Characterization of glucosylceramides in Pseudallescheria boydii and ...

Glycobiology vol. 12 no. 4 pp. 251–260, 2002

Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation

Marcia R. Pinto3, Marcio L. Rodrigues4,5, Luiz R. Travassos4, Rosa M. T. Haido6, Robin Wait1,7 and Eliana Barreto-Bergter2,3 3Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro–UFRJ, CCS-Cidade Universitária, Rio de Janeiro, 21941-590, Brazil; 4Disciplina de Biologia Celular, Universidade Federal de São Paulo, São Paulo, Brazil; 5Disciplina de Microbiologia Médica, Faculdade de Medicina, Universidade Estácio de Sá, Rio de Janeiro, Brazil; 6Instituto Biomédico, Universidade do Rio de Janeiro, Rio de Janeiro, Brazil; and 7Centre for Applied Microbiology and Research, Salisbury, SP4 0JG, UK

Received on July 6, 2001; revised on August 24, 2001; accepted on August 27, 2001

Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment–mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.

1Present 2To

address: Kennedy Institute of Rheumatology, W8 8LH, London, UK whom correspondence should be addressed

© 2002 Oxford University Press

Key words: ceramide monohexosides/fungal differentiation/ Pseudallescheria boydii

Introduction Glycosphingolipids (GSLs) are amphipathic molecules consisting of a ceramide lipid moiety linked to a glycan chain of variable length and structure. These molecules have been implicated in many fundamental cellular processes, including growth, differentiation, and morphogenesis. GSLs may also modulate cell signaling by controlling the assembly and specific activities of plasma membrane proteins (Hakomori, 1990; Kasahara and Sanai, 2000). GSLs are present in fungi of the most primitive class of phycomycetes (Weiss et al., 1973) as well as in the most complex basidiomycetes (Barreto-Bergter and Villas-Boas, 1996). The ceramide monohexosides (CMHs) gluco- and galactosylceramides are the main neutral glycosphingolipids expressed in fungal pathogens such as Paracoccidioides brasiliensis (Toledo et al., 1995), Candida albicans (Matsubara et al., 1987), Cryptococcus neoformans (Rodrigues et al., 2000), Aspergillus fumigatus (Villas-Boas et al., 1994a) Fusarium solani (Duarte et al., 1998), Sporothrix schenckii (Toledo et al., 2000), and Histoplasma capsulatum (Toledo et al., 2001). The long chain base 9-methyl-4,8-sphingadienine, first described in monohexosylceramides (cerebrosides) from the plant pathogen Fusicoccum amygdali (Ballio et al., 1979) and subsequently isolated from Schizophyllum commune (Kawai and Ikeda, 1983), A. oryzae (Fujino and Ohnishi, 1977), and the edible fungi Clitocybe geotrope and C. nebularis (Fodegal et al., 1986) is present in almost all pathogenic fungi so far studied (Levery et al., 2000). Structural modifications of the ceramide moiety in these GSLs include different sites of unsaturation as well as the varying length of fatty acid residues. Although the structure of CMHs from several fungi has been elucidated, the biological relevance of these molecules is not fully understood. Fungal cerebrosides induce cell differentiation in S. commune with formation of fruiting bodies (Kawai and Ikeda, 1982). Seemingly, the 8E-double bond and the methyl group at C-9 in the sphingoid base are essential for differentiation. More recently, Umemura and co-workers (Umemura et al., 2000) showed that monohexosylceramides isolated from the rice pathogen Magnaporthe grisea have elicitor activity in plants. Monohexosylceramides are also associated with fungal growth, as reported by our group in a recent work (Rodrigues et al., 2000). We showed that the human pathogen C. neoformans synthesizes a cell wall–associated glucosylceramide that accumulates at regions of cell division, and that antibodies 251

