The Candida albicans Phospholipomannan Is a Family of Glycolipids Presenting Phosphoinositolmannosides with Long Linear Chains of β-1,2-Linked Mannose Residues*

In a series of studies, we have shown thatCandida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for β-1,2-oligomannosides and was biosynthetically labeled by [3H]mannose, [3H]palmitic acid, and [32P]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 β-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 β-1,2-oligomannosides in a glycoconjugate other than the C. albicansphosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.

The yeast Candida albicans is a normal component of the human endogenous microflora, but it can cause frequent and severe disseminated infections among hospitalized patients (1). Basic progress has been made in the elucidation of C. albicans characteristics linked to switching (2), dimorphism (3), adhesion (4), and enzyme secretion (5,6) that could explain the mechanism by which this fungus can be an opportunistic pathogen. However, the mechanisms that direct susceptibility and resistance to C. albicans infection are as yet unclear, and current extensive research concerns C. albicans molecules interacting with the host immune system. Among these studies, research has gradually focused on ␤-1,2-linked oligomanno-sides. Oligomannnosides with this unusual type of linkage were first described by Shibata et al. (7) as associated with the C. albicans cell wall phosphopeptidomannan by phosphodiester bridges. ␤-1,2-oligomannosides are immunogenic and elicit specific antibodies in animals (8 -10) and humans (11). Anti-␤-1,2oligomannosides antibodies have been shown to be protective against C. albicans in rodent models of systemic and vaginal candidosis (5,12). ␤-1,2-oligomannosides derived from C. albicans phosphopeptidomannan have also been shown to induce TNF 1 -␣ synthesis from cells of the macrophage lineage through a phosphotyrosine kinase-dependent pathway (13) and to bind to macrophage cell membranes (14,15).
In previous studies, we have shown by use of specific monoclonal antibodies (16) that ␤-1,2-oligomannosides are present (in the absence of accessible ␣-linked mannose residues) on a polydispersed low molecular weight antigen and that this antigen is a glycolipid. This glycolipid has been named a phospholipomannan (PLM) on the basis of its composition (17). The PLM is a strong TNF-␣ inducer in vitro and in vivo (18). When C. albicans comes into contact with macrophages, large amounts of PLM are rapidly shed by C. albicans, which trigger intense signaling and secretory responses from these target cells (19). Similar signaling events induced in host cells have been described as induced by GPI-related glycolipids from pathogens of the genera Leishmania, Trypanosoma, and Mycobacteria (20 -23). In this study, we have further purified and chemically analyzed the C. albicans PLM to establish the relationship of PLM with these microbiolglycolipids and to provide a structural basis for the understanding of the immunochemical and immunomodulatory properties of PLM. MATERIALS (Isotopchim). The purification of high amounts of PLM for physicochemical analysis was performed from yeast cells grown at 28°C in a bioreactor as described for the preparation of phosphopeptidomannan (24).
Cell Extracts-Whole cell extracts, designated as Fr. A, were ob-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Purification of PLM and Release of Its Glycan Moiety-The main purification steps are summarized in Fig. 1. Dried French press extracts (Fr. A), either labeled or not, were subjected to three extractions with chloroform/methanol (2:1, by volume), three extractions with chloroform/methanol/water (10:10:1, by volume), and finally to three extractions with chloroform/methanol/water (10:10:3, by volume; C/M/W3). The three last extracts containing the PLM were pooled, filtered on a GF/F membrane (Whatman) and designated as Fr. B. Fr. B was then evaporated and partitioned at least three times between water and water-saturated butanol. The water phase was designated as Fr. C. Improved purification of unlabeled PLM for physicochemical analyses was performed from Fr. C using additional chromatographic steps. Fr. C was dissolved in 0.1 M ammonium sulfate, 1% ethanol and subjected to hydrophobic interaction chromatography on a phenyl-Sepharose (Amersham Pharmacia Biotech) column (1 ϫ 10 cm). The column was successively washed with 1, 5, and 10% ethanol. PLM elution was obtained with 40% ethanol. The PLM fractions were concentrated, dissolved in C/M/W3, and subjected to chromatography on a silica gel 60 column (1.5 ϫ 20 cm) at 4°C. Components were first eluted with C/M/W3, and after the recovery of PLM, with chloroform/methanol/ water (2:3:1, by volume). These PLM fractions were concentrated and frozen until used and were designated as Fr. D.
