Beta-1,2-mannosylation of Candida albicans mannoproteins and glycolipids differs with growth temperature and serotype.

Increasing the growth temperature from 28 to 37 degrees C reduced the expression of beta-1,2-oligomannoside epitopes on mannoproteins of Candida albicans serotypes A and B. In contrast, beta-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (M(r)) of this compound. At all growth temperatures investigated, serotype A PLM displayed an M(r) and an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.

Contribution of phospholipomannan to the surface expression of beta-1,2-oligomannosides in Candida albicans and its presence in cell wall extracts.

beta-1,2-Oligomannosides (beta-1,2-Man) derived from Candida albicans mannan have been shown to act as adhesins and to induce protective antibodies. We used monoclonal antibodies specific for beta-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of beta-1,2-Man epitopes. These monoclonal antibodies were also used for Western blotting of cell surface extracts to study the nature of the molecules expressing the beta-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of beta-1,2-Man epitopes at the cell wall surfaces of yeasts grown on solid media. PLM was present in intercellular matrixes of colonies grown on agar and was detected as a contaminant in mannan batches prepared by conventional methods.

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.

Role of extracellular signal-regulated protein kinase cascade in macrophage killing of Candida albicans.

The pathogenic yeast Candida albicans and its derived molecules stimulate a wide range of macrophage secretory functions and may adapt to escape being killed by this phagocyte. In this study, phagocytosis of C. albicans and of the nonpathogenic yeast Saccharomyces cerevisiae was shown to be associated with phosphorylation of the mitogen-activated protein kinase (MAPK)/extracellularly regulated kinase (ERK) pathway in the absence of significant activation of either p38MAPK or stress-activated protein kinase/c-Jun N-terminal kinase. However, although 80% of endocytosed C. albicans survived after 1 h, 80% of S. cerevisiae cells were killed. Considerable quantitative differences were observed between the two species in the sequential phosphorylation of MAPK/ERK kinase (MEK), extracellularly regulated kinase-1, and 90-kDa-ribosomal S6 kinases. A lower level of activation of the pathway by C. albicans was associated with a species-specific overexpression of the MEK phosphatase MAPK phosphatase (MKP)-1. Killing of both C. albicans and S. cerevisiae could be reduced using PD98059, which mimics MKP-1 and inhibits MEK phosphorylation, suggesting that specific MKP-1 activation by C. albicans could contribute to its ability to escape the yeast lytic potential of macrophages.

Candida albicans-derived beta-1,2-linked mannooligosaccharides induce desensitization of macrophages.

Candida albicans beta-1,2-oligomannosides stimulate macrophage tumor necrosis factor alpha (TNF-alpha) but not NO release. This stimulation desensitized macrophages by altering beta-1, 2-oligomannoside-dependent TNF-alpha production and lipopolysaccharide-dependent TNF-alpha and NO secretion. Desensitization was not related to tyrosine phosphorylation signal transduction but was transferred by culture supernatants in which arachidonic acid derivatives were evidenced.

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.

Early signal transduction induced by Candida albicans in macrophages through shedding of a glycolipid.

Cell wall beta-1,2-oligomannosides are involved in Candida albicans binding to macrophages and in their stimulation to produce cytokines. The nature of signaling events occurring during initial interaction of macrophage J774 cell line and C. albicans, together with the nature of molecules containing beta-1,2-oligomannosides released by the yeasts, was examined. Cocultivation led to a herbimycin A-sensitive production of tumor necrosis factor-alpha. Immunofluorescence and Western blotting confirmed tyrosine phosphorylation and revealed an accumulation of 90- to 120-kDa phosphoproteins. Antibodies specific for beta-1,2-oligomannosides showed that these epitopes were shed at an early stage from the yeasts to the macrophage membrane, in association with a glycolipid previously described as C. albicans phospholipomannan. Incubation of macrophages with purified phospholipomannan alone led to a signal transduction pathway identical to that observed with living yeasts. All of these results demonstrate that C. albicans phospholipomannan shedding is involved in C. albicans-macrophage interaction through beta-1,2-oligomannosides.

Definitive chemical evidence for the constitutive ability of Candida albicans serotype A strains to synthesize beta-1,2 linked oligomannosides containing up to 14 mannose residues.

