Multiple antibiotic resistant bacteria continue to be a threat to the health of the world's population. Glycopeptide antibiotics are one type of drug that are used to treat these serious pathogens. Increased usage over the years has led to the emergence of bacteria which are resistant to these glycopeptide antibiotics and now the need for altered antibiotics with an increased effectiveness has arisen. 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 𝘵𝘰𝘺𝘰𝘤𝘢𝘦𝘯𝘴𝘪𝘴 NRRL 15009 produces the glycopeptide antibiotic A47934. Here, the biosynthetic gene cluster for A47934 was sequenced in its entirety. All enzymes encoded by assigned open reading frames were analyzed and functions assigned where possible. The resulting biosynthesis cluster encodes all the enzymes necessary to produce A47934, as well as confer resistance and regulate the resistance response. In addition to sequencing the biosynthetic gene cluster, enzymatic studies were attempted on the two-component regulatory system (VanR and VanS) which confers resistance to A47934. Finally, inactivation of 𝘴𝘵𝘢𝘓 was attempted. Overall, the results presented here should help us to further understand how these chemically complex glycopeptide antibiotics are made and lend further insight into how we can attempt to produce new semi-synthetic versions. / Thesis / Master of Science (MSc)

Le cancer est l’une des principales causes de mort dans le pays développés ; bien que les opérations chirurgicales, la radiothérapie et la chimiothérapie représentent aujourd’hui les principales options de traitement des patients souffrants de tumeurs malignes, leurs effets secondaires sévères ont ouvert la voie au développement de l’immunothérapie antitumorale. A part l’immunothérapie passive, qui est basée sur les anticorps ou tout autre composant du système immunitaire synthétisés en dehors du corps dont la potentielle menace de réactions immunes a été prouvée, nous avons concentré nos efforts sur l’immunothérapie active, qui réside dans la stimulation du système immunitaire du patient pour éradiquer sélectivement les cellules malignes. L’identification d’antigènes carbohydrates associés aux tumeurs (TACAs), surexprimés à la surface des cellules cancéreuses, a permis le développement de vaccins spécifiques à cet antigène. Il est connu depuis plus de 40 ans que la majorité des cancers chez l’homme sont caractérisés par une glycosylation aberrante. Les cellules tumorales peuvent surexprimer des versions tronquées d’oligosaccharides, une séquence terminale inhabituelle ou une augmentation de la sialylation des glycolipides et des glycoprotéines de surface. Un oligosaccharide d’une glycoprotéine tronqué peut rendre une partie de la chaîne principale du peptide, d’habitude caché par le sucre, plus accessible au système immunitaire. Parmi les différents TACAs, nous avons concentré notre attention sur les antigènes Tn et Tf, qui peuvent être trouvés sur des glycoprotéines comme MUC-1, surexprimés sur plus de 90% des carcinomes du sein. Bien que la conception de ces immunomodulateurs repose toujours sur des règles empiriques, il est important de déclencher à la fois la réponse humorale et cellulaire, ainsi qu’un effet de mémoire. Ce défi peut être relevé en combinant, sur une seule molécule, l’antigène carbohydrate exprimés à la surface des tumeurs (épitope des cellules B), les peptides capables de stimuler les cellules CD4+ et CD8+ (épitopes des cellules T) et un adjuvant, pour recueillir tous les éléments du système immunitaire au niveau du site d’injection et renforcer l’absorption des antigènes. De précédentes études faites dans notre groupe de recherche ont publié pour la première fois la synthèse et l’évaluation immunologique d’un prototype de vaccin anticancéreux à quatre composant capable d’induire une réponse immunitaire de longue durée sur des modèles murins. Dans mon travail de thèse, nous avons voulu synthétiser des prototypes de vaccin anticancéreux basés sur les TACAs avec des propriétés immunologiques accrues. Notre stratégie de conception a été guidée par (i) l’importance d’une haute densité de carbohydrates pour promouvoir une capture d’antigène plus efficace et un traitement par les cellules présentatrices d’antigène, et (ii) l’expression hétérogène des TACAs au cours de la maladie et parmi différents patients. En respectant ces deux aspects, il sera possible de déclencher une réponse immunitaire plus forte et à plusieurs facettes. / Cancer is one on the leading causes of death in developed countries; although surgical resection, direct irradiation and cytotoxic chemotherapy represent nowadays the main treatment options for patients suffering with malignancies, their severe side effects paved the way for the rise in popularity of antitumoral immunotherapy. Apart from passive immunotherapy, which is comprised of antibodies or other immune system components that are made outside of the body and has been shown to be associated to potentially life threatening immune reactions, we focused our efforts towards active immunotherapy, which purpose is stimulate the patient immune system to selectively eradicate malignant cells. The identification of tumor-associated carbohydrate antigens (TACAs) on the surface of cancer cells has allowed the development of antigen-specific vaccines. It has been known for over four decades that the majority of human cancers are characterized by aberrant glycosylation. Tumor cells may over-express truncated versions of oligosaccharides, unusual terminal oligosaccharide sequences, or increase sialylation of cell-surface glycolipids and O- and N-linked glycoproteins. A truncated oligosaccharide of a glycoprotein may render a part of the peptide backbone, which is normally shielded by the glycan, more accessible to the immune system. Among the assortment of TACAs we focussed our attention on Tn and TF-antigens, which can be found in membrane-bound glycoproteins like MUC-1, over-expressed in more than 90% of breast carcinomas. Although the design of such immuno-modulators still relies on empiric rules, it is noteworthy important to trigger both humoral and cellular responses, and a memory effect. This challenge can be achieved by combining, within a single molecule, carbohydrate antigen expressed on the surface of tumors (B-cell epitope), peptides capable to stimulate both CD4+ and CD8+ T-cells (T-cell epitopes) and an adjuvant, to gather immune system elements in the injection site and boost the antigen uptake. Previous studies of our research group reported for the first time the synthesis and immunological evaluation of a four-component anticancer vaccine prototype capable of inducing a long-lasting immune response in mice models. In my PhD work we aimed to synthesize TACA-based anticancer vaccine prototypes with improved immunological properties. The principles which guided our design strategies rely on (i) the importance of a high density of carbohydrate epitopes to promote a more effective antigen capture and processing by antigen-presenting cells, and (ii) the evidence of heterogenic expression patterns of TACAs during the course of the disease and among different individuals. Addressing these two aspects would provide a stronger and multifaceted immune response.

