Crohn’s disease (CD) is an inflammatory bowel disease characterised by chronic inflammation with a complex pathophysiology involving host, environmental, and microbial factors. The intestinal microbiota is an important regulator of inflammation within the intestine, and a disruption of the interplay between gut bacteria and host immunity is a key factor in CD development. Intestinal inflammation itself is known to cause changes to the intestinal physiology that affect the ability of various bacteria to survive. Additionally, certain environmental risk factors for CD such as antibiotics are also known for their ability to impact the intestinal microbiota. CD is associated with various changes in the intestinal microbiome including increased colonisation with a group of bacteria known as adherent-invasive Escherichia coli (AIEC). The purpose of this study is to investigate how AIEC interact with antibiotics and intestinal inflammation in vivo. Multiple classes of antibiotics were found to increase the colonisation of AIEC and to increase its persistence. These antibiotics caused a loss diversity in the intestinal microbiome, but this did not explain the increased infectivity of AIEC. Antibiotic-induced inflammation was found to produce metabolites that benefitted AIEC growth in the intestine and similar results were found with chemically-induced inflammation. These results show that AIEC can benefit from both antibiotics and other sources of inflammation through inflammation-derived metabolites, which contributes to a greater understanding of the interactions between AIEC and CD. / Thesis / Doctor of Philosophy (PhD) / Crohn’s disease is a type of inflammatory bowel disease that affects many young adults in Canada. It causes a wide range of symptoms including nausea, pain, and diarrhea. While the disease can be treated with surgery and medications, it is considered incurable and affects most individuals for life. The exact cause of Crohn’s disease is not known, but it is thought to be caused by a combination of factors including genetics, environmental exposures, and changes in the number and types of bacterial species in the intestine. Intestinal bacteria play an important role in preventing inflammation in the intestine. An unusual strain of bacteria called adherent-invasive E. coli is found more commonly in Crohn’s disease patients than in healthy individuals. This strain does not cause the disease on its own, but it may interact with other environmental factors that are also associated with Crohn’s disease, such as taking antibiotics. Antibiotic use is a risk factor for developing Crohn’s disease later in life and antibiotics have previously been shown to promote the growth of other E. coli strains in the intestine. In a mouse model of Crohn’s disease, we found that antibiotics made mice more vulnerable to infection with this E. coli strain. This increased vulnerability was because the antibiotics caused inflammation, and we also found that other sources of inflammation benefitted this E. coli strain. These findings help us understand how gut bacteria and other Crohn’s disease risk factors might interact to cause the disease.

PspE, encoded by the last gene of the phage shock protein operon, is one of the nine proteins of Escherichia coli that contain a rhodanese homology domain. PspE is synthesized as a precursor with a 19-amino acid signal sequence and secreted to the periplasm. Mature PspE is the smallest rhodanese of E. coli (85 amino acids) and catalyzes the transfer of sulfur from thiosulfate to cyanide forming thiocyanate and sulfite. Cation exchange chromatography of a freeze-thaw extract of a PspE-overexpressing strain yielded two major peaks of active, homogeneous PspE. The two peaks contained two forms of PspE (PspE1 and PspE2) of distinct size and/or charge that were distinguished by native polyacrylamide gel electrophoresis and gel chromatography. PspE2 was converted to the more compact PspE1 by treatment with thiosulfate, which suggested that PspE1 is the persulfide form. One equivalent of cyanizable sulfur was associated with PspE1, with much less present in PspE2. Consistent with the conclusion that the single active site cysteine of PspE1 contains a persulfide sulfur was the observation that this form was much more tolerant to chemical inactivation by thiol-specific modifying reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)). Rhodanese activity was subject to inhibition by anions (sulfite, sulfate, chloride, phosphate and arsenate), suggesting PspE has a cationic site for substrate binding. Kinetic analysis revealed that PspE employs a double-displacement mechanism, as is the case for other rhodaneses. The Kms for SSO32- and CN- were 3.0 and 43 mM, respectively. PspE exhibited a kcat of 72 s-1. To aid in understanding the physiological role of PspE, a strain with a pspE gene disruption was constructed. Comparison of rhodanese activity in extracts of wild-type and mutant strains revealed that PspE is a major contributor of rhodanese activity in E. coli. The pspE mutant displayed no obvious growth defect or auxotrophies, and was capable of molybdopterin biosynthesis, indicating that pspE is not essential for production of sulfur-containing amino acid or cofactors. Growth of wild-type and mutant strains deficient in pspE and other sulfurtransferase paralogs in medium with cyanide or cadmium was compared. The results indicated that neither PspE nor other E. coli rhodanese paralogs play roles in cyanide or cadmium detoxification. The physiological role of PspE remains to be determined. / Master of Science

In this study a library of EcoRI fragments encompassing the entire TOL region of the Pseudomonas putida TOL plasmid pDK1 was constructed in the Escheria coli cloning vector pBR325.