Fr. D was subjected to alkaline hydrolysis to release the PLM glycan moiety. 2 mg of purified PLM (Fr. D) estimated by its sugar content were incubated for 90 h at 37°C in 1 N KOH, 30% methanol and then neutralized with 1 N acetic acid, dried, and subjected to hexane extraction. The hydrolysate was then solubilized in 1% propanol, 0.1 M ammonium acetate and applied to a octyl-Sepharose (Amersham Pharmacia Biotech) column (1 ϫ 11 cm). Hydrolyzed PLM was eluted with the same buffer and desalted on a Bio-gel P2 (Bio-Rad SA, Ivry sur Seine, France) column (0.8 ϫ 15 cm). This material was designated as Fr. Dg.
Thin Layer Chromatography (TLC) Analysis-Thin layer chromatography of unlabeled material was performed primarily using aluminumbacked silica 60 TLC plates (Merck, Darmstadt, Germany). Plates were developed at room temperature using chloroform/methanol/water (2: 3:1, by volume) as the solvent system, and spots were revealed with an orcinol stain. TLC of radiolabeled extracts were developed on glassbacked silica gel SI 60 plates at room temperature using C/M/W3 or chloroform/methanol/0.1% KCl (2:3:1, by volume) as solvent systems. Each lane was then scanned for radioactivity profile with a Berthold LB 2842 automatic TLC scanner. To further identify the PLM peaks on TLC, the radioactive areas were scraped, and the glycolipids were extracted successively with methanol, 10% and C/M/W3 and then subjected to a dot-blot analysis involving the monoclonal antibody (mAb) DF9-3 (25), a mouse IgM with specificity for ␤-1,2-linked oligomannosides (17). This mAb was kindly provided by Dr. M. Borg-von-Zepelin (Göttingen, Germany).
Methanolysis of the Glycan Moiety of PLM-Carbohydrate analysis of Fr. Dg was performed by methanolysis (0.5 M HCl in anhydrous methanol for 24 h at 80°C) followed by N-reacetylation and trimethylsilylation according to the procedure of Kamerling et al. (26).
Methylation Analysis-Fr. Dg was submitted to two runs of permethylation (27). After methanolysis of the permethylated material and O-acetylation of the resulting free hydroxyl groups, the acetylated and methylated glycosides were analyzed by GC/MS according to the method of Fournet et al. (28).
Inositol and Glucosamine Analysis-Alkaline-treated PLM (Fr. Dg) was submitted to a strong acid hydrolysis (6 M HCl, 20 h at 110°C) according to Ferguson (29). After trimethylsilylation, inositol was analyzed by GC/MS using the chemical ionization mode with NH 3 as the reactant gas. An aliquot of the hydrolysate was submitted to N-reacetylation and trimethylsilylation for detection of glucosamine.
Nuclear Magnetic Resonance (NMR) Analysis-The NMR experiments were performed on a Bruker ASX 400 spectrometer in D 2 O at 25°C. Chemical shifts (␦) were referenced to internal acetone (␦ ϭ 2.225 ppm under experimental conditions). Two-dimensional homonuclear COSY (correlation spectroscopy) and HMQC (heteronuclear multiple quantum correlation) experiments were performed by using standard Bruker pulse programs.