<p> Glycopeptide Antibiotics (GPAs) such as vancomycin are often used clinically as antibiotics of last resort against infections due to Gram-positive bacteria that are resistant to more commonly used antibiotics such as methicillin. The clinical emergence of vancomycin resistant enterococci (VRE) and vancomycin resistantS. aureus (VRSA) necessitates methods to evade this resistance. </p> <p> GP As consist ofa heptapeptide backbone that is cross-linked to create a pocket that binds the D-Alanyl-D-Alanine terminus of peptidoglycan intermediates, inhibiting strengthening ofthe cell wall and resulting in susceptibility to osmotic stress. Resistance to GPAs occurs when D-Ala-D-Lactate replaces D-Ala-D-Ala and the GPA pocket can no longer bind effectively. In order to create novel binding pockets, we must understand the specificity ofthe P450 monooxygenase enzymes that have been shown to catalyze the cross-links. The 4 P450-encoding genes ofthe GPA A47934 biosynthetic cluster of Streptomyces toyocaensis as well as genes encoding electron transport proteins necessary for P450 function from Streptomyces coelicolor were cloned in Escherichia coli for heterologous expression and characterization. One P450, StaJ was purified and shown to bind CO as expected using spectrophotometric tests. </p> <p> The genes responsible for GP A resistance are regulated by a two component regulatory system consisting ofa sensor kinase (VanS) and a response regulator (V an.R). In order to probe the events leading to VanS autophosphorylation and ultimately resistance activation we utilize a series of GP A derivatives harbouring the photo labile group benzophenone as well as the fluorescent and affinity moieties BODIPY and biotin. Benzophenone permits light controlled covalent binding of the GP A to proteins that bind them while BODIPY allows fluorescence detection and biotin allows enrichment and detection by Western analysis. We report that this system was insufficient to clearly identify vancomycin binding proteins due to background signals despite multiple rounds of troubleshooting. It must be our conclusion that under the conditions tested, there are no proteins that bind the GP A derivative used in this study. </p> / Thesis / Master of Science (MSc)