We have previously reported the presence of phosphate bound beta-1,2 linked oligomannosides with unusually high degrees of polymerization (DP > 7) in the mannan of Candida albicans strain VW32. To confirm this observation, we have prepared these oligomannosides from the mannan of C. albicans strain NIH A 207. Gel filtration chromatography and TLC analysis revealed DP up to 14. For both strains, NMR analysis confirmed the exclusive presence of beta-1,2 linkages in the pools of oligomannosides with a DP higher than 6 which presented an average DP of 10.6 (VW32) and 10.4 (NIH A 207). These results are important to consider in relation with the ability of these C. albicans derived oligomannosides to trigger TNFalpha synthesis according to their DP.

Evidence for different mannosylation processes involved in the association of beta-1,2-linked oligomannosidic epitopes in Candida albicans mannan and phospholipomannan.

A monoclonal antibody specific for beta-1,2-linked oligomannosides was used to study the association of these residues with Candida albicans mannan and phospholipomannan (PLM) in relation to growth conditions and in mannan mutant strains. Double immunofluorescence assays performed on cells grown under standard conditions indicated a highly heterogeneous cell surface expression of these epitopes in comparison with the homogeneous expression of alpha-linked oligomannosidic epitopes. Growth in the presence of tunicamycin, which inhibits mannan N-glycosylation, resulted in an absence of beta-1,2-oligomannosidic epitopes on the cell surface, although PLM synthesis still occurred as shown by autoradiography. Similarly, growth in acidic conditions, which inhibits the incorporation of beta-1,2-oligomannosides in mannan, resulted in an absence of beta-1,2-oligomannosidic epitopes at the cell surface, although they still associated with PLM as shown by Western blotting. Western blots of C. albicans mutant strains with reduced amounts or an absence of phosphorus and acid-labile beta-1,2-oligomannosides in their mannan confirmed that the association of beta-1,2-linked oligomannosides with mannan and with PLM involves different mannosylation processes.

Mapping of beta-1,2-linked oligomannosidic epitopes among glycoconjugates of Candida species.

The distribution of beta-1,2-linked oligomannosides among glycoconjugates of various Candida species was investigated by Western blotting, using monoclonal and polyclonal antibodies which react with these epitopes. Expression of beta-1,2-linked oligomannosidic epitopes on a 14-18 kDa polydisperse antigen nonreactive with concanavalin A (ConA), previously identified as a C. albicans serotype A phospholipomannan (PLM), appeared to be restricted to C. albicans serotypes A and B (including var. C. stellatoidea types I and II) and C. tropicalis. In C. albicans, beta-1,2-linked oligomannosidic epitopes also appeared to be slightly associated with high molecular mass (> 100 kDa) polydisperse ConA-reactive mannoproteins. For all the other Candida strains investigated, belonging to the species C. parapsilosis, C. krusei, C. glabrata and C. robusta (S. cerevisiae), beta-1,2-linked oligomannosidic epitopes were found to be present in association with medium molecular mass (18-100 kDa) and high molecular mass ConA-reactive mannoproteins, giving reproducible labelling profiles that varied between species.

Beta-1,2-linked oligomannosides from Candida albicans act as signals for tumor necrosis factor alpha production.

Different cell wall components from Candida albicans have been shown to stimulate murine macrophages for tumor necrosis factor alpha (TNF-alpha) secretion. All of these molecules contain beta-1,2-oligomannosides. In order to examine their role in TNF-alpha production, acid-labile oligosaccharides, released from C. albicans VW32 cell wall phosphopeptidomannan by mild acid hydrolysis, and previously shown to correspond to homopolymers of beta-1,2-linked mannopyranosyl units, were separated by gel filtration chromatography according to their degree of polymerization. Murine macrophages incubated with purified oligomannosides (M2 to M8) released TNF-alpha to an extent which was dependent on, although not directly correlated with, the length of the mannosyl chain. Slight activity was observed with M4 and M5; M6 and M7 had virtually no effect, whereas M8 was associated with strong TNF-alpha release. This effect of M8 was dose dependent and was not altered by polymyxin B, known to interfere with lipopolysaccharide-induced TNF-alpha production. These results suggest that stimulation of TNF-alpha release by C. albicans glycoconjugates containing beta-1,2-linked oligomannosides may be due, at least in part, to the presence of these components.

The Candida albicans phospholipomannan induces in vitro production of tumour necrosis factor-alpha from human and murine macrophages.

