ABSTRACT
The fungal pathogen Cryptococcus neoformans is distinguished by a cell-wall-anchored 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 Δsbh1 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 Δsbh1 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. IMPORTANCE Cryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell-wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.
Subject(s)
Cryptococcosis , Cryptococcus neoformans , Saccharomyces cerevisiae Proteins , Animals , Mice , Cryptococcosis/microbiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Mammals/metabolism , Polysaccharides/metabolism , Protein Transport , Proteomics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , SEC Translocation Channels/genetics , Translocation, Genetic , Virulence , Virulence Factors/genetics , Virulence Factors/metabolism , Endoplasmic Reticulum/metabolismABSTRACT
ß-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. ß-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 ß-mannosides is under the control of a family of nine genes coding for putative ß-mannosyltransferases. Two of them, CaBmt1 and CaBmt3, have been shown to initiate and prime the elongation of the ß-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 ß-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 ß-1,2 mannosides and for monovalent DNJ derivative as a first target for the design of future ß-mannosyltransferase inhibitors.
Subject(s)
Candida albicans/enzymology , Enzyme Inhibitors/pharmacology , Fungal Proteins/antagonists & inhibitors , Glucosamine/analogs & derivatives , Imino Sugars/pharmacology , Mannosyltransferases/antagonists & inhibitors , 1-Deoxynojirimycin/chemical synthesis , 1-Deoxynojirimycin/pharmacology , Candida albicans/drug effects , Candida albicans/genetics , Cell Wall/drug effects , Cell Wall/enzymology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Glucosamine/chemical synthesis , Glucosamine/pharmacology , Imino Sugars/chemical synthesis , Kinetics , Mannosides/metabolism , Mannosyltransferases/genetics , Mannosyltransferases/metabolism , Pichia/genetics , Pichia/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1â2) and ß (1â2) 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 ß-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (ß Man (1,2) ß Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies.
Subject(s)
Candida albicans/enzymology , Fluorescent Dyes/metabolism , Mannosides/metabolism , Mannosyltransferases/metabolism , Fluorescent Dyes/chemistry , Mannosides/chemistry , Molecular Conformation , Substrate SpecificityABSTRACT
ß-1,2-Linked mannosides are expressed on numerous cell-wall glycoconjugates of the opportunistic pathogen yeast Candida albicans. Several studies evidenced their implication in the host-pathogen interaction and virulence mechanisms. In the present study, we characterized the in vitro activity of CaBmt3, a ß-1,2-mannosyltransferase involved in the elongation of ß-1,2-oligomannosides oligomers onto the cell-wall polymannosylated N-glycans. A recombinant soluble enzyme Bmt3p was produced in Pichia pastoris and its enzyme activity was investigated using natural and synthetic oligomannosides as potential acceptor substrates. Bmt3p was shown to exhibit an exquisite enzymatic specificity by adding a single terminal ß-mannosyl residue to α-1,2-linked oligomannosides capped by a Manß1-2Man motif. Furthermore, we demonstrated that the previously identified CaBmt1 and CaBmt3 efficiently act together to generate Manß1-2Manß1-2[Manα1-2]n sequence from α-1,2-linked oligomannosides onto exogenous and endogenous substrates.
Subject(s)
Candida/enzymology , Fungal Proteins/metabolism , Mannans/metabolism , Mannosyltransferases/metabolism , Phosphopeptides/metabolism , Candida/metabolism , Cell Wall/metabolism , Substrate SpecificityABSTRACT
Glycosylation is a crucial step in the modification of proteins or sphingolipids that then play a prominent role in fungal biology. Glycosylation controls the structure and plasticity of the fungal cell wall and fungi-host interactions. Non-pathogenic and pathogenic yeasts, such as Saccharomyces cerevisiae and Candida albicans, respectively, have been useful models for analyzing the mannosylation of proteins and sphingolipids, which mainly takes place in the Golgi apparatus. Studies of these yeasts have identified different mannosyltransferases that belong to separate families of glycosyltransferases. The characterization of mannosyltransferases and their activities is essential for deciphering cell wall biogenesis, for identifying mannosides involved in virulence and for designing inhibitors that target specific mannosylation processes.
Subject(s)
Candida albicans/metabolism , Fungal Proteins/metabolism , Glycoconjugates/metabolism , Golgi Apparatus/metabolism , Mannose/metabolism , Saccharomyces cerevisiae/metabolism , Sphingolipids/metabolism , Candida albicans/enzymology , Glycosylation , Golgi Apparatus/enzymology , Mannosyltransferases/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
The presence of ß-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (ß-mannosyltransferase 1), a member of the recently identified ß-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic α-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of α-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of α-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal ßMan (ß-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific ß-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.