Genetic and biochemical studies were conducted on S. toyocaensis NRRL 15009, a gram-positive spomlating filamentous bacterium, and producer of the glycopeptide antibiotic A47934. This compound is structurally similar to the clinically important antibiotic vancomycin, and the recent spread of vancomycin-resistant enterococci (VRE) in North American hospitals has driven the need for new glycopeptides with enhanced activities. Studies were aimed at developing an understanding of the mechanism of A47934 biosynthesis inS. toyocaensis NRRL 15009, as well as the mechanism of resistance employed by this organism. Two cosmid clones, containing a partial A47934 biosynthesis gene cluster on a total of65 kilobases of S. toyocaensis NRRL 15009 chromosomal DNA, were isolated for study. Preliminary sequencing indicates the presence of several genes predicted in glycopeptide assembly, such as peptide synthetases and glycosyltransfe~ases. Furthermore, using a oligonucleotide probe designed to identify D-alanine-D-alanine ligases, an 8.1 kilobase chromosomal fragment was isolated from S. toyocaensis NRRL 15009 and found to contain genes very similar to VRE vanH, vanA and vanX. Phylogenetic analysis of the predicted products of these genes showed them to be more similar to the VRE enzymes than any other in each enzyme class. These genes were also found in the vancomycin producer A. orienta/is C329.2 and several other glycopeptide antibiotic producing organisms. Not only does this imply that these organisms employ a mechanism of resistance similar to clinical VRE, it also suggests that these organisms may have been the source of the VRE genes. The enzymes VanHst and DdlN were studied in some detail and found to have biochemical properties similar to their corresponding VRE enzymes VanH and VanA, respectively. Given that the latter group of enzymes has physical properties that have impeded detailed analysis of enzyme mechanism, these new enzymes could find use as model systems in drug development programs. / Thesis / Doctor of Philosophy (PhD)

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: G.D. Wright. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: G.D. Wright. Includes bibliographical references. Also available via World Wide Web.

Endogenous peptides modulate a wide range of physiological conditions in the central and peripheral nervous systems, but have not been harnessed to perform similar functions in pharmaceutical roles due to their ease of degradation and difficulty in introducing into the neurovascular unit. We report herein advances that evidence the wide applicability that glycosylation provides as a pathway for improving the drug-like properties of peptides. This is demonstrated by utilizing novel sugar-amino acids to modify the potent mu opioid receptor agonist DAMGO to provide antinociception, and serine glycosides to modify secretin family peptides for neuroprotection and angiotensin-(1-7) to both reduce cognitive impedance following myocardial infarction and as a treatment for peripheral neuropathy. Evidence is presented via a series of in vitro and in vivo models and assays, and demonstrates the advantageous effects of glycosylation through increased persistence in serum, greatly improved blood-brain barrier penetration, and the tolerance of receptor interactions to the addition of a carbohydrate.

Cette thèse développe de nouvelles séquences réactionnelles divergentes vers les lactones sesquiterpéniques, ainsi que leurs analogues. La réactivité multiple d’un substrat linéaire face à divers catalyseurs a tout d’abord permis d’obtenir différentes structures polycyliques dont la fonctionnalisation a permis d’isoler plusieurs produits naturels et des analogues. De nouvelles méthodologies ont été étudiées pour accéder aux gamma-butyrolactones, une fonctionnalité prépondérante dans les lactones sesquiterpéniques, ainsi qu’au noyau hydroazulène contenu dans les guaianes. Enfin, une synthèse divergente courte et performante a été mise au point pour accéder à divers analogues de la déoxyéléphantopine, un sesquiterpène aux propriétés anti-cancéreuses, afin de moduler et étudier son activité biologique. En parallèle de ces travaux sur les sesquiterpènes, une autre chimie a été explorée visant à réaliser la glycosylation de cystéines avec des carbohydrates non protégés. / The main thread throughout this thesis is to develop reaction sequences that could provide facile access to the sesquiterpene lactones, or analogs thereof, using strategies that would be compatible with divergent reaction pathways. A first project harnessed the multiple reactivity mode of a linea rsubstrate to obtain different polycyclic frameworks found in sesquiterpenes whose functionalisation led to several natural products and their analogs. New methodologies were studied to access gamma-butyrolactones, a preponderant functionality in sesquiterpene lactones, and hydroazulene core, the bicyclic framework of guaianes. Finally, a short divergent pathway was designed to access diverse analogs of deoxyelephantopin, a sesquiterpene showing anti-cancer effects, so as to modulate and study its biological activity. In parallel to this work on sesquiterpenes, a different chemistry was explored aiming at performing glycosylation of cysteines with unprotected carbohydrates.