Mass Spectrometry-Mass measurement was first performed by matrix-assisted laser desorption and time-of-flight (MALDI-TOF) mass spectrometry on a Vision 2000 instrument (Finnigan Mat, Hemel) in reflection mode (nitrogen laser, 337 nm). Fr. Dg was dissolved in water at a concentration of 50 -100 pmol⅐l Ϫ1 , and then 1 l was mixed with 1 l of matrix (3-aminochinoline in 1 mM CH 3 COONH 4 ϩ ) and allowed to crystallize onto the target at room temperature.
The electrospray (ES) ionization mass spectrometry was carried out on a Quatro II triple-quadruple mass spectrometer (Micromass). Fr. Dg was dissolved in 50% methanol at an approximate concentration of 50 pmol/l, and the solution was infused into the electrospray ion source by a Harvard syringe pump. The voltage difference between the needle tip and the source electrode was Ϫ3.2 kV.

TLC Analysis of C. albicans Extracts Showed that PLM Corresponds to a Family of Glycolipids Labeled with [ 3 H]Mannose and [ 3 H]Inositol-Preliminary characterization of C. albicans
PLM was made through chloroform/methanol/water extraction procedure and Western blot analysis (30). When the same extraction procedure was applied to [ 3 H]mannose-labeled Fr. A and analyzed by thin layer chromatography (Fig. 2, a-c), PLM corresponded to an heterogeneous peak with an average relative migration (R f ) of 0.147, which was present only in the more polar extract of the last extraction step (Fr. B) (Fig. 2c). This was confirmed by dot-blot analysis, which revealed its reactivity with mAb DF9-3 (data not shown). Subsequent butanol/ water partitions of Fr. B resulted in the presence of PLM in the water phase (Fr. C) (Fig. 2d) in contrast to other peaks that were separated in the butanol phases (Fig. 2e).
The heterogeneity of the PLM preparation was clearly demonstrated by thin layer chromatography analysis of [ 3 H]mannose-labeled Fr. C using 0.1% KCl instead of water in the solvent system (Fig. 3a). Moreover, the profiles of [ 3 H]mannose-and [ 3 H]inositol-labeled Fr. C (Fig. 3, a and b) were strikingly superimposed, with the exception of the peak at R f 0.69, which was observed only with the inositol labeling. Analysis by dot-blot procedure of the reactivity of the peaks with mAb DF9-3 (Fig. 3c) confirmed that peaks 1-5, observed in both profiles, were related to a family of glycolipids expressing ␤-1,2-oligomannosidic epitopes. Peaks 6 and 7 did not react with the antibody. Moreover, these two peaks displayed properties quite different from the others when tested on octyl-Sepharose column (data not shown) and thus appeared unrelated to PLM.
Alkaline Hydrolysis of Purified PLM Released a Soluble Fraction Containing Mannose Residues and Inositol-To obtain large quantities of PLM for physicochemical analysis, Fr. C was prepared from C. albicans grown in a bioreactor. Orcinol staining of this fraction also revealed the main contaminant previously observed with [ 3 H]inositol labeling (Fig. 3b). Successive chromatographic purifications of Fr. C on phenyl-Sepharose and silica gel 60 columns were thus performed and resulted in a PLM preparation (Fr. D) that produced a single spot on TLC (Fig. 4a, lane 1). Fr. D displayed a low solubility in most solvent systems, leading to opalescent solutions. This suggested a micellar conformation of PLM at a high concentration. This hypothesis was confirmed by the exclusion of concentrated Fr. D from the Ultrogel AcA34 column (Biosepra, Cergy-Pontoise, France), for which the exclusion limit was 750 kDa (data not shown). According to these properties, mass spectrometry

FIG. 3. Comparative TLC analysis of [ 3 H]mannose-labeled (panel a) and [ 3 H]inositol-labeled (panel b) Fr. C of C. albicans.
