógenos de maior ocorrência e o S. aureus, a espécie de estafilococos mais pesquisada. Dessa forma, pouco se conhece sobre a patogenicidade de SCN e outros estafilococos coagulase positiva (SCP), além do S. aureus. Também há poucos estudos sobre a variação da viabilidade celular na mastite subclínica de cabras. Assim, o objetivo do presente estudo foi determinar os fatores de virulência de adesão e produção de biofilme de estirpes de Staphylococcus spp. isoladas de amostras de leite de cabras, verificando possíveis associações com a viabilidade celular. Para realizar a colheita das amostras, primeiramente, foi efetuada um exame físico da glândula mamária, com posterior realização dos testes da caneca de fundo preto e California mastitis test (CMT). A colheita do leite foi efetuada em três alíquotas: análises microbiológicas, contagem de células somáticas (CCS) e viabilidade celular. Realizou-se a identificação, teste de antibiograma e PCR (Reação em cadeia polimerase) dos Staphylococcus spp. isolados nas amostras de leite. Os genes de virulência pesquisados no PCR foram: cna, eno, ebpS, fnbA, fnbB, fib e bap. Avaliou-se a quantidade de CCS, em equipamento de citometria de fluxo, e a viabilidade celular, após centrifugações das amostras de leite e visualização das células em microscópio, utilizando o corante azul de Trypan. Os resultados foram: 122 amostras com crescimento bacteriano e dessas, 110 (90,2%) foram identificadas como Staphylococcus spp., sendo 90 (73,8%) de SCN e 12 (16,4%) de SCP. As espécies mais isoladas de estafilococos foram: S. epidermidis (24,55%), S. lugdunensis (15,40%) e S. intermedius (13,64%). As amostras apresentaram maior resistência aos antimicrobianos: penicilina (81,8%), oxacilina (60,0%) e ampicilina (55,5%). Observou-se maior sensibilidade para: enrofloxacina (99,1%), eritromicina (98,2%), gentamicina (98,2%) e vancomicina (98,2%). Com relação aos fatores de virulência pesquisados, foram encontradas amostras positivas para todos os genes, com exceção do gene fnbB: eno (53,6%), bap (43,7%), ebpS (19,1%), fnbA (18,2%) e fib (16,4%). Mais de um gene foi detectado em algumas estirpes, sendo que as associações de maior ocorrência foram: bap/eno em SCN e ebpS/eno/fib/fnbA em SCP. Os valores da CCS das amostras de leite com isolamento de Staphylococcus spp., SCN e SCP foram maiores do que nas amostras sem isolamento bacteriano e as estirpes com presença da associação de genes ebpS/eno/fib/fnbA apresentaram maior CCS do que bap/eno. Com relação à viabilidade celular, as amostras com isolamento de Staphylococcus spp. apresentaram maior viabilidade celular do que as amostras sem isolamento bacteriano e não houve associação dos genes identificados nas estirpes com a viabilidade celular. Concluiu-se que os genes eno e bap apresentaram maior ocorrência nas estirpes de Staphylococcus spp., sendo os mais encontrados nos isolados de SCN e os genes ebpS, fib e fnbA foram os mais detectados nos SCP. A viabilidade celular foi maior nas amostras com isolamento de Staphylococcus spp. em relação as sem isolamento bacteriano e não houve associação entre os fatores de virulência das estirpes de Staphylococcus spp. e a viabilidade celular. / Subclinical mastitis in goats is mainly caused by Staphylococcus spp., coagulase-negative staphylococci (CNS) is the most frequent pathogens and S. aureus, the most researched specie of staphylococci. Thus, little is known about the pathogenicity of SCN and other coagulase-positive staphylococci (CPS), beyond the S. aureus. There are few studies on the variation of cell viability in subclinical mastitis of goats. The aim of the present study was to determine the virulence factors of adhesion and biofilm production of Staphylococcus spp. strains isolated from milk samples of goats, verifying for possible associations with cell viability. To collect samples, firstly, was performed a physical examination of the mammary gland, with subsequent tests for mug of black background and California mastitis test (CMT). Three aliquots of milk were collected: microbiological analysis, somatic cell count (SCC) and cell viability. Identification, antibiotic susceptibility testing and PCR (polymerase chain reaction) were performed of Staphylococcus spp. isolated from milk samples. Virulence genes researched in PCR were: cna, eno, ebpS, fnbA, fnbB, fib and bap. Evaluation of CCS, using flow cytometry equipment, and cell viability, after centrifugation of milk samples and visualization the cells in the microscope using Trypan blue dye, were performed. The results were: from 122 samples with bacterial growth, 110 (90.2%) were identified as Staphylococcus spp., 90 (73.8%) of CNP and 12 (16.4%) of the CNP. The most isolated staphylococci species were: S. epidermidis (24.55%), S. lugdunensis (15.40%) and S. intermedius (13.64%). Samples showed higher resistance to antimicrobials: penicillin (81.8%), oxacillin (60.0%) and ampicillin (55.5%). We observed higher sensitivity to: enrofloxacin (99.1%), erythromycin (98.2%), gentamicin (98.2%) and vancomycin (98.2%). Regarding virulence factors researched, positive samples were found for all genes, except fnbB gene: eno (53.6%), bap (43.7%), ebpS (19.1%), fnbA (18.2%) and fib (16.4%). More than one gene were detected in some strains, with the most frequent associations were bap/eno in CNS and ebpS/eno/fib/fnbA in CNP. The values of SCC of milk samples with isolation of Staphylococcus spp., CNS and CNP were higher than samples without bacterial isolation and the isolation of strains with combination of ebpS/eno/fib/fnbA genes showed higher SCC than bap/eno. Regarding cell viability, samples with isolation of Staphylococcus spp. showed higher cell viability than samples without bacterial isolation and there was no association of the genes identified in strains with cell viability. In conclusion, eno and bap genes were more frequent in Staphylococcus spp. strains, eno and bap genes were mostly found in isolated CNS and ebpS, fib and fnbA genes were more detected in CNP. Cell viability was higher in samples with isolation of Staphylococcus spp. compared those without bacterial isolation and there was no association between the virulence factors of Staphylococcus spp. strains and cell viability.