Staphylococcus aureus est une bactérie pathogène de l'homme impliquée dans de nombreuses infections nosocomiales et communautaires. Comme elle acquiert régulièrement de nouvelles résistances à diverses classes d'antibiotiques, il devient urgent de proposer de nouvelles cibles thérapeutiques. Certains ARN régulateurs (ARNrég) sont importants dans le contrôle de la virulence et de la pathogénie de la bactérie. Au cours de ma thèse, nous avons étudié la fonction d'un ARNrég, appelé SprX (alias RsaOR), exprimé par Staphylococcus aureus. Dans un premier temps, nous avons montré que, dans les souches N315 et HG001, l'expression de SprX varie au cours de la croissance et lors de différentes conditions expérimentales. Dans un second temps, nous avons identifié, par une analyse comparative du protéome, plusieurs protéines dont l'expression est dépendante de SprX et découvert le mécanisme de régulation de l'une de ces protéines par SprX. En effet, SprX interagit avec l'ARNm yabJ-spoVG au niveau des signaux d'initiation de la traduction de SpoVG par un mécanisme antisens qui conduit à la répression de sa traduction. Une boucle accessible de SprX, qui contient un motif riche en C, est impliquée dans la régulation de l'expression de SpoVG et est nécessaire à la modulation de la résistance aux antibiotiques de S. aureus. Nous avons également étudié l'effet des modifications dans la séquence des différentes copies de SprX sur la régulation de l'expression de SpoVG. Ainsi, parmi les deux copies de SprX dans la souche HG001, SprX2 possède une meilleure affinité pour l'ARNm yabJ-spoVG que la copie SprX1. L'ensemble de ces résultats suggèrent que les ARNrég peuvent altérer la résistance des bactéries aux antibiotiques et il est a prévoir que d'autres exemples seront découverts prochainement. / Staphylococcus aureus is a serious human pathogen responsible for both hospital and community-acquired infections. As it becomes alarmingly and increasingly resistant to antibiotics, studies on the mechanisms involved in its virulence is a promising path to develop new treatments. Some, small regulatory RNAs (sRNAs) are important actors in bacterial virulence and pathogenicity. During my thesis, we investigated the functions and the mechanisms of action of a sRNA, named SprX (also known as RsaOR), expressed by the Staphylococcus aureus. First, we demonstrated that, in strains N315 and HG001, SprX expression varies through the growth and among numerous environmental conditions. By a comparative proteomic study, we identified several proteins whose expressions are ‘SprX-dependent’ and elucidated the mechanism of SprX action on one of those proteins. Indeed, SprX interacts specifically with the SpoVG translational initiation site of the yabJ-spoVG mRNA by an antisense mechanism inhibiting its expression. An accessible loop within SprX structure contains a C-rich domain involved in SpoVG regulation and is required and sufficient to modulate bacterial antibiotic resistance. We also studied whether the nucleotides changes between SprX sequence copies could influence SpoVG regulation triggered by SprX. Therefore, among the two copies of SprX in strain HG001, SprX2 has a higher affinity for yabJ-spoVG mRNA than SprX1. Altogether, our results showed that a regulatory RNA can alter bacterial resistance to antibiotics, and additional examples will probably be detected in the near future for more sRNAs and antibiotics.

The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.