Fr. C were obtained as described in the legend to Fig. 1 from metabolically labeled cells and were analyzed on the same TLC plate. Following radioactivity scanning on a Berthold scanner, areas 1-7 of the silica gel corresponding to the main peaks were scraped off the plates, extracted with solvents and their reactivity to the mAb DF9-3 specific for ␤-1,2oligomannosides was analyzed by a dot-blot procedure (panel c). Solvent system, chloroform/methanol/KCl 0.1% (10:15:5, by volume). O, origin; F, front. and NMR spectrometry analyses of Fr. D were unsuccessful, and assays were therefore performed to obtain a soluble derivative from Fr. D. Treatment of Fr. D in 1 N KOH in 30% methanol for 90 h at 37°C was found to gradually increase its solubility through the release of a highly water-soluble fraction that no longer interacted with an octyl-Sepharose column. TLC analysis of this fraction (Fr. Dg) showed that it displayed an R f different from that of Fr. D (Fig. 4a, lanes 2 and 1). Fr. Dg behaved as a highly polymerized glycan when compared with phosphopeptidomannan-derived ␤-1,2-oligomannosides, with DP ϭ 13 and 14 (Fig. 4a, lanes 4 and 3); these oligomannosides were used as controls in relation to the expected glycan structure of Fr. Dg. Improved TLC analysis following five consecutive migration runs revealed a co-migration of this unlabeled Fr. Dg (Fig. 4b, lane 3) with a Fr. Dg (Fig. 4b, lane 1) prepared from PLM of C. albicans metabolically labeled with [ 3 H]inositol (Fig. 4b, lane 2). These experiments, which showed the radiolabeling still present in Fr. Dg, suggested that hydrolysis had occurred between inositol and the PLM hydrophobic moiety.  Table I. The proton resonance region (Fig. 5a) is dominated by two major signals at ␦ 5.046 and 4.384 ppm, which were assigned, respectively, to the H-1 and H-2 atom resonances of ␤-mannose. Three other resonances at ␦ 4.908, 4.934, and 5.05 ppm also possessed the characteristics of ␤-mannose anomeric protons. The set of these H-1 and H-2 resonances was identical to the NMR data obtained for the ␤-1,2-Man homopolymers released from C. albicans phosphopeptidomannan by mild acid hydrolysis (24) and allowed us to ascertain the mannose units on the spectrum from 1 to n. However, the absence of reducing terminal ␣-Man can be notified in Fr. Dg and results in the modification of H-1 and H-2 resonances of Man 1 to Man 3. The integration value of the main peak at ␦ 5.046, which included the additive contributions of Man 3 to Man n-1, also revealed a higher degree of polymerization of the ␤-1,2-oligomannosides from PLM with an average DP of 14 mannose residues. Signals relative to inositol and glycerol units were not detected, and the two minor atom resonances at ␦ 5.557 and 5.140 ppm, respectively, seemed to possess the characteristics of the anomeric protons of ␣or ␤-Man-1-phosphate ( 3 J H,P Х 7.5), but these correlations remain to be verified. Moreover, the heteronuclear NMR spectrum (Fig. 5b) clearly indicates that all C-2 atoms resonate at ␦ 80.6 -80.9 ppm, with the exception of the C-2 atom of the mannose unit n (␦ 71.66 ppm) in the terminal nonreducing position, and definitively confirms the presence of a linear chain of ␤-1,2-linked mannose residues in PLM.