We have previously identified a Candida albicans 14,000-18,000 MW antigen reacting with anti-beta-1,2-linked oligomannosides antibodies as being a phospholipomannan (PLM). Because of the structural similarities between the C. albicans PLM and lipophosphoglycans from various microbial pathogens known to be potent tumour necrosis factor-alpha (TNF-alpha) inducers, we investigated the PLM ability to induce TNF-alpha. Incubation of human monocytic cells THP-1 with PLM led to dose-dependent production of TNF-alpha that was significantly increased by prestimulation of the cells with interferon-gamma (IFN-gamma). Production of TNF-alpha by macrophages under PLM stimulation was confirmed by using macrophages elicited from the mouse peritoneal cavity. In all investigated conditions, PLM-induced TNF-alpha production differed significantly in both kinetics and dose dependence from lipopolysaccharide (LPS) induction used as control. It appears, therefore, that the C. albicans PLM shares functional homologies with microbial lipophosphoglycans identified as pathogenicity factors, although prestimulation of the target cells was required for the PLM-derived opportunistic pathogen to trigger the cytokine network.

Isolation and preliminary characterization of the 14- to 18-kilodalton Candida albicans antigen as a phospholipomannan containing beta-1,2-linked oligomannosides.

Western blot (immunoblot) analysis of Candida albicans germ tube extracts has demonstrated the probable presence of beta-1,2-linked oligomannosides acting as epitopes distributed over a 14- to 18-kDa antigen unreactive to concanavalin A. These conclusions about the existence of these non-mannan-associated oligomannoside species were reinforced in the present study by the demonstration of reactivity of factor serum 5 (Iatron Laboratories) with the same antigen. A monoclonal antibody which reacted in an enzyme immunoassay with beta-1,2-linked oligomannosides converted into neoglycolipids and in Western blotting with the 14- to 18-kDa antigen from yeast and germ tubes, through metaperiodate-sensitive epitopes, was used for further characterization of the molecule. Reducing agents and strong protease digestion, which have deleterious effects on C. albicans proteins and mannoproteins, affected neither the antigenicity nor the relative molecular weight of the molecule. Western blots performed after migration of protease-treated extracts in polyacrylamide gels without sodium dodecyl sulfate (SDS) showed that the 14- to 18-kDa antigen could be negatively charged, whereas metabolic radiolabeling demonstrated that these charges could originate, at least in part, from the presence of phosphorus within the molecule. Chloroform-methanol-water extraction of protease-resistant material led to purification of the 14- to 18-kDa antigen, as determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Metabolic radiolabeling with mannose confirmed the presence of these sugar residues within the purified 14- to 18-kDa antigen (despite its nonreactivity to concanavalin A), whereas radiolabeling with palmitic acid demonstrated its lipopolysaccharidic nature. Together, these results led to the conclusion that the 14- to 18-kDa antigen is a phospholipomannan.

Probable presence of beta(1-2)-linked oligomannosides that act as human immunoglobulin G3 epitopes and are distributed over a Candida albicans 14- to 18-kilodalton antigen.

Kinetic analysis of candidosis patients' immunoglobulin G3 response has shown that reactivity towards beta(1-2)-linked mannan-derived oligomannosides was associated with the recognition through metaperiodate-sensitive epitopes of a 14- to 18-kDa Candida albicans antigen unreactive with concanavalin A.

Qualitative and quantitative differences in recognition patterns of Candida albicans protein and polysaccharide antigens by human sera.

Cytoplasmic and cell wall proteins and glycoproteins extracted from Candida albicans germ tubes were screened by Western blotting for their ability to differentiate between the serological responses of patients with candidosis and healthy individuals. Molecules of 114, 74 and 65 kDa were not recognized by any sera. Qualitative differences were observed for responses to proteins and glycoproteins from 29 to 60 kDa. Conversely, only quantitative differences were found to high molecular mass glycoproteins. Their recognition by control sera was invariably associated with reactivity against a 14-18 kDa antigen. However, despite a high level of antibodies against high molecular mass mannoproteins, some patients sera failed to react with the 14-18 kDa antigen, or lost this reactivity during the course of the disease.

Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies.