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against these molecules prevent cell budding and fungal growth in vitro. In the present work, we characterized monohexosylceramides from Pseudallescheria boydii, a fungal pathogen that has emerged as an agent of disseminated mycosis in immunocompromised patients (Berenguer et al., 1989) and causes a clinical syndrome known as human white-gray mycetoma (Rippon, 1998). Structural analyses revealed the presence of conserved CMHs in P. boydii that are antigens recognized by antibodies from a rabbit infected with this fungus. Based on previous information describing an association between monohexosylceramides and differentiation, we also evaluated the influence of binding of anti-CMH antibodies in the germ tube formation of P. boydii and C. albicans. Our results indicate that fungal monohexosylceramides may be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells. Results Purification of GSL The purification steps of GSLs from P. boydii are shown in Figure 1. An orcinol-reactive band was detected by highperformance thin-layer chromatography (HPTLC), with a chromatographic mobility corresponding to a standard CMH from bovine brain. Gas chromatography–mass spectrometry (GC-MS) analysis of the fatty acids from P. boydii GC-MS analysis of the methanolyzed and trimethylsilylated CMH from P. boydii revealed two peaks. Their electron ionization mass spectra showed weak molecular ions (data not shown) at m/z 386 and 358, ions at m/z 371 (M-15) and m/z 343 (M-15) and base peaks at m/z 327 (M-59) and m/z 299 (M-59). The latter fragments, originating from facile cleavage between the carboxyl group and carbon 2, is characteristic of 2-hydroxy fatty acid methyl esters (Eglinton et al., 1968; Ryhage and Stenhagen, 1960), thus identifying the compounds as 2-hydroxyoctadecanoic and 2-hydroxy-hexadecanoic acids.

Fig. 1. Isolation and purification of P. boydii glucosylceramides. Steps of purification (left) and their corresponding fractions (right) are shown.

252

Fast-atom bombardment mass spectrometry (FAB-MS) analysis of CMH FAB-MS analysis indicated that the glycolipid from P. boydii consisted of two components that differ in their fatty acid compositions. In the negative ion spectrum deprotonated molecules were observed at m/z 726 and 754, consistent with deprotonated molecules of monohexosyl ceramides containing hydroxyhexadecanoic and hydroxyoctadecanoic acids and C19 sphingadiene (Figure 2A). Elimination of hexose results in the doublet of Yo ions at m/z 564 and 592. On peracetylation with acetic anhydride/pyridine, [M+Na]+ ions were observed at m/z 1030 and 1002, indicating addition of six acetyl groups to the mass of the underivatized glycolipids, consistent with hydroxy acid–containing monohexosylceramides. Additional [M+H-60] fragments at m/z 948 and 920 were observed (Figure 2B). Collisional activation of these ions resulted in the spectra shown in Figure 3 (A, B). An ion at 331 indicated terminal hexose and this assignment is supported by the presence of the expected secondary fragments at m/z 229, 169 and 109. The base derived ion at m/z 276 was also observed. Evidence for the location of the double bonds in the long chain base was obtained by collisional activation of the sphingadiene fragments at m/z 276. Cleavages of the alkyl chain (Costello and Vath, 1990; Adams and Ann, 1993) indicated the presence of a 4,8 sphingadiene structure, with a methyl substituent on carbon 9 (Sawabe et al., 1994) (Figure 4). An identical daughter ion spectrum was obtained from collisional activation of m/z 276 in the FAB spectrum of the CMH from F. solani (Duarte et al., 1998) and A. fumigatus, which have been shown by nuclear magnetic resonance (NMR) spectroscopy to contain 9-methyl4,8-sphingadiene (Villas-Boas et al., 1994a). Monosaccharide identification The constituent monosaccharide was liberated by hydrolysis and identified by TLC and GC-MS as glucose (not shown). The chemical shifts and coupling constants of the sugar protons of the peracetylated glycolipid were identical to those of an authentic sample of peracetylated glucosylceramide derived from Gaucher spleen. The 7.9 Hz coupling constant of

Fig. 2. FAB spectra of monohexosylceramides from P. boydii. (A) Negative-ion spectrum of the native CMHs [M-H]–. (B) Positive-ion spectrum of the peracetylated CMHs [M+Na]+.

Glucosylceramides in P. boydii and fungal differentiation

Fig. 3. Helium collisional activation spectrum of m/z 948 (A) and m/z 920 (B) from the CMHs of P. boydii.

Fig. 4. Helium collisional activation spectrum of the sphingadienine fragment at m/z 276 from the CMH of P. boydii.

