Candida albicans phospholipomannan,a new member of the fungal mannose inositol phosphoceramide family

The pathogenic yeast Candida albicans has the ability to synthesize unique sequences of beta-1,2-oligomannosides that act as adhesins, induce cytokine production, and generate protective antibodies. Depending on the growth conditions, beta-1,2-oligomannosides are associated with different carrier molecules in the cell wall. Structural evidence has been obtained for the presence of these residues in the polysaccharide moiety of the glycolipid, phospholipomannan (PLM). In this study, the refinement of purification techniques led to large quantities of PLM being extracted from Candida albicans cells. A combination of methanolysis, gas chromatography, mass spectrometry, and nuclear magnetic resonance analyses allowed the complete structure of PLM to be deduced. The lipid moiety was shown to consist of a phytoceramide associating a C(18)/C(20) phytosphingosine and C(25), C(26), or mainly C(24) hydroxy fatty acids. The spacer linking the glycan part was identified as a unique structure: -Man-P-Man-Ins-P-. Therefore, in contrast to the major class of membranous glycosphingolipids represented by mannose diinositol phosphoceramide, which is derived from mannose inositol phosphoceramide by the addition of inositol phosphate, PLM seems to be derived from mannose inositol phosphoceramide by the addition of mannose phosphate. In relation to a previous study of the glycan part of the molecule, the assignment of the second phosphorus position leads to the definition of PLM beta-1,2-oligomannosides as unbranched linear structures that may reach up to 19 residues in length. Therefore, PLM appears to be a new type of glycosphingolipid, which is glycosylated extensively through a unique spacer. The conferred hydrophilic properties allow PLM to diffuse into the cell wall in which together with mannan it presents C. albicans beta-1,2-oligomannosides to host cells.     

The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by [(3)H]mannose, [(3)H]palmitic acid, and [(32)P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units. NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of beta-1, 2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.     

Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on β-mannosylation of the phospholipomannan cell-wall glycosphingolipid

A family of nine genes encoding proteins involved in the synthesis of β-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add β-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to β-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated β-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM β-mannosylation. β-Mannose surface expression was not affected, confirming that β-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.     

Candida albicans phospholipomannan promotes survival of phagocytosed yeasts through modulation of bad phosphorylation and macrophage apoptosis

The surface of the pathogenic yeast Candida albicans is coated with phospholipomannan (PLM), a phylogenetically unique glycolipid composed of beta-1,2-oligomannosides and phytoceramide. This study compared the specific contribution of PLM to the modulation of signaling pathways linked to the survival of C. albicans in macrophages in contrast to Saccharomyces cerevisiae. C. albicans endocytosis by J774 and disregulation of the ERK1/2 signal transduction pathway was associated downstream with a reduction in Bad Ser-112 phosphorylation and disappearance of free Bcl-2. This suggested an apoptotic effect, which was confirmed by staining of phosphatidylserine in the macrophage outer membrane. The addition of PLM to macrophages incubated with S. cerevisiae mimicked each of the disregulation steps observed with C. albicans and promoted the survival of S. cerevisiae. Externalization of membranous phosphatidylserine, loss of mitochondrial integrity, and DNA fragmentation induced by PLM showed that this molecule promoted yeast survival by inducing host cell death. These findings suggest strongly that PLM is a virulence attribute of C. albicans and that elucidation of the relationship between structure and apoptotic activity is an innovative field of research.     

Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition

The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans.     

Surface hydrophobicity changes of two Candida albicans serotype B mnn4delta mutants

Cell surface hydrophobicity (CSH) of Candida species enhances virulence by promoting adhesion to host tissues. Biochemical analysis of yeast cell walls has demonstrated that the most significant differences between hydrophobic and hydrophilic yeasts are found in the acid-labile fraction of Candida albicans phosphomannoprotein, suggesting that this fraction is important in the regulation of the CSH phenotype. The acid-labile fraction of C. albicans is unique among fungi, in that it is composed of an extended polymer of beta-1,2-mannose linked to the acid-stable region of the N-glycan by a phosphodiester bond. C. albicans serotype A and B strains both contain a beta-1,2-mannose acid-labile moiety, but only serotype A strains contain additional beta-1,2-mannose in the acid-stable region. A knockout of the C. albicans homolog of the Saccharomyces cerevisiae MNN4 gene was generated in two serotype B C. albicans patient isolates by using homologous gene replacement techniques, with the anticipation that they would be deficient in the acid-labile fraction and, therefore, demonstrate perturbed CSH. The resulting mnn4delta-deficient derivative has no detectable phosphate-linked beta-1,2-mannose in its cell wall, and hydrophobicity is increased significantly under conditions that promote the hydrophilic phenotype. The mnn4delta mutant also demonstrates an unanticipated perturbation in the acid-stable mannan fraction. The present study reports the first genetic knockout constructed in a serotype B C. albicans strain and represents an important step for dissecting the regulation of CSH.     

Existence of novel beta-1,2 linkage-containing side chain in the mannan of Candida lusitaniae, antigenically related to Candida albicans serotype A.