Les Escherichia coli pathogènes extra-intestinaux (ExPEC) sont responsables d’une grande variété de maladies. Plus particulièrement, certaines souches ExPEC, du sous-groupe d’E. coli uropathogènes, sont porteuses de fimbriae de type P. Cette famille d’adhésines est soumise à une régulation transcriptionnelle appelée variation de phase; un mécanisme du tout ou rien. Il s’agit d’une compétition entre deux protéines régulatrices : la Dam méthylase et la nucléoprotéine Lrp. Ce mécanisme est aussi soumis à l’influence des régulateurs locaux PapB et PapI, deux régulateurs essentiels. Afin d’étudier PapI et ses homologues ainsi que leur impact sur la variation de phase des fimbriae F1651, Pap et CS31A. Grâce à une fusion chromosomique entre la région régulatrice de clp et les gènes lacZYA, nous avons étudié l’effet, en trans, de PapI et FooI qui ont pu restaurer la variation de phase avec une forte tendance pour la phase OFF. Pour étudier l’action de ces protéines sur foo et pap, nous avons utilisé un système utilisant gfp comme gène rapporteur de l’activité des promoteurs des opérons pap et foo. Cela a permis d’observer la variation de phase au niveau cellulaire par cytométrie en flux et en temps réel par microscopie à fluorescence. Ces expériences ont confirmé que la population de cellules F1651 positives a un phénotype d’expression de F1651 partielle alors que les cellules Pap sont en majorité en phase OFF. PapI et FooI n’ont pas la même influence sur la variation de phase, puisque FooI favorise une plus grande fréquence de variation de phase. / Escherichia coli extra-intestinal pathogenic (ExPEC) are responsible for a wide variety of diseases. Particularly ExPEC strains from the subset called uropathogenic E.coli (UPEC) are carrying fimbriae type P. This adhesin family is subject to transcriptional regulation called phase variation, an all or nothing mechanism. It is a competition between two regulatory proteins: the Dam methylase and the nucleoprotein Lrp. This mechanism is also under the influence of the local regulators PapB and PapI. These two regulators are essential to the phase variation. We therefore sought to investigate PapI and its homologs and their impact on the phase variation of fimbriae F1651, Pap, and CS31A. By means of a chromosomal fusion between the regulatory region of clp gene and lacZYA, we studied the effect in trans of PapI and FooI which could restore the phase variation with a strong tendency to phase OFF. To study the action of PapI and FooI, we used a system with gfp as a reporter gene in operons pap and foo. This allowed the observation of the phase variation at the cellular level by flow cytometry and real-time fluorescence microscopy. These experiments confirmed that the population of F165 positive cells have a partial expression state whereas Pap cells mostly have an OFF expression state. We also confirmed that FooI and PapI do not have the same influence on phase variation and that FooI promotes greater frequency of phase variation.

Peptide fusion tags are fundamental for the identification, detection, and capture of proteins in biological assays. Commonly used peptide fusion tags rely on temporary non-covalent interactions for binding, which can put constraints on assay sensitivity. Here, peptide fusion tags were developed that could specifically interact with protein binding partners via spontaneous and irreversible isopeptide bond formation. To develop covalently interacting peptide-protein pairs, outer-membrane proteins from Gram-positive bacteria that form autocatalyzed intramolecular isopeptide bonds were dissected to generate a short peptide fragment and a protein binding partner. Initially, the major pilin subunit Spy0128 from Streptococcus pyogenes was split to develop the 16 residue isopeptag peptide and the 31 kDa pilin-C protein partner. The isopeptag:pilin-C pair were able to react via spontaneous isopeptide bond formation between an Asn residue in isopeptag and a Lys residue in pilin-C without the requirement for any accessory factors, and with a yield of 60% after a 72 hr reaction. Reconstitution between the isopeptag:pilin-C pair was robust and occurred under all biologically relevant conditions tested, and also in the complex environment of a bacterial cytosol and on the surface of mammalian cells. A similar approach was also used to dissect the small CnaB2 domain that is part of the large FbaB fibronectin-binding protein from S. pyogenes. This led to the development of a more efficient peptide-protein pair, which was rationally modified to generate the highly optimized SpyTag:SpyCatcher pair. SpyTag is a 13 amino acid peptide with a reactive Asp that forms a spontaneous intermolecular isopeptide bond with a Lys present in the 12 kDa SpyCatcher binding partner. In a reaction with SpyTag, over 40% of SpyCatcher was depleted after 1 min and SpyCatcher could no longer be detected after 2 hr. The SpyTag and SpyCatcher reaction did not require any accessory factors and proceeded efficiently at a range of biologically relevant temperatures, pH values, concentrations, buffer compositions, and in the presence of commonly used detergents. The SpyTag:SpyCatcher technology was also used for specific cell surface labelling on mammalian cell membranes. SpyTag and SpyCatcher are both composed of the regular 20 amino acids and can therefore be genetically encoded as fusion constructs for a variety of in vitro and in vivo applications. Potential applications of the SpyTag:SpyCatcher technology include specific cell surface labelling, the development of novel protein architectures, and the covalent and irreversible capture of target proteins in biological assays.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal