Structure-function interactions of β-1,2-mannosyltransferases of Candida albicans : toward a better understanding of phosphopeptidomannan's β-mannosylation

Candida albicans est une levure saprophyte présente dans la flore digestive humaine. Elle peut néanmoins devenir pathogène chez des individus immunodéficients et causer des infections sévères associées à de forts taux de mortalité. La paroi de C. albicans, en contact avec l’hôte, contient des β-1,2 oligomannosides (β-Man) liés à de multiples molécules pariétales telles que le phospholipomannane (PLM) ou le phosphopeptidomannane (PPM). Ces β-Man sont présents dans les espèces les plus pathogènes de Candida (principalement C. albicans, mais également C. glabrata et C. tropicalis) et sont considérés comme des facteurs de virulence. L’identification d’une famille de 9 gènes codant pour des β-mannosyltransférases (CaBmt) a permis une meilleure compréhension du rôle de 6 de ces enzymes. Des études de génétique inverse ont montré que la β-1,2-mannosylation du PPM était assurée par les enzymes CaBmt1 à 4, tandis que CaBmt5 et 6 étaient impliquées dans celle du PLM. Une première enzyme responsable de l’initiation de la β-mannosylation du PPM, CaBmt1, a donc été caractérisée dans l’équipe grâce à l’étude de l’activité d’une forme recombinante soluble.L’objectif de cette thèse est de caractériser l’activité et la structure de CaBmt3, l’enzyme qui initie la polymérisation des β-Man suite à l’action de CaBmt1, pour mieux comprendre le mécanisme catalytique des β-1,2-mannosyltransférases. Ainsi, nous avons précisément identifié le substrat accepteur de CaBmt3 et défini ses paramètres enzymatiques. Par une approche combinant la diffusion des rayons X aux petits angles (SAXS), la modélisation moléculaire in silico et la mutagenèse dirigée de protéines recombinantes, nous proposons un modèle structural et catalytique de CaBmt3 qui pourrait être étendu à l’ensemble de la famille. En parallèle, nous avons montré que des iminosucres mono- et multivalents étaient capables de moduler l’activité des β-mannosyltransférases. Enfin, nous avons amorcé le travail sur une dernière enzyme, CaBmt4, qui est susceptible de polymériser le β-Man initié par CaBmt1 et CaBmt3. Pour conclure, ces travaux offrent une meilleure compréhension de la β-mannosylation du PPM de C. albicans. Ces études fonctionnelles, couplées aux avancées structurales, pourraient conduire à l’élaboration d’inhibiteurs de CaBmt et développer ainsi de nouvelles approches thérapeutiques contre les candidoses invasives.Candida albicans is a saprophytic yeast of human gastro-intestinal tract. It can however become pathogenic in immunocompromised individuals and cause severe infections associated with high mortality rates. The cell wall of C. albicans, in contact with the host, contains β-1,2 oligomannosides (β-Man) linked to several parietal molecules such as phospholipomannan (PLM) and phosphopeptidomannan (PPM). These β-Man are found in the most pathogenic Candida species (primarily C. albicans, but also in non-albicans species such as C. glabrata and C. tropicalis) and are considered as virulence factors. The identification of a family of 9 genes coding for β-mannosyltransferases (CaBmt) led to a better understanding of the role of 6 of these enzymes. Reverse genetics studies showed that CaBmt1-4 were responsible for the β-1,2-mannosylation of PPM, whereas CaBmt5-6 were involved in the β-1,2-mannosylation of PLM. A first enzyme responsible for the initiation of the PPM’s β-mannosylation, CaBmt1, was characterized in the lab by studying the activity of its recombinant soluble form.The goal of this thesis is to characterize both activity and structure of CaBmt3, the enzyme initiating β-Man polymerization following CaBmt1’s activity, in order to further the understanding of β-1,2-mannosyltransferases catalytic mechanism. Thus, we precisely identified CaBmt3 acceptor substrate and characterized its enzymatic parameters. Combining small angle X-ray diffraction (SAXS), in silico molecular modelization and site-directed mutagenesis of recombinant proteins, we propose a structural and catalytic model of CaBmt3 which could be extended to the whole family. In parallel, we showed that mono- and multivalent iminosugars were able to modulate β-mannosyltransferases activity. Finally, we started to work on one last enzyme, CaBmt4, which could potentially polymerize the β-Man initiated by CaBmt1 and CaBmt3.In conclusion, the synthesis of these investigations offers a better understanding of the β-mannosylation processes occurring on the PPM of C. albicans. Functional studies, in conjunction with structural advances could ultimately lead to the development of CaBmt inhibitors in order to design new therapeutic approaches for the management of invasive candidiasis

Relations structure-fonction des β-1,2-mannosyltransférases de Candida albicans : vers une meilleure compréhension de la β-mannosylation du phosphopeptidomannane