The antigenicity of Candida lusitaniae cells was found to be the same as that of Candida albicans serotype A cells, i.e. both cell wall mannans react with factors 1, 4, 5, and 6 sera of Candida Check. However, the structure of the mannan of C. lusitaniae was significantly different from that of C. albicans serotype A, and we found novel beta-1,2 linkages among the side-chain oligosaccharides, Manbeta1-->2Manbeta1--> 2Manalpha1-->2Manalpha1-->2Man (LM5), and Manbeta1-->2Man-beta1-->2Manbeta1-->2Manalpha1-->2Manalpha1-->2Man (LM6). The assignment of these oligosaccharides suggests that the mannoheptaose containing three beta-1,2 linkages obtained from the mannan of C. albicans in a preceding study consisted of isomers. The molar ratio of the side chains of C. lusitaniae mannan was determined from the complete assignment of its H-1 and H-2 signals and these signal dimensions. More than 80% of the oligomannosyl side chains contained beta-1,2-linked mannose units; no alpha-1,3 linkages or alpha-1,6-linked branching points were found in the side chains. An enzyme-linked immunosorbent inhibition assay using oligosaccharides indicated that LM5 behaves as factor 6, which is the serotype A-specific epitope of C. albicans. Unexpectedly, however, LM6 did not act as factor 6.     

Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells.

Cell surface hydrophobicity of the opportunistic fungal pathogen Candida albicans has been linked to the level of cell wall protein glycosylation. Previous work demonstrated that outer chain mannosylation, rather than overall glycosylation, correlated with cell surface hydrophobicity. These studies further suggested that the phosphodiester-linked, acid-labile beta-1,2-mannan was the correlating element. The present work tests this hypothesis and extends the previous results. The composition of bulk mannan from hydrophobic and hydrophilic yeast cells, and the acid-labile mannan from both cell types are compared. Compositional analysis shows that the protein, hexose, and phosphorus content of bulk mannan is similar between the two phenotypes. Electrophoretic separation of acid-released and fluorophore-labeled mannan shows that the acid-labile oligomannosides from hydrophobic cells are longer and potentially in greater abundance than those from hydrophilic cells. These results suggest that regulation of a single step in cell wall protein outer chain mannosylation affects the cell surface ultrastructure and phenotype of C.albicans.     

Peptides that mimic Candida albicans-derived beta-1,2-linked mannosides

Beta-1,2-linked mannosides from Candida albicans phosphopeptidomannan (PPM) bind to macrophages through a receptor independent from the macrophage alpha-linked mannose receptor and stimulate these cells to secrete immune mediators. Anti-beta-1,2-linked mannoside but not anti-alpha-linked mannoside antibodies produced after immunization with neoglycoproteins protect animals from disseminated candidiasis. In this study, peptides that mimic beta-1,2-linked mannosides were isolated using phage display methodology. A phage library expressing random peptides was panned with an anti-beta-1,2-linked mannoside monoclonal antibody (mAb). After three rounds of biopanning, the isolated phages were able to inhibit recognition of C. albicans by the mAb. Sixty percent of the phages had an identical DNA insert corresponding to the peptide sequence FHENWPS that was recognized specifically by the mAb. Injection of KLH-coupled peptide into mice generated high titers of polyclonal antibodies against C. albicans yeast cell walls. The anti-FHENWPS antibodies bound to C. albicans PPM and were inhibited by soluble beta-1,2-mannotetraose. Together, these data provide evidence for mimotopic activity of the peptide selected by biopanning with the anti-beta-1,2-oligomannoside mAb.     

Toll-like receptors and innate antifungal responses

The mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in the mediation of innate immunity and the activation of adaptive immunity. Analysis of infection with human pathogenic fungi Candida albicans and Aspergillus fumigatus implicated TLR2 and TLR4 in elicitation of immune responses. Cryptococcus neoformans is recognized by a process that uses TLR4. C. albicans induces immunostimulation through causative agents, such as mannan or its structural derivatives (e.g. phospholipomannan), which are recognized by the immune system as pathogen-associated molecular patterns and are located in the cell wall of fungi. Secreted aspartic proteinases represent a key virulence factor that contributes to the ability of C. albicans to cause mucosal and disseminated infections, and might be a further potential stimulator of TLRs. Simultaneous activation of other pattern recognition receptors collaborating with TLRs illustrates the cooperation of various chains within ligand-specific receptor complexes for the recognition of fungal pathogens and their cell wall components.     

Candida albicans serotype B strains synthesize a serotype-specific phospholipomannan overexpressing a beta-1,2-linked mannotriose

Candida albicans strains consist of serotypes A and B depending on the presence of terminal beta-1,2-linked mannose residues in the acid-stable part of serotype A phosphopeptidomannan (PPM). The distribution of C. albicans serotypes varies according to country and human host genetic and infectious backgrounds. However, these epidemiological traits have not yet been related to a phenotypically stable molecule as cell surface expression of the serotype A epitope depends on the growth conditions. We have shown that C. albicans serotype A associates beta-mannose residues with another molecule, phospholipomannan (PLM), which is a member of the mannoseinositolphosphoceramide family. In this study, PLM from serotype B strains was analysed in order to provide structural bases for the differences in molecular mass and antigenicity observed between PLMs from both serotypes. Through these analyses, carbon 10 was shown to be the location of a second hydroxylation of fatty acids previously unknown in fungal sphingolipids. Minor differences observed in the ceramide moiety appeared to be strain-dependent. More constant features of PLM from serotype B strains were the incorporation of greater amounts of phytosphingosine C20, a twofold reduced glycosylation of PLM and overexpression of a beta-1,2 mannotriose, the epitope of protective antibodies. This specific beta-mannosylation was observed even when growth conditions altered serotype A PPM-specific epitopes, confirming the potential of PLM as a phenotypically stable molecule for serotyping. This study also suggests that the regulation of beta-mannosyltransferases, which define specific immunomodulatory adhesins whose activity depends on the mannosyl chain length, are part of the genetic background that differentiates serotypes.     

Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus

The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.     

Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1Delta contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1Delta did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream beta-1,2 mannosylation, mit1Delta represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1Delta was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1Delta was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced beta-mannosylation of phosphopeptidomannan. Increased beta-mannosylation of mannoproteins was observed under growth conditions that prevented the association of beta-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating beta-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of beta-oligomannoses on the host response need to be defined.     

Effect of aculeacin A, a wall-active antibiotic, on synthesis of the yeast cell wall

A wall-active, amphophilic antibiotic aculeacin A significantly but incompletely inhibited in vitro the activity of beta-(1,3)glucan synthase prepared from highly susceptible yeasts Saccharomyces cerevisiae and Candida albicans. In contrast, comparable cell-free preparations from S. cerevisiae active in chitin synthase or mannan synthase were insensitive to the antibiotic, suggesting selectivity of its action in synthesis of the yeast cell wall. An electron microscopic study of the effects of aculeacin A at 0.31 micrograms/ml, the optimally active concentration, on osmotically stabilized C. albicans cells revealed morphological alterations in both cell walls and cell membranes. Deformation in contour and derangement of the layered structure of the cell wall were prominent. In addition, massive fibrous material of beta-glucan-like microfibrils was occasionally extruded from the cell surface. Accompanying this effect on the cytology of the cell wall, ultrastructural and functional impairment of the cell membrane was demonstrated by transmission and freeze-fracture electron microscopic techniques. These data suggest that aculeacin A affects synthesis of the yeast cell wall through not only selective blockage of beta-(1,3)glucan synthase, as a result of a primary interaction with the cell membrane, but also inhibition of the fabrication of beta-glucan or other wall components into well-organized cell walls.     

Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms

The structure of the cell-wall mannan from the J-1012 (serotype A) strain of the polymorphic yeast Candida albicans was determined by acetolysis under mild conditions followed by HPLC and sequential NMR experiments. The serotype A mannan contained beta-1,2-linked mannose residues attached to alpha-1,3-linked mannose residues and alpha-1,6-linked branching mannose residues. Using a beta-1,2-mannosyltransferase, we synthesized a three-beta-1,2-linkage-containing mannoheptaose and used it as a reference oligosaccharide for 1H-NMR assignment. On the basis of the results obtained, we derived an additivity rule for the 1H-NMR chemical shifts of the beta-1,2-linked mannose residues. The morphological transformation of Candida cells from the yeast form to the hyphal form induced a significant decrease in the phosphodiesterified acid-labile beta-1,2-linked manno-oligosaccharides, whereas the amount of acid-stable beta-1,2 linkage-containing side chains did not change. These results suggest that the Candida mannan in candidiasis patients contains beta-1,2-linked mannose residues and that they behave as a target of the immune system.     

Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells

Candida glabrata owes its success as a pathogen, in part, to a large repertoire of adhesins present on the cell surface. Our current knowledge of C. glabrata adhesins and their role in the interaction between host and pathogen is limited to work with only a single family of epithelial adhesins (Epa proteins). Here, we report on the identification and characterization of a family of glycosylphosphatidylinositol-anchored cell wall proteins in C. glabrata. These proteins are absent in both Saccharomyces cerevisiae and Candida albicans, suggesting that C. glabrata has evolved different mechanism(s) for interaction with host cells. In the current study, we present data on the characterization of Pwp7p (PA14 domain containing Wall Protein) and Aed1p (Adherence to Endothelial cells) of this family in the interaction of C. glabrata with human umbilical vein endothelial cells. The deletion of C. glabrata genes PWP7 and AED1 results in a significant reduction in adherence to endothelial cells compared with the wild-type parent. These data indicate that C. glabrata utilizes these proteins for adherence to endothelial cells in vitro.     

C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100%) and a decrease in cell wall mannan (5.7-17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.     

Acetolysis of Pichia pastoris IFO 0948 strain mannan containing alpha-1,2 and beta-1,2 linkages using acetolysis medium of low sulfuric acid concentration

To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages. By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose. However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose. Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose. Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.