L'enzyme glutamyl-queuosine-ARNt synthétase, présente chez plusieurs bactéries et codée par le gène yadB chez Escherichia coli, catalyse la formation de glutamylqueuosine- ARNtAsp. Le rôle de cet aminoacyl-ARNt est inconnu, mais la cotranscription de yadB et de dksA suggère une implication dans la réponse générale au stress. yadB a été remplacé dans une souche de Escherichia coli [delta]lac par lacZ, sans modification des éventuels éléments transcriptionnels de yadB. L'activité [béta]-galactosidase a été utilisée comme rapporteur pour déterminer le niveau d'expression de yadB dans diverses conditions de croissance et de stress. Les différences phénotypiques entre les souches sauvage et [delta]yadB ont été investiguées. Nous avons découvert que yadB est un gène non-essentiel chez Escherichia coli. Sa faible expression suggère un rôle dans le métabolisme basal de la cellule. Cette expression est sensiblement plus élevée pendant la phase stationnaire de croissance. Aucune différence phénotypique n'a été découverte entre les souches sauvage et [delta]yadB.

L'enzyme étudiée est la glutamyl-ARNt synthetase (GluRS) de la bactérie Escherichia coli. Son activité consiste principalement à charger l'acide glutamique sur l'ARNtGlu. Cette GluRS se compose de 5 domaines structuraux, dont les deux derniers (#4 et 5) situés dans la partie C-terminale ont été acquis durant l'évolution et sont responsables de la reconnaissance de la boucle de l'anticodon de l'ARNtGlu. Le domaine 4 est lié au domaine 5 par une charnière mobile (4-5) permettant à ce dernier de s'incliner et de s'adapter à l'anticodon de l'ARNtGlu (Nureki et al., 1995). Cette GluRS bactérienne, qui joue un rôle essentiel dans la biosynthèse des protéines, est considérée comme une nouvelle cible de l'antibiothérapie grâce à l'existence d'analogues du glutamyl-AMP qui inhibent plus des GluRS bactériennes que la GluRS cytopiasmique des mammifères (Balg et al., 2007). La première partie de ce projet consiste à étudier le rôle de la charnière 4-5 sur l'activité de la GluRS en produisant par mutagenèse dirigée du gène gltX de E. coli des variants E366A, E455R et D333A, altérés dans les résidus qui pourraient influencer les orientations relatives des domaines 4 et 5. Les propriétés cinétiques de ces variants dans la réaction de formation du glutamyl-ARNt montrent qu'il n'y a pas d'effet majeur de ces substitutions sur les Km sauf pour D333A. La constante de spécificité de l'enzyme sauvage reste plus grande que celle des variants ce qui suggère que la flexibilité de la charnière n'est pas grandement affectée par la substitution d'un seul de ces résidus. Accidentellement, un variant double charnière (#dc) a été obtenu dont le Km pour l'ARNtGlu est 7,7 fois plus grand que celui de la GluRS sauvage, et dont la constante de spécificité est 15 fois plus faible. Ce résultat suggère que la longueur de la charnière a peut-être autant d'influence que la nature de ces résidus pour le bon fonctionnement de la GluRS. La deuxième étape du projet est de tester l'hypothèse selon laquelle la flexibilité de la charnière 4-5 de la GluRS est importante pour l'activité catalytique de cette enzyme. Des formes tronquées de ces variants ne contenant que les 2 derniers domaines 4 et 5 ont été surproduites et purifiées pour la mesure de la flexibilité de la charnière 4-5 par résonance magnétique nucléaire (RMN). Une analyse préliminaire de la GluRS tronquée sauvage par RMN a montré qu'elle est bien repliée. Notre étude structure-fonction de la charnière 4-5 facilitera le design rationnel de nouveaux inhibiteurs plus efficaces de la GluRS. Ceux-ci pourraient être des composés de départ (« lead compounds ») pour la conception d'un nouvel antibiotique.

Les aminoacyl-ARNt synthetases (aaRS) sont des enzymes clés dans le mécanisme de biosynthèse des protéines. Chacun des membres de cette famille d'enzymes reconnaît et lie les éléments d'identités propres à chaque acide ribonucléique de transfert (ARNt) permettant ainsi leur aminoacylation par un acide aminé spécifique. Une fois l'ARNt correctement chargé, celui-ci peut lier un facteur protéique (EF-Tu pour les aminoacyl-ARNt élongateurs) qui le conduit au ribosome pour la biosynthèse des protéines. Plus particulièrement la glutamyl-ARNt synthetase (GluRS) d'Escherichia coli est une protéine capable d'estérifier l'ARNtGIU par un résidu glutamate. Cette enzyme monomérique est subdivisée en 5 domaines structuraux, chacun capable de lier une portion de l'ARNt qui lui est propre. La région formant la boucle d'anticodon de l'ARNtG'u est reconnue par les domaines 4 et 5 séparés par une charnière, et situés en C-terminal de l'enzyme. L'étude d'une GluRS tronquée, délétée de son domaine 5, a montré l'importance de ce domaine sur l'activité catalytique de cette enzyme. Afin de comprendre les mécanismes mis en place entre ces deux derniers domaines lors de la liaison du substrat ARNtGly, des variants protéiques visant à altérer les interactions existant entre les domaines 4 et 5 et la charnière ont été synthétisés, et leur constantes catalytiques mesurées. L'étude du simple variant, substitué au niveau du second résidu chargé de la charnière de la GluRS de E. coli a montré l'importance de ce dernier dans les interactions mises en place entre le domaine 5 et la charnière. Par ailleurs l'altération des constantes cinétiques (en comparaison avec les valeurs trouvées pour l'enzyme sauvage), de double et triple variants, substitués au niveau des résidus chargés trouvés sur la charnière séparant les domaines 4 et 5, ont révélé l'importance de certain de ces résidus dans les interactions existant entre ces deux domaines.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 152-162).

The prevalence and antibiotic resistant profiles of shiga-toxin producing Escherichia coli 0157 strains isolated from faeces samples of cattle, pigs and human stool samples were determined. The strains were further characterised by molecular methods for the presence of shiga-toxin virulence genes and antibiotic resistant genes. Seventy-six Escherichia coli 0157 strains were isolated and the prevalence was higher among E. coli isolated from faeces from pigs (44.2% to 50%) than those from cattle faeces (5.4% to 20.0%) or human stool samples (7 .5%). On testing E. coli 0157 isolates for their resistance to 9 antimicrobial agents, multiple antibiotic resistance (MAR) was observed in all of the isolates arising from resistance to three or more antibiotics. Seventy (92.1 %) of the E. coli 0157 isolated from humans, cattle and pigs were resistant to tetracycline. 73 (96.1 %) were resistant to sulphamethoxazole, 63 (82.9%) were resistant to erythromycin. 40 (52.6%) were resistant to streptomycin and 26 (34.2%) were resistant to ampicillin. The highest frequency of resistance was observed among the human isolates (n=3 ), where 3 (I 00%) of the isolates were resistant to tetracycline, sulphamethoxazole, erythromycin and ampicillin. Furthermore, among the pig isolates (n=60), 58 (96. 7%) were resistant to tetracycline, 57 (95%) were resistant to sulphamethoxazole, 47 (78.3%) were resistant to erythromycin. 38 (63.3%) were resistant to streptomycin and 22 (36. 7%) were resistant to ampicillin. The MAR phenotypes S-Smx-T-E, Smx-T-Ap and Smx-T-E were the dorminant phenotypes among the E. coli 0157 isolated from the faeces samples of communal pigs in 30.4%, 21 .7% and 17.4% of these isolates, respectively. However, phenotypes Smx-T -E and S-Smx-T-E-Ne were identified at I6.2% and 10.8%, respectively within the isolates obtained from commercial pig faeces. The phenotype Smx-T-E was the only MAR phenotype identified among the E. coli 0157 isolated from the faecal samples of commercial cattle at Lichtenburg. Furthermore, MAR phenotypes Smx-T-E-C, K-S-Smx-T-E, S-Smx-T-E and Smx-T-E-Ap were obtained at 25%, respectively for the isolates obtained from communal cattle at Mogosane while Smx-T-E-Ap was the dorminant (66.7%) phenotype among the isolates of human origin. The phenotype Smx-T fom1ed the basis of all the MAR phenotypes obtained and this was similar to the percentage antibiotic resistance data. The distribution of the resistant determinants for tetracycline was determined by PCR analysis in resistant isolates. A tetB gene was detected in E. coli 0157 of pig origin. Based on the characterisation of 30 isolates for the presence of STEC virulence genes by PCR, 18 (60%) possessed the hlyA gene, 7 (23.7%) possessed the eae gene and 5 ( 16. 7%,) harboured both genes. The average MAR indices for pig, cattle and human E. coli 0157 isolates were 0.4n2, 0.3419 and 0.4814, respectively. Among the cattle isolates, the group MAR index was highest for the communal (Mogosane) population while the values for the commercial populations at Lichtenburg and Rustenburg were 0.33 and 0.22, respectively. £. coli 0157 isolated from pigs revealed MAR index results that were 0.508 and 0.415 for the commercial and communal populations respectively and 0.1851 for the E. coli control strains. Characterisation by cluster analysis to determine the commonness and resolve differences between the E. coli 0157 isolated from the Various sources revealed a close association between pig (Tlapeng and Mareetsane), cattle (Mogosane) and human isolates. Interestingly, E. coli 0157 isolated from pigs occurred at the highest frequency in all the clusters. which suggested their role in the dissemination of resistant determinants. / MSc. (Agric.) North-West University, Mafikeng Campus, 2006

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 111-127).

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 81-88.