H-1 in the CMH from P. boydii (data not shown) indicated that glucose residue was β-linked. The presence of glucose as the terminal residue was also confirmed by sequential mass spectra of perdeuteroacetate labeled CMH, according with Guevremont and Wright (1988). Fragment ion spectrum of m/z 343 (formed by loss of CD3-COOH from pentatrideuteroacetylated glucose) was obtained (Figure 5). Fragment ions at m/z 280 and 217 were present. The last one corresponds to the loss of CD3COOH. The ions observed at m/z 172 (major) and m/z 173 correspond to the loss of CD3-COOH, followed by loss of perdeuteroketene (CD2CO), plus CD3COOD or CD3COOH, respectively. 253

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Table I. 1H and 13C-NMR chemical shifts (ppm) for the peracetylated glucosylceramide from P. boydii obtained by one- and two-dimensional NMR spectroscopy Carbona

δ-1H

δ-13C

1″

4.49

100.5

2″

4.95

71.19

3″

5.19

72.68

4″

5.08

68.23

5″

3.67

71.93

6a″

4.15

61.82

6b″

4.23

1a

3.60

1b

3.92

—

2

4.23

50.65

NH

6.35

—

3

5.31

73.07

NMR analysis of the CMH from P. boydii

4

5.36

124.51

Results of one- and two-dimensional NMR analysis of the peracetylated glucosylceramide from P. boydii are summarized in Table I. The HH–correlation spectroscopy spectrum enabled the connectivity of the long-chain base protons to be traced between C-1 and C-11 and those of the fatty acid from C-2′ to C-5′ (see Figure 6 for numbering of the carbon atoms). The resonances at 5.31 ppm assigned to the long chain base H-3 is typical of protons directly bonded to O-acetylated carbon adjacent to olefinic bond. The resonance at 5.36 ppm, assigned as H-4 of the long chain base, exhibited a coupling constant (2J H, H) of 15.3 Hz to H-5, consistent with trans-double bonds. The β configuration of the glucose residue was evident from the coupling constant of H-1″ (7.9 Hz).

5

5.80

136.82

6

2.09

32.58

7

2.09

27.39

8

5.07

122.94

9a

1.57

15.98

10

1.94

39.73

11

1.25

28.03

2′

5.43

73.95

3′

5.43

32.58

4′

5.85

24.70

5′

2.02

29.80

Aliphatic–CH3

0.85–2.17

14.5–32.6

Fig. 5. Fragment ion spectrum of ions of m/z 343 from FAB ionization of perdeuteroderivative of CMH of P. boydii.

Antibody specificity Antibodies to CMH were obtained from serum of a rabbit previously immunized with P. boydii. The rabbit serum was

Fig. 6. Structures of ceramide monohexosides from P. boydii.

254

9

136.25

1′

aCarbon

67.17

169.28

numbers are specified in Figure 6.


tested for reactivity against CMH in enzyme-linked imunosorbent assay (ELISA) experiments (not shown), confirming that antibodies to glucosylceramide are produced during the contact of fungal cells with the animal host. The reactive serum was then immunoadsorbed on the solid-phase fixed glucosylceramide, and acid-eluted antibodies were recovered and identified as IgG. The specificity of these antibodies was addressed using HPTLC-immunostaining and western blot analyses. Figure 7A shows the incubation of a crude lipid mixture from P. boydii cells in the presence of rabbit antibodies to CMH. A single band from the P. boydii extract with RF similar to that of CMH was recognized by these antibodies, which excluded the possibility of cross-reactivity with other fungal lipid components. Additionally, the standard CMH from bovine brain was not reactive. To evaluate whether antiCMH antibodies would nonspecifically bind to P. boydii proteins, their reactivity against a crude protein extract was assayed by western blot analysis. Recognition of P. boydii proteins by purified antibodies was not detectable (Figure 7B). In addition, similar profiles of reactivity were observed when the protein extract was incubated in the presence of the rabbit immune serum before or after depletion of antibodies to CMH (data not shown), confirming that the purified antibodies reacted specifically with P. boydii CMHs. Immunofluorescence Antibodies to CMH were incubated with different forms of P. boydii and analyzed by fluorescence microscopy. As shown in Figure 8, mycelial forms and pseudo-hyphae were strongly reactive with anti-CMH antibodies. In contrast, the reaction of conidial forms with antiglucosylceramide antibodies was absent or very weak, suggesting a possible differential expression of glucosylceramide in P. boydii.

Fig. 7. Reactivity of antibodies to CMH against lipid (A) or protein (B) extracts of P. boydii. (A) A crude lipid mixture from P. boydii (a) and a standard CMH purified from bovine brain (b) were separated in HPTLC plates and visualized by reaction with orcinol-H2SO4. These preparations were incubated in the presence of antibodies to CMH, showing that the fungal CMH (c) but not the bovine glycolipid (d) was specifically recognized. (B) A crude protein extract from P. boydii mycelia was obtained and separated on 11% SDS–PAGE. Proteins were stained with Coomassie brilliant blue (a) or transferred to nitrocellulose membranes and incubated with antibodies to CMH, which were unable to recognize proteins from the fungal extract (b). M, molecular weight standards.

Fig. 8. Binding of anti-CMH antibodies to P. boydii cells. Mycelial, pseudo-hyphal, and conidial forms of P. boydii were incubated in the presence of antiglucosylceramide antibodies and analyzed under differential interferential contrast microscopy (DIC) and indirect immunofluorescence (IF). In control systems, in which no antiglucosylceramide antibodies were added prior to incubation with FITC-anti-rabbit IgG, no detectable fluorescence was observed (not shown). Bars represent 10 µm.

Antibodies to CMH inhibit germ tube formation in P. boydii and C. albicans The previous reports suggesting the involvement of CMHs on fungal growth and differentiation (Kawai and Ikeda, 1985; Kawai et al., 1986; Rodrigues et al., 2000) led us to investigate the influence of anti-CMH antibodies on growth and germ tube formation in P. boydii. Conidia from P. boydii in RPMI were incubated at 37°C in culture medium and aliquots of 10 µl were taken at 3-h intervals for observation in a light microscope. Germ tube induction in P. boydii was clearly observed in periods longer than 24 h, with the maximum differentiation rate being reached after 48 h (Figure 9A and C). Cultures that were supplemented with antibodies to CMH, however, failed to form germ tubes (Figure 9B and D). The anti-CMH antibodies inhibited cell differentiation to give rise to mycelium but did not affect the latter once it was formed (Figure 10). Generation of germ tubes in C. albicans is as a crucial event for the invasive growth of the fungus in vivo (reviewed by Gow, 1997). Because C. albicans is also able to synthesize a glucosylceramide with a structure extremely similar to that currently described for P. boydii (Matsubara et al., 1987), the effect of anti-CMH antibodies in Candida germ tube formation was also investigated. As shown in Figure 9, the addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. This could be observed at early (Figure 9G–H) and prolonged periods of incubation (Figure 9I–J). Fungal differentiation was not affected in the presence of control antibodies (Figure 9E–F and K–L). Discussion In the present work, glycosphingolipids were isolated from mycelia of P. boydii and purified to homogeneity by Iatrobeads 255

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Fig. 9. Antibodies to CMH inhibit germ tube formation in P. boydii and C. albicans. Panels show germ tube formation observed after 24 (A) and 48 (C and E) h of incubation of P. boydii in the RPMI medium, and after 3 (G) and 21 (I and K) h of incubation of C. albicans in the same medium. Addition of antibodies to CMH inhibits differentiation of P. boydii (B and D) and C. albicans (H and J). In contrast, addition of control antibodies to the differentiation systems of P. boydii (F) and C. albicans (L) did not affect mycelia or germ tube formation. Bars represent 10 µm.

Fig. 10. Growth of P. boydii mycelium is not affected by anti-CMH antibodies. Closed circles, control mycelium growth; open circles, growth in the presence of anti-CMH antibodies.

chromatography. Their structures were determined by chemical, spectrometric, and spectroscopic methods. From our results it can be concluded that the major species of glycosphingolipids produced by P. boydii have the chemical structure indicated in Figure 6. Similar structures that were isolated from the rice pathogenic fungus M. grisea (Koga et al., 1998) and S. commune (Kawai and Ikeda, 1983) are involved in the hypersensitive response in rice and cell differentiation, respectively. As previously observed in A. fumigatus and A. versicolor (Villas-Boas et al., 1994a), yeast and mycelial forms of H. capsulatum (Toledo et al., 2001) and mycelial forms of S. schenckii (Toledo et al., 2000), P. boydii appears to synthesize only glucosylceramides containing 9-methyl-4,8sphingadienine as the long chain base. The different molecular species can thus be attributed to CMH molecules differing only in the chain length of the hydroxylated fatty acids (16:0 and 18:0). Although an (E)-∆3-unsaturation in the 2-hydroxy fatty N-acyl moiety is found in CMHs of other fungal species 256

(Toledo et al., 2001), it has not been observed in the CMH from P. boydii. Lacking the (E)-∆3-unsaturation is also a common feature for other fungi, such as C. neoformans (Rodrigues et al., 2000), C. albicans (Matsubara et al., 1987), S. commune (Kawai and Ikeda, 1983) and fruiting bodies of two basidiomycetes, Clitocybe geotropa and C. nebularis (Fodegal et al., 1986). In several fungal infections, sera from affected individuals react with purified cerebrosides (Toledo et al., 1995; Rodrigues et al., 2000). Due to the high similarity observed between the structures of fungal CMHs, the occurrence of cross-reactive anti-CMH antibodies is expected. In fact, a recent study by our group (Rodrigues et al., 2000) demonstrated that a purified CMH from C. neoformans reacted with sera from patients with cryptococcosis, histoplasmosis, aspergillosis, and paracoccidioidomycosis, indicating that antibodies against similar structures are produced during the course of these mycoses. The glucosylceramide obtained from P. boydii is very similar to other fungal cerebrosides in that they all contain a 9-methyl-4,8-sphingadienine in combination with N-2′-hydroxy fatty acids (Levery et al., 2000; Toledo et al., 2001). This molecule was recognized by serum of an infected rabbit (not shown), confirming that antibodies to cerebrosides are produced during infection by P. boydii. Hydroxylation at position 2 of the fatty acid is apparently important for antigenicity of the CMH (Young et al., 1981; Nakakuma et al., 1989), and possible epitopes involve both glucose and the hydroxylated fatty acid, with modulation by the sphingosinederived base. Conformer 4 of glucosylceramide—as studied by Nyholm and Pascher (1993a),b), which is allowed in a membrane layer, and further stabilized by a hydrogen bond between the 2-OH group on the fatty acid and the 6-OH group on the glucose residue, in addition to the hydrogen bond between glucose O5 and the amide hydrogen—is a candidate for epitopes reactive with anti-CMH antibodies. In agreement with these observations, the recognition of the P. boydii CMH by rabbit immune serum was only partially inhibited by β-methylglucopyranoside (data not shown), showing that the cerebroside antigenicity is dependent on a specific conformation that involves both carbohydrate and the lipid moiety. Antibodies to CMH were purified and used in immunofluorescence analysis. Interestingly, reactions of these antibodies and P. boydii conidial forms were absent or very weak, whereas mycelia and pseudo-hyphae were strongly reactive. These results suggest that CMHs are differentially expressed in P. boydii according with morphological phase. Biosynthesis, expression, or chemical structures of CMHs seem to be modified during the conidium → mycelium transition, which suggests a role for CMHs in fungal differentiation. In accordance with this is the observation that antibodies to CMH were able to inhibit the formation of germ tube–like structures in P. boydii, although they did not influence mycelial growth. We have shown (unpublished data) that germ tubes are induced after the contact of P. boydii conidia with animal cells, a step preceding efficient fungal invasion. Germ tube formation is also recognized as a crucial event in tissue invasion by C. albicans (Gow, 1997), a fungus that synthesizes CMHs (Matsubara et al., 1987) structurally similar to those previously described in other fungi (Rodrigues et al., 2000; Toledo et al., 2001) and to the currently characterized molecule from P. boydii. In this context, the influence of antibodies to CMH on C. albicans


differentiation was also evaluated. As with P. boydii, antiCMH antibodies inhibited germ tube formation in C. albicans. Such information can be correlated with previous results demonstrating that cerebrosides are involved in fungal differentiation (Kawai and Ikeda, 1982). Additionally, we have recently shown that CMHs are distributed over the cell wall of C. neoformans and that antibodies against these molecules inhibited fungal growth (Rodrigues et al., 2000). The mechanisms by which anti-CMH antibodies inhibit fungal growth and/or differentiation remain to be established, but there is a possibility that CMHs are associated with enzymes involved in the hydrolysis and synthesis of the cell wall and/or with glycosylphosphatidylinositol–anchored precursors during cell differentiation and division. In this context, binding of antibodies to CMHs could impair the action of CMH-associated functional proteins inhibiting cell wall synthesis. Although fungal infections represent an increasing health problem, efficient therapies for their control are few (Garber, 2001). The study of additional targets for action of antifungal agents is extremely relevant, specially those associated with the fungal cell wall, a vital structure for fungal growth and differentiation. Materials and methods Microorganism and growth conditions P. boydii, isolated from eumycotic mycetoma, was kindly supplied by Bodo Wanke from the Evandro Chagas Hospital, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. It was maintained in (g L–1): Difco peptone, 10; Difco yeast extract, 5; Difco agar, 20; glucose, 40. Cells were grown on Sabouraud solid slants and inoculated into Erlenmeyer flasks (500 ml) containing culture medium (200 ml) and incubated for 7 days at 25°C with shaking. Cultures were then transferred to the same medium (3 L) and incubated for 7 days at the same temperature with shaking; the mycelium was filtered, washed with distilled water, and stored at –20°C. Lipid extraction Lipids were successively extracted from intact hyphae of P. boydii at room temperature with 10 volumes of chloroform/ methanol (2:1 and 1:2 v/v). Evaporation of these combined extracts yielded a crude lipid mixture, which was subjected to mild alkaline methanolysis (Wells and Dittmer, 1965) and partitioned according to Folch et al. (1957). The upper phase was carefully removed and the lower phase was washed twice with Folch’s theoretical upper phase for further vacuum concentration. Isolation and purification of neutral glycosphingolipids The lipids recovered from the lower layer of the Folch extract were purified by silica gel chromatography; neutral lipids, glycolipids, and phospholipids were recovered by elution with chloroform, acetone, and methanol. The acetone and methanol fractions, containing the glycosphingolipids, were further purified on a silica gel column, which was sequentially eluted with chloroform/methanol with increasing concentrations of methanol (95:5, 9:1, 8:2 and 1:1 v/v) and finally with 100% methanol. Fractions eluted with chloroform/methanol (9:1 and 8:2 v/v) were further purified by chromatography on

Iatrobeads RS 2060 (Macherey & Nagel, Düren, Germany), using the same elution system, yielding a purified glycosphingolipid fraction. Some of this material was peracetylated with pyridine/acetic anhydride (1:1 v/v) overnight at room temperature. After removal of the reagents by evaporation under nitrogen, the peracetylated glycolipid was purified on a small Iatrobeads column (0.5 × 5 cm) as previously described (Villas-Boas et al., 1994a). HPTLC Native and peracetylated neutral glycolipids were analyzed on HPTLC plates developed with chloroform/methanol/water (65:25:4 v/v) and 1,2-dichloroethane/acetone (8:2 v/v), respectively. The separated glycolipids were visualized with iodine vapor and by spraying with orcinol/sulfuric acid. Sugar analysis Glycosphingolipids were hydrolyzed with 3 M trifluoroacetic acid at 100°C for 3 h, and the resulting monosaccharides were characterized by TLC and quantified by GC as alditol-acetate derivatives (Sawardeker et al., 1965) using an 0V-225 fused silica capillary column (30 m × 0.25 mm ID), with temperature programmed from 50°C to 220°C at 50°C/min. Characterization of fatty acids Fatty acid methyl esters were prepared by acid methanolysis using 1 ml toluene:methanol (1:1 v/v) containing 2.5% concentrated sulfuric acid (overnight at 70°C). Samples were diluted in 0.5 ml deionized water and extracted twice with hexane/chloroform (4:1 v/v). The combined extracts were dried by vacuum centrifugation and trimethylsilylated by treatment with 100 µl of bis-(trimethylsilyl)trifluoracetamide/ pyridine(1:1 v/v; 30 min at 60°C). The reagent was removed by vacuum centrifugation, and the samples were dissolved in hexane for GC-MS analysis. GC-MS GC-MS was performed with a Kratos MS80 RFA spectrometer (Kratos, Manchester, UK) directly interfaced to a Carlo Erba 5160 chromatograph. Helium (0.7 ml/min) was used as the carrier gas, and samples were introduced by splitless injection (splitless time 30 s) into a BPX-5 fused silica column (25 m × 0.2 mm; SGE, Milton Keynes, UK). The injector and interface oven were maintained at 250°C. One minute after injection, the column oven temperature was programmed from 60°C to 200°C at 40°C/min, then at a rate of 3°C/min to 230°C, with a final 8°C ramp to 265°C, then held for 10 min. Electron ionization spectra were recorded at an ionization energy of 70 eV, trap current of 100 µÅ, and a source temperature of 220°C. Chemical ionization spectra were obtained using isobutane as the reagent gas with an emission current of 250 mÅ. The magnet was scanned at 0.6 s/decade over the range 550–40. FAB-MS FAB spectra were obtained with a Kratos MS80 spectrometer, fitted with an Ion Tech saddle-field atom gun supplied with high-purity xenon gas. Spectra of the underivatized glycolipids were recorded in both the positive and negative ion modes using either 3-nitrobenzyl alcohol, or a 1:19 v/v) mixture of glycerol and dithiotreitol-dithioerythritol (5:1, w/w) as liquid matrices. Approximately 10 µl of each sample was loaded onto 257

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the FAB probe. The instrument was operated at an accelerating voltage of 4 kV and a resolution of 1000 (10% valley), and the magnet was scanned at 10 s/decade of mass over the range of 2000–200. Peracetyl derivatives (1–5 µg) were analyzed from 3-nitrobenzyl alcohol matrix. Collisional activation spectra were recorded by scanning the magnetic field (B) and the electrostatic analyzer voltage (E), while maintaining a constant ratio of B to E. The helium collision gas pressure was adjusted to give 50% attenuation of the ion beam. 1H-

and 13C-NMR spectroscopy

The peracetylated sample was dissolved in 0.5 ml CdCl3. Spectra were recorded at 75.45 MHz and 300.14 MHz using a Bruker AC 300 P spectrometer equipped with an Aspect 3000 computer operated in the Fourier transform mode. Rabbit immune sera One milliliter of a cell suspension containing 2 mg of freezedried mycelium of P. boydii was mixed with the same volume of complete Freund’s adjuvant, and 1 ml of this mixture was injected intradermally in a white male rabbit at weekly intervals for 3 weeks. Then, during a 1-week period, cell suspensions at the same concentration were injected intravenously at 2-day intervals. The hyperimmune serum obtained was used in ELISA experiments (Villas-Boas et al., 1994b) and for antibody purification. Purification of anti-glucosylceramide antibodies Antibodies to CMH were purified as previously described (Rodrigues et al., 2000). Briefly, the purified glucosylceramide (500 µg) from P. boydii was dissolved in 50 µl of methanol and spotted on a strip of polyvinylidene difluoride membrane, which was blocked with phosphate buffered saline (PBS) containing 0.1% Tween 20 and 10% bovine serum albumin (BSA) for 2 h at room temperature. The membrane was washed four times in PBS-Tween and then incubated overnight at 4°C in the presence of a rabbit hyperimmune serum (at 1:2), obtained as described. After repeated washing for removal of unbound proteins, bound antibodies were eluted using 3 ml of 100 mM glycine acid buffer, pH 3.0, and immediately neutralized with 1 M Tris–HCl, pH 9.0. This process was repeated three times, and the unbound depleted fraction contained antibodies that gave absorbance readings by ELISA similar to those from normal rabbit serum at the same low dilution. Eluted antibodies were further ultrafiltered in a Centricon 10 micropartition system from Amicon and concentrated by vacuum centrifugation. The recovery of antiglucosylceramide antibodies was monitored by analyzing the eluted samples by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE). Antibody specificity To exclude the possibility of unspecific recognition of P. boydii molecules by antibodies to CMH, their reactivity against protein and lipid extracts from mycelial cells was examined by using western blot and HPTLC-immunostaining analyses. The crude protein extract of P. boydii was obtained from a thick suspension of mycelial cells in a lysis buffer (0.01 M PBS, pH 7.4; 1% Triton X-114, 1 mM ethylenediamine tetra-acetic acid, 1 mM E-64, 5 mM dithiothreitol, 1 µg/ml aprotinin, and 1 µg/ml pepstatin). Equivalent volume of glass beads (0.3 mm in diameter) was then added to the 258

suspension, and cells were broken in a cell disrupter (type 853023/8, B. Braun Biotech International, Germany) by alternating 1-min shaking periods and 2-min cooling intervals. After removal of the glass beads, the suspension was centrifuged (10,000 × g, 30 min, 4°C). Proteins present at the supernatant were separated on 11% SDS–PAGE and then stained with Coomassie brilliant blue or transferred to nitrocellulose membranes. The membranes were blocked for 2 h with 10% skimmed milk in PBS and incubated for 1 h at room temperature in the following systems: (1) rabbit immune serum (at 1:5000 dilution); (2) rabbit immune serum that has been depleted of antibodies to CMH by immunoadsorption (at 1:5000 dilution); and (3) purified antibodies to CMH (at 1:100 dilution). Membranes were then incubated in the presence of a peroxidaselabeled anti-rabbit antibody followed by immunodetection with diaminobenzidine. For immunostaining analysis, the crude lipid extract of P. boydii was separated by HPTLC, the plate being air-dried, soaked in 0.5% polymethacrylate in diethyl ether, and blocked for 2 h with 10% skimmed milk in PBS. The plate was then incubated for 2 h in presence of the preparation of antibodies to CMH (at 1:100 dilution) followed by sequential incubation with peroxidase-conjugated anti-rabbit antibody and diaminobenzidine. Immunofluorescence analysis Mycelial or conidial forms of P. boydii were fixed in 4% paraformaldehyde cacodylate buffer 0.1 M, pH 7.2, for 1 h at room temperature. Fixed cells were washed twice in PBS and incubated sequentially for 30 min in PBS containing 150 mM NH4Cl and then in 1% BSA in PBS for 1 h. Fungal cells were washed in PBS and sequentially incubated with purified antibodies to CMH (at 1:10 dilution) and fluorescein isothiocyanate (FITC)–labeled anti-rabbit IgG (at 1:100 dilution) for 12 h at room temperature. Control cells, which had not been incubated with antiglucosylceramide antibodies, were also prepared. Fungal cells were finally washed and microscopically observed with Axioplan 2 (Zeiss, Germany) fluorescence microscope. Fungal differentiation Previous reports have demonstrated the involvement of fungal CMHs on growth (Rodrigues et al., 2000) and differentiation (Kawai and Ikeda, 1985; Kawai et al., 1986). We therefore evaluated the influence of antibodies to CMH on cell differentiation of P. boydii and C. albicans. Conidia (106, P. boydii) or yeast cells (C. albicans) were transferred to sterile microcentrifuge tubes containing 500 µl of RPMI, a medium that has previously shown to induce germ tube formation in C. albicans. Purified antibodies to CMH were then added at 10 µg/ml and the suspensions incubated at 37°C. Aliquots of 10 µl from each culture were taken at 3-h intervals, and the germ tube formation was assayed in a light microscope. Experiments were also performed in the presence control antibodies, which were obtained after depletion of anti-CMH antibodies from rabbit sera followed by purification of IgG in Protein G Sepharose 4 Fast Flow. Cell growth To evaluate the effects of antibodies to CMH on the mycelial growth of P. boydii, the fungus was cultivated for 7 days at


room temperature on plates containing Sabouraud-dextrose agar. After this period, a preparation of conidial cells was obtained by washing plate surface with PBS. Cells were then washed three times with PBS, counted in a Neubauer chamber, and resuspended in Sabouraud’s liquid medium at a final concentration of 105 conidia per ml. These suspensions were incubated for 24 h at room temperature, which allowed a complete conversion of conidial to mycelial cells, as assessed by light microscopy. After differentiation to mycelia, antibodies to CMH were added at 10 µg/ml, and fungal suspensions were further incubated for additional periods varying from 24 to 96 h. After 24-h intervals, the mycelial cells were recovered by filtering the suspensions through 0.22-µm membranes and extensive washing with water. For quantitative determinations of growth, the cells were dehydrated by heating and the mycelial dry weight measured.

Acknowledgments We thank Maria de Fatima F. Soares for technical assistance. This work was supported by Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ), Conselho de Ensino e Pesquisa da UFRJ (CEPG), and Programa de Apoio a Núcleos de Excelência (PRONEX). This work is dedicated to the memory of Eduardo A. Leitão, a talented glycobiologist whose promising career was prematurely ended.

Abbreviations BSA, bovine serum albumin; CMH, ceramide monohexoside; ELISA, enzyme-linked immunosorbent assay; FAB, fast atom bombarment; FITC, fluorescein isothiocyanate; GC, gas chromatography; GSL, glycosphingolipid; HPTLC, high performance thin-layer chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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