Mass Spectrometry Revealed that the Heterogeneity of PLM Alkali-labile Fraction Was Due to Different Chain Lengths of    Table III).  the Phosphoinositolmannosides-The MALDI-TOF mass spectrum (Fig. 6a), recorded in the negative mode, exhibited a series of main peaks with an alternatively spaced m/z ratio of 80 and 82 Da, which may be clustered in different families (Table II). The first family of pseudo-molecular ions with m/z increments of 162 from 745 to 3499 Da corresponded to molecules containing n mannoses ϩ 1 inositol ϩ 1 phosphate group, with n varying from 3 to 19, whereas the second series with the same m/z increments of 162 Da from 663 to 3417 Da corresponded to molecules containing n mannoses ϩ 1 inositol ϩ 2 phosphate groups, n also varying from 3 to 19. A third series of minor peaks close to the second one (⌬m ϭ ϩ14) was also observed, but at the moment their masses did not fit to any reasonable molar composition. This analysis revealed the distribution and high degrees of polymerization of ␤-Man residues from PLM. These DP mainly comprised between 8 and 18 sugar units because phosphoinositolmannosides with a mass lower than 1200 Da seemed to be overestimated by this method according to the TLC analysis of the chloroform/methanol/water extracts (Figs. 2 and 3) and NMR results. It also evidenced the heterogeneity of PLM. These observations were confirmed by ES mass spectrometry ( Fig. 6b and Table III), which indicated, in addition to the previous phosphoinositolmannosides, the presence of a family of minor molecules derived from the second family through the formation of a presumed cyclic phosphodiester bridge during the alkaline hydrolysis (⌬m ϭ Ϫ18).

DISCUSSION
Unlike ␣-mannosides, which are widely expressed on glycoconjugates, the rarity of ␤-mannosides may be explained by a less favorable stereochemistry. To date, homopolymers of ␤-1,2linked mannose have been chemically characterized only in imperfect yeasts of the genus Candida (related to Ascomycetes). They are associated to side chains of the cell wall phosphopeptidomannan of C. albicans and C. tropicalis through phosphodiester bridges (7,31). NMR analysis of these residues released from C. albicans phosphopeptidomannan by mild acid hydrolysis has also shown changes in their ratios and degrees of polymerization, depending on the strains (7,31), the cell form (32,33), and the growth conditions (34,35).
In relation to the pathogenic character of C. albicans, several groups have investigated the recognition of ␤-1,2-oligomannosides by immune systems. These molecules have been shown to elicit specific antibodies in mice (36), rats (37), rabbits (9), and humans (11,38). The construction of neoglycolipids with phosphopeptidomannan-released ␤-1,2-oligomannosides has demonstrated that they can act as epitopes for a large number of anti-C. albicans phosphopeptidomannan monoclonal antibodies (16), suggesting that C. albicans mannoglycoconjugate(s) expressing these residues are strong immunogens. Two anti-␤-1,2-oligomannosides monoclonal antibodies have been described as protective against experimental C. albicans infection. The first one, reacting with a ␤-1,2-linked mannotriose, protected mice in a systemic model of candidosis (12). The second one protected rats in a model of vaginal infection (5). When we analyzed C. albicans molecules expressing ␤-1,2-oligomannosidic epitopes, we observed that all polyclonal or monoclonal antibodies specific for these residues bound to a 14 -18-kDa antigen that did not display accessible ␣-linked mannose residues (16). This antigen, named phopholipomannan (17), is expressed only in C. albicans and C. tropicalis, which are the most pathogenic Candida species (25). PLM is synthesized by C. albicans including under growth conditions that prevent association of ␤-1,2-linked oligomannosides to phosphopeptidomannan (30). Recent experiments have shown that C. albicans, in contact with macrophages, shed large amounts of PLM, which triggers an intense phosphotyrosine kinase-dependent signaling pathway and the secretion of TNF-␣ (19). The synthesis of surface glycolipids that protect from host defenses and/or disturb host cell immune functions is recognized as a pathogenic characteristic of eukaryotic protozoa of the genera Leishmania and Trypanosoma (20) and of prokaryotes of the genus Mycobacteria (21)(22)(23).
In this study we analyzed PLM to obtain chemical evidence for the presence of ␤-1,2-oligomannosides and to assess the possible structural relationships with surface glycolipids of these other microbial pathogens. Like these glycolipids, the C. albicans PLM was metabolically labeled by [ 3 H]mannose and [ 3 H]inositol, and both labeling profiles were superimposed in a family of hydrophilic glycolipids with ␤-1,2-oligomannosidic epitopes. The physicochemical analysis of the PLM sugar moiety confirmed the presence of mannose and inositol and evidenced the absence of glucosamine. The absence of this residue is consistent with the PLM resistance to nitrous acid treatment (29) and its unlabeling with [ 3 H]glucosamine (unpublished data). Glucosamine linking inositol to the sugar moiety is a common feature of GPI and GPI-related glycolipids (39) of eukaryotic cells. Its absence has only been reported to date in lipoarabinomannan, a GPI-like structure from prokaryotes of the genus Mycobacteria. Another peculiarity of PLM lies in the exclusive presence of ␤-1,2-linked mannose residues in its sugar moiety, which were found to be organized in linear chains with degrees of polymerization ranging from 8 to 18. Confirmation of the probable presence of a Man-1-phosphate linkage in the molecule, as deduced from NMR spectrum, will require further studies.
The average mass of PLM may be estimated, from the present study, to be about 4 kDa. This mass is different from the former description of the PLM as corresponding to a C. albicans 14 -18-kDa antigen in Western blotting (16). By using more reticulated gels (7-20% acrylamide) and migration conditions favoring the progressive blockage of the molecules in the gel rather than their migration speed, we observed that the PLM relative molecular mass upon SDS-PAGE decreased to 7 kDa (data not shown).
In Fig. 7 we suggest a structural model for the PLM glycan moiety, based on the first chemical evidence for the presence of ␤-1,2-linked oligomannosides in a glycoconjugate other than the yeast phosphopeptidomannan. Very little is known about ␤-1,2-mannosyltransferases of C. albicans, their activation and substrate specificity, but the presence of such linear chains of up to 18 mannose residues represent quite unusual structures (24). It has been suggested that a consequence of coating parasite surfaces with long sugar chains is the triggering of host effector mechanisms at a distance too great for efficient antimicrobial activity on the parasite. The recent demonstration for the presence of PLM at the C. albicans cell wall surface (19) suggests that these mechanisms may play a role during host-C. albicans interaction. Moreover, ␤-1,2-oligomannosides have been shown to act as C. albicans adhesins for the macrophage membrane (15) and to stimulate macrophages to produce high levels of TNF-␣ (13, 18). The stimulating activity of ␤-1,2-FIG. 7. Proposed structure for the family of glycolipids found in PLM of C. albicans VW32, serotype A. The structure of the glycan moiety was deduced from our results, and its linkage to the lipid moiety was postulated both from our results and from the usual structures of inositol and phosphorus containing glycolipids; n may vary from 5 to 15. (a), the position of this branch in the molecule and the percentage of molecules displaying this branch are still unknown.
oligomannosides was found to depend on the length of the mannosyl chain and maximum activity was observed for DPs of 8 or higher (13). Interestingly, these high DPs are present mainly in the C. albicans PLM but correspond to minor components among the ␤-1,2-oligomannosides released from the mannan of the same species (24).
In conclusion, we have shown that the pathogenic yeast C. albicans synthesizes inositol-labeled glycolipids that have glycan moieties devoid of glucosamine. These C. albicans glycolipids are thus structurally more similar to lipoarabinomannans of Mycobacteria than to the glycosylinositolphospholipids of parasitic protozoa or the lipophosphoglycan of Leishmania. Recently, as well as being B cell antigens, mannose sequences of lipoarabinomannan have been implicated in the presentation to T cells by CD1b nonclassical major histocompatibility complex molecules (40). Whether or not this property is shared by C. albicans PLM remains to be investigated. An important PLM structural peculiarity lies in the presence of long chains of ␤-1,2-linked mannose residues. There is now considerable experimental evidence that these sugar residues are involved in virulence and immunomodulation and can elicit protective antibody responses. Therefore, PLMs are molecules that must be considered for a comprehensive analysis of host-C. albicans relationships. A complete elucidation of their structure and biosynthetic pathways will be necessary to provide a structural basis for understanding their immunochemical properties and some aspects of the pathogenesis of C. albicans infections.