Six monoclonal antibodies (MAbs) from various laboratory sources (EB-CA1, EB-CA2, H5, AF1, C6, and 5B2), reacting with the polysaccharidic moieties of Candida albicans mannoproteins, were used for epitope mapping by an enzyme-linked immunosorbent assay (ELISA) with neoglycolipids and by Western blotting (immunoblotting) of a C. albicans germ tube extract. The ELISA involved neoglycolipids constructed from three families of oligomannosides released by sequential depolymerization of C. albicans phosphopeptidomannan by acid hydrolysis (NGLH), beta-elimination (NGLO), and acetolysis (NGLA). All of the MAbs exhibited low reactivities against NGLO. MAbs EB-CA1, EB-CA2, and H5 reacted mainly against NGLA, and MAbs C6 and AF1 recognized mainly NGLH, whereas MAb 5B2 reacted with both families of neoantigens. When this method was compared with Western blotting, strong reactivity to NGLA was associated with the presence of epitopes shared by high-molecular-weight mannoproteins, whereas strong reactivity to NGLH was associated with a reactivity to a family of 14- to 18-kDa antigens. The reactivity of MAb 5B2 was associated with both high-molecular-weight mannoproteins and the 14- to 18-kDa antigens. In relation to the present knowledge about the structure of the C. albicans phosphopeptidomannan oligomannosidic repertoire, these results provide preliminary data concerning the molecular basis of the recognition of mannopyranosyl sequences by MAbs and their distribution among C. albicans mannoproteins.

Immunological relationship among glyceraldehyde-3-phosphate dehydrogenases in the genera Enterobacter and Escherichia.

The comparative immunological study of glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) among Enterobacteriaceae carried out with an anti-Enterobacter cloacae G-3-PDH serum pointed out the large heterogeneity of the genera Enterobacter and Escherichia. The use of two-dimensional maps integrating our new data and previously acquired quantitative data confirmed these results.

[Comparative immunological study of glyceraldehydephosphate dehydrogenase in Enterobacteriaceae: contribution of an anti-glyceraldehydephosphate dehydrogenase antiserum of Enterobacter intermedium]. / Etude immunologique comparative de la glycéraldéhyde-3-phosphate-déshydrogénase chez les Entérobactéries: apports d'un sérum anti-glycéraldéhyde-3-phosphate-déshydrogénase de Enterobacter intermedium.

A comparative immunological study of glyceraldehyde-3-phosphate dehydrogenase among Enterobacteriaceae was carried out with an antiserum against Enterobacter intermedium G-3-PDH. Results of immunodiffusion experiments and microcomplement fixation studies showed E. intermedium to be a homogeneous species. The genera Enterobacter and Escherichia were found to be quite heterogeneous.

Antigenic specificity of Escherichia coli alkaline phosphatase studied with monoclonal antibodies: immunological characterization of E. coli and Shigella strains.

Monoclonal antibodies (MoAb) to the alkaline phosphatase of Escherichia coli were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of E. coli and SP2O/Ag-14 myeloma cells. Five stable clones were established. They all produced antibodies which reacted by enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase of all E. coli (25 strains) independently of their origin (drinking water, saline water, surface water, faecal or clinical origin), and with that of four Shigella species (7 strains) tested. Four of these MoAb gave a positive reaction with 52% (MoAb 4G10), 73% (MoAb 4F8, MoAb 4G6) and 89% (MoAb 3C8) of 14 other bacterial species (30 strains) studied, while one (MoAb 2E5) did not react with alkaline phosphatase of these unrelated bacterial strains and thus appeared specific for E. coli and Shigella species. This MoAb was still detectable in ascitic fluids at 1/500,000 in ELISA, and detected all E. coli strains in an indirect immunofluorescence assay at 1/100. It could therefore be used as a reagent for routine detection of E. coli in drinking water, foods or clinical specimens.

[Immunological study of glyceraldehyde-3-phosphate dehydrogenase in Enterobacteriaceae; taxonomic value]. / Etude immunologique de la glycéraldéhyde-3-phosphate-déshydrogénase chez les Entérobactéries; intérêt taxonomique.

The antigenic structure of glyceraldehyde-3-phosphate dehydrogenase of the most representative Enterobacteriaceae species were compared with an antiserum to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase. The results of the immunodiffusion experiments were confirmed and specified by micro-complement fixation studies. They demonstrated a total immunological identity between the E. coli enzyme and the enzymes of Alcalescens-dispar and the Shigella species, a marked relatedness of the Salmonella species enzyme and a more or less significant relation of the enzymes of the other Enterobacteriaceae species. Moreover, the micro-complement fixation had the same sensitivity and a better selectivity than the DNA/DNA hybridizations. The results show that, like DNA polymerase, this enzyme has evolved more slowly than the other enzymes studied at this time.