Characterization of the enzymatic activity of beta-1,2-mannosyltransferases CaBmt1 and CaBmt3, the initiation and elongation enzymes of the beta-mannosylation of phosphopeptidomannan of Candida albicans

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

Characterization of the enzymatic activity of beta-1,2-mannosyltransferases CaBmt1 and CaBmt3, the initiation and elongation enzymes of the beta-mannosylation of phosphopeptidomannan of Candida albicans

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

Evaluation of monovalent and multivalent iminosugars to modulate Candida albicans β-1,2-mannosyltransferase activities.

International audienceβ-1,2-Linked oligomannosides substitute the cell wall of numerous yeast species. Several of those including Candida albicans may cause severe infections associated with high rates of morbidity and mortality, especially in immunocompromised patients. β-1,2-Mannosides are known to be involved in the pathogenic process and to elicit an immune response from the host. In C. albicans, the synthesis of β-mannosides is under the control of a family of nine genes coding for putative β-mannosyltransferases. Two of them, CaBmt1 and CaBmt3, have been shown to initiate and prime the elongation of the β-mannosides on the cell-wall mannan core. In the present study, we have assessed the modulating activities of monovalent and multivalent iminosugar analogs on these enzymes in order to control the enzymatic bio-synthesis of β-mannosides. We have identified a monovalent deoxynojirimycin (DNJ) derivative that inhibits the CaBmt1-catalyzed initiating activity, and mono-, tetra- and polyvalent deoxymannojirimycin (DMJ) that modulate the CaBmt1 activity toward the formation of a single major product. Analysis of the aggregating properties of the multivalent iminosugars showed their ability to elicit clusterization of both CaBmt1 and CaBmt3, without affecting their activity. These results suggest promising roles for multivalent iminosugars as controlling agents for the biosynthesis of β-1,2 mannosides and for monovalent DNJ derivative as a first target for the design of future β-mannosyltransferase inhibitors

Candida albicans β-1,2 mannosyl transferase Bmt3: Preparation and evaluation of a β (1,2), α (1,2)-tetramannosyl fluorescent substrate.

International audienceWe describe for the first time the chemical synthesis of a tetramannoside, containing both α (1 → 2) and β (1 → 2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies