2 Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006-Madrid, Spain; 3 Centro de Microbiología y Biología Celular, IVIC, P.O. Box 21827, Caracas 1020A, Venezuela; and 4 Instituto de Química Orgánica, CSIC, Juan de la Cierva 3, 28006-Madrid, Spain
Received on January 13, 2003; revised on March 13, 2003; accepted on March 13, 2003
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Abstract |
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Key words: cell wall polysaccharides / fungi / NMR spectroscopy / Paracoccidioides brasiliensis
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Introduction |
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P. brasiliensis has been subjected to a number of chemical studies intended to analyze cell wall components (proteins, glycoproteins, glycolipids, and oligo- or polysaccharides) or metabolites whose presence would modulate hostparasite relationships through interactions with the host immune system (Toledo et al., 1999; Levery et al., 1998
, Almeida et al., 1996
; Azuma et al., 1974
; Kanetsuna and Carbonell, 1970
; Kanetsuna et al., 1969
).
The alkali-extractable and water-soluble fungal polysaccharides (F1SS), which are minor components of the cell wall (28%), are the glycosidic moieties of peptido-polysaccharides. They differ in composition and structure among genera and, in certain cases, among species within a genus (Leal et al., 2001). These polysaccharides are antigenically relevant (Ahrazem et al., 2000a
; Domenech et al., 1996
, 1999
; Mischnick and De Ruiter, 1994
; Latgé et al., 1991
; De Ruiter et al., 1991
) and are probably involved in cellcell and/or cellhost recognition mechanisms.
The aim of the present study was to ascertain whether the structures of P. brasiliensis polysaccharides F1SS differ in the mycelial and yeast phases, a fact that might have pathobiological relevance.
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Results |
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To find the connections among residues, we performed a heteronuclear multiple bond correlation (HMBC) experiment, which gives cross-peaks between a proton and the carbons placed at two or three bonds from it. In addition to expected intraring connections, peaks corresponding to H-1A/C-6A', H-1B/C-6C, and H-1C/C-2A could be observed. We denote a second unit of A as A'.
The NMR spectral data, together with those of the methylation analyses, suggest the following main structure for the cell wall polysaccharide of the mycelial form of P. brasiliensis.
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The 1H-NMR spectrum (Figure 3a) showed a severely crowded anomeric region, from which very little information could be inferred. The 13C-NMR spectrum (Figure 3b) contained four main and one small anomeric singlets. The signal at 108.7 ppm suggests a ß-galactofuranose moiety, and those around 103 ppm are characteristic of mannopyranose units. The small singlet appeared at identical position to that of the -galactofuranose unit (101.2 ppm) found in the spectrum of the mycelial phase. The 2D 1H-13C correlation (HMQC) spectrum contained six anomeric cross-peaks, which were labelled DI. By using 2D homo- and hetero-NMR experiments, we were able to assign most proton and carbon signals of the six main residues present in the polysaccharide (Table III). Again, by comparison of the chemical shifts values with those of model compounds (Bock and Pedersen, 1983
) and consideration of the methylation results, we could deduce the glycosylation sites as being D and E 2,6-di-O-substituted mannopyranoses; F and G, terminal galactofuranoses; H, terminal mannopyranose; and I, 6-O-substituted mannopyranose.
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To discriminate among the various possibilities of arrangement of the different fragments, we recorded a long-range protoncarbon correlation HMBC experiment that, in addition to trivial cross-peaks, showed signals for H-1D/C-6E (D'), H-1E/C-6D (E'), H-1F/C-6I, H-1G/C-6I, H-1H/C-2D (E), and H-1I/C-2E (D). Second units of D and E are labeled D' and E'.
All the NMR spectral data, in agreement with the methylation analyses, allow us to propose the main structure of the galactomannan from the yeast phase of P. brasiliensis as being n, m, and p in a proportion around 1:1:0.2, as deduced from integration of the anomeric singlets of the carbon spectrum.
