No description available.

In this work, we examine two disparate aspects of glycosylation. The first project involves the elucidation of the glycosylation of the novel tetronolide natural product, kijanimicin. The biosynthesis of the deoxysugar TDP-L-digitoxose from the kijanimicin natural product pathway was achieved in vitro. The genes were identified from the cluster, cloned, expressed and the products were purified. Activity was demonstrated for the novel enzymes and the pathway was reconstructed in vivo using Streptomyces lividans. These strains of S. lividans were used to examine kijanimicin glycosyltransferase activity. We were able to demonstrate activity for 3 of 4 digitoxosyltransferases in the biosynthetic pathway and propose a biosynthetic scheme by which the tetrasaccharide chain is formed. We identified two putative glycosidases with novel folds, and one glycosyltransferase that appears to have unprecedented activity, attaching 2 if not 3 sugars in sequence. In the second portion of this work, we attempted to identify the eukaryotic C-mannosyltransferase enzyme and demonstrate its activity in vitro and in vivo. Here, we describe our efforts to identify the CMT. Through in silico analysis, putative C-mannosyltransferase genes were identified. These genes were expressed in E. coli and S. cerevisiae, however gene expression was apparently toxic to E. coli. S. cerevisiae expression was acceptable, but extraction proved to be somewhat problematic. We describe our efforts to develop a CMT assay for use in vitro by expressing the putative CMT in insect cells, which was much more promising. We also attempted to knock down the putative CMT genes using shRNA, which demonstrated that the genes of unknown function that were identified were essential for cellular viability. This work has contributed to the fields of both C-mannosylation and natural product glycosylation. We have elucidated the biosynthetic pathway of a novel deoxysugar, and identified potentially valuable tools for glycoengineering including a glycosyltransferase that appears to exhibit novel polymeric activity, as well as identifying two glycosyltransferase proteins that are apparent glycosidases. Our attempts to identify the CMT provided valuable insight into the future development of a C-mannosylation assay, and we have identified several promising protein candidates that are apparently essential for H. sapiens cellular viability.

Candida albicans est une levure de la flore digestive humaine, pouvant dans certaines conditions s’avérer être un pathogène opportuniste. Différents glycoconjugués de la paroi de la levure, en particulier le phosphopeptidomannane (PPM), sont impliqués dans l’interaction hôte-pathogène notamment via des beta-1,2 oligomannosides (β-Mans) terminaux intervenant dans les mécanismes de virulence de la levure. Les travaux de thèse visent à mieux comprendre les voies de biosynthèse des β-Mans par l’étude des enzymes impliquées (CaBmts). Dans ce contexte, la première enzyme étudiée a été CaBmt1, impliquée dans l’initiation de la β-mannosylation du PPM. La stratégie d’étude a consisté d’une part en la préparation d’un panel important de substrats accepteurs potentiels et leur caractérisation structurale par spectrométrie de masse et RMN, et d’autre part à l’étude de l’activité enzymatique de Bmt1p, enzyme recombinante produite chez Pichia pastoris, en présence des substrats naturels et de substrats de synthèse. Nous avons démontré que Bmt1p était capable in vitro de transférer successivement deux résidus de mannose en β-1,2 sur un tri-ou tétramannoside lié en α-1,2. Dans la seconde partie, nous nous sommes intéressés à la caractérisation de l’activité de CaBmt3, enzyme impliquée dans l’élongation des β-Mans du PPM ; la même démarche a été retenue pour l’étude. Les résultats obtenus montrent que Bmt3p transfère in vitro un seul résidu mannose en β-1,2 sur un tétramannoside constitué d’une chaine en α-1,2 avec un mannose terminal lié en β-1,2. L’ensemble de ces travaux sont un préalable à l’élaboration de nouvelles drogues anti-fongiques ciblant la synthèse de la paroi. / Candida albicans is a commensal yeast present in human digestive flora; nevertheless, this opportunistic pathogen may cause severe infections. Several cell wall components including phosphopeptidomannan (PPM) are involved in C. albicans-host cells interaction especially by terminal beta-1,2 oligomannosides (β-Mans) known as implicated in the yeast virulence mechanisms. The aim of our work is to better understand biosynthetic pathways of β-Mans by the characterization of CaBmts individual activities. In this context, the first enzyme to be studied was CaBmt1, involved in the initiation of β-mannosylation of PPM. The strategy is based firstly on the preparation of a large panel of potential acceptor substrates and their structural characterization by mass spectrometry and NMR. On the other hand, the study of enzymatic activity of Bmt1p, a recombinant soluble form produced in Pichia pastoris, was performed in the presence of the natural substrates and synthetic substrates. We established that Bmt1p can sequentially transfer in vitro two -1,2-mannosyl units onto a α1-2 linked tri-or tetramannoside. The second part of this work focused on the characterization of the activity of CaBmt3, the enzyme involved in the elongation of the β-Mans chain on the PPM; the same approach was used for the study. Our results demonstrated that Bmt3p can catalyse the in vitro transfer of one -1,2-mannosyl unit onto a tetramannoside containing a terminal β-1,2-Man linked to a α(1-2)Man chain. These data are a prerequisite for the design of new potential antifungal drugs that target the biosynthesis of cell wall.

No description available.

La microsporidie, Encephalitozoon cuniculi, parasite intracellulaire, est un pathogène opportuniste. Des reconstructions tridimensionnelles à partir de coupes sériées ont permis de visualiser les différents stades cellulaires au cours de la sporogenèse. L'immunolocalisation de protéines pariétales couplée à l'hybridation in situ des ARNm correspondants ont révélé leur expression différentielle durant le développement intracellulaire. L'étude sur la glycosylation des protéines a permis de démontrer l'absence de N-glycosylation et l'existence d'une voie de O-mannosylation. Semblables à celles des champignons, les chaînes sont linéaires d'une longueur maximale de 8 mannoses liés en alpha1,2 et les mannoprotéines sont localisées dans le capuchon polaire. Des protéines fucosylées sont présentes dans la paroi sporale. La mise au point d'un protocole de séparation des stades sporogoniques en gradient de densité, offre des perspectives d'analyses biochimiques comparatives