Candida albicans is a saprophytic yeast of human gastro-intestinal tract. It can however become pathogenic in immunocompromised individuals and cause severe infections associated with high mortality rates. The cell wall of C. albicans, in contact with the host, contains β-1,2 oligomannosides (β-Man) linked to several parietal molecules such as phospholipomannan (PLM) and phosphopeptidomannan (PPM). These β-Man are found in the most pathogenic Candida species (primarily C. albicans, but also in non-albicans species such as C. glabrata and C. tropicalis) and are considered as virulence factors. The identification of a family of 9 genes coding for β-mannosyltransferases (CaBmt) led to a better understanding of the role of 6 of these enzymes. Reverse genetics studies showed that CaBmt1-4 were responsible for the β-1,2-mannosylation of PPM, whereas CaBmt5-6 were involved in the β-1,2-mannosylation of PLM. A first enzyme responsible for the initiation of the PPM’s β-mannosylation, CaBmt1, was characterized in the lab by studying the activity of its recombinant soluble form.The goal of this thesis is to characterize both activity and structure of CaBmt3, the enzyme initiating β-Man polymerization following CaBmt1’s activity, in order to further the understanding of β-1,2-mannosyltransferases catalytic mechanism. Thus, we precisely identified CaBmt3 acceptor substrate and characterized its enzymatic parameters. Combining small angle X-ray diffraction (SAXS), in silico molecular modelization and site-directed mutagenesis of recombinant proteins, we propose a structural and catalytic model of CaBmt3 which could be extended to the whole family. In parallel, we showed that mono- and multivalent iminosugars were able to modulate β-mannosyltransferases activity. Finally, we started to work on one last enzyme, CaBmt4, which could potentially polymerize the β-Man initiated by CaBmt1 and CaBmt3.In conclusion, the synthesis of these investigations offers a better understanding of the β-mannosylation processes occurring on the PPM of C. albicans. Functional studies, in conjunction with structural advances could ultimately lead to the development of CaBmt inhibitors in order to design new therapeutic approaches for the management of invasive candidiasis.Candida albicans est une levure saprophyte présente dans la flore digestive humaine. Elle peut néanmoins devenir pathogène chez des individus immunodéficients et causer des infections sévères associées à de forts taux de mortalité. La paroi de C. albicans, en contact avec l’hôte, contient des β-1,2 oligomannosides (β-Man) liés à de multiples molécules pariétales telles que le phospholipomannane (PLM) ou le phosphopeptidomannane (PPM). Ces β-Man sont présents dans les espèces les plus pathogènes de Candida (principalement C. albicans, mais également C. glabrata et C. tropicalis) et sont considérés comme des facteurs de virulence. L’identification d’une famille de 9 gènes codant pour des β-mannosyltransférases (CaBmt) a permis une meilleure compréhension du rôle de 6 de ces enzymes. Des études de génétique inverse ont montré que la β-1,2-mannosylation du PPM était assurée par les enzymes CaBmt1 à 4, tandis que CaBmt5 et 6 étaient impliquées dans celle du PLM. Une première enzyme responsable de l’initiation de la β-mannosylation du PPM, CaBmt1, a donc été caractérisée dans l’équipe grâce à l’étude de l’activité d’une forme recombinante soluble.L’objectif de cette thèse est de caractériser l’activité et la structure de CaBmt3, l’enzyme qui initie la polymérisation des β-Man suite à l’action de CaBmt1, pour mieux comprendre le mécanisme catalytique des β-1,2-mannosyltransférases. Ainsi, nous avons précisément identifié le substrat accepteur de CaBmt3 et défini ses paramètres enzymatiques. Par une approche combinant la diffusion des rayons X aux petits angles (SAXS), la modélisation moléculaire in silico et la mutagenèse dirigée de protéines recombinantes, nous proposons un modèle structural et catalytique de CaBmt3 qui pourrait être étendu à l’ensemble de la famille. En parallèle, nous avons montré que des iminosucres mono- et multivalents étaient capables de moduler l’activité des β-mannosyltransférases. Enfin, nous avons amorcé le travail sur une dernière enzyme, CaBmt4, qui est susceptible de polymériser le β-Man initié par CaBmt1 et CaBmt3. Pour conclure, ces travaux offrent une meilleure compréhension de la β-mannosylation du PPM de C. albicans. Ces études fonctionnelles, couplées aux avancées structurales, pourraient conduire à l’élaboration d’inhibiteurs de CaBmt et développer ainsi de nouvelles approches thérapeutiques contre les candidoses invasives

The ER Protein Translocation Channel Subunit Sbh1 Controls Virulence of Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is distinguished by a cell-wallanchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37°C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in Dsbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors. Consistent with these observations, the C. neoformans Dsbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity

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

The impact of heteromultivalency in lectin recognition and glycosidase inhibition: an integrated mechanistic study

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.This study was supported by the Spanish Ministerio de Economía y Competitividad (contract numbers SAF2016‐76083‐R and CTQ2015‐64425‐C2‐1‐R), the Junta de Andalucía (contract number FQM2012‐1467) and the European Regional Development Funds (FEDER and FSE)