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Discussion |
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It has been shown (Kanetsuna and Carbonell, 1970; Kanetsuna et al., 1969
) that both yeast and mycelial forms have chitin as a common structural polysaccharide, but an
-(1
3) glucan was found in the yeast phase, whereas a ß-(1
3) glucan was encountered in the mycelial form. This difference has been suggested as a possible contribution to the distinct morphology of both forms (San Blas and San Blas, 1994
). However, a study on the structure of glucans and F1SS polysaccharides in Eupenicillium, Penicillium, and Talaromyces species grown at 25°C (Leal and Bernabé, 1998
) revealed that the mycelium from these microorganisms contained either
-(1
3) or ß-(1
3) glucans, regardless of their common mycelial shape. On the other hand, studies on mutants of P. brasiliensis have suggested a direct relationship between virulence and the presence of variable amounts of
-(1
3) glucan in the cell walls of the mutant strains (San-Blas, 1982
and references therein). Major quantities of this glucan determine enhancement of the virulence, whereas a lower amount of the glucan results in a decreased virulence. This behavior has been explained as the result of
-glucan working as a protection mechanism of the fungus against host defences (San-Blas, 1982
).
Structural variations have also been observed by Mendonça et al. (1976) in the alkali-extractable cell wall polysaccharides from both morphological types of the dimorphic fungus Sporotrix schenckii. Nevertheless, they dissociated the effect of the temperature on morphological phase transition because 100% yeast was obtained in a synthetic medium either at 25°C or 37°C. Therefore they concluded that the variations in the structure of the polysaccharides observed must be due to differences in the morphology of both phases and not to modification on growth temperatures.
Nickerson (1948 and references therein) attributed the changes in morphology to reversible denaturation or activation of enzyme processes due to the changes in temperature. In this context, it has been shown that exogenous cAMP inhibits the yeast to mycelial transitions, thus favoring the pathogenic yeast form (Borges-Walmsley et al., 2002
).
It is not currently possible to interpret the meaning and evaluate the importance of the structural modifications of F1SS polysaccharides observed in the transition from the mycelial to the yeast phase of P. brasiliensis. The antigenic relevance of the ß-galactofuranose domains of polysaccharides from several pathogenic fungi (Latgé et al., 1991; Notermans et al., 1988
; Azuma et al., 1974
) is known, yet little or nothing is known of the role of
-galactofuranoses because these residues have only been described in cell wall polysaccharides from a few species belonging to Onygenales (Bernabé et al., 2002
).
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Material and methods |
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Wall material preparation and fractionation
Cell walls from P. brasiliensis were prepared according to Kanetsuna et al. (1969). Fractions and polysaccharides F1SS were obtained and purified following Ahrazem et al. (2000b)
.
Chemical analysis
For analysis of neutral sugars the polysaccharides were hydrolyzed with 3 M trifluoracetic acid (TFA) (1 h at 121°C), converted into their corresponding alditol acetates (Laine et al., 1972), and identified and quantified by GLC using a SP-2380 fused silica column (30 m x 0.25 mm ID x 0.2 µm film thickness) with a temperature program (210°C to 240°C, initial time 3 min, ramp rate 15°C min-1, final time 7 min) and a flame ionization detector.
The monosaccharides released after hydrolysis were derivatized according to Gerwig et al. (1979) and their absolute configuration was determined by gas chromatography mass spectrometry of the tetra-O-TMSi-(+)-2-butylglycosides obtained.
Methylation analyses
The polysaccharides (15 mg) were methylated according to the method of Ciucanu and Kerek (1984). The methylated material was treated and processed according to Ahrazem et al. (2000b)
, with the exception that the partially methylated polysaccharide was hydrolysed with TFA 1.5 M (100°C, 30 min).
Partial hydrolysis of the polysaccharide F1SS from the mycelial phase
Eighty milligrams of the polysaccharide were hydrolyzed as described by Prieto et al. (2001).
NMR analysis
1D and 2D 1H- and 13C-NMR experiments were carried out at 40°C on a Varian Unity 500 spectrometer with a reverse probe and a gradient unit or a Varian INOVA-300 spectrometer (1H, 300 MHz). Proton chemical shifts refer to residual HDO at 4.61 ppm. Carbon chemical shifts refer to internal acetone at
31.07 ppm. The polysaccharides F1SS (
20 mg) were dissolved in D2O (1 ml) followed by centrifugation (10,000 x g, 20 min) and lyophilization. The process was repeated twice, and the final samples were dissolved in D2O (0.6 ml, 99.98% D).
The 2D NMR experiments (DQCOSY, TOCSY, HMQC, HSQC-TOCSY, and HMBC) were performed by using the standard Varian software.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Ahrazem, O., Gómez-Miranda, B., Prieto, A., Bernabé, M., and Leal, J.A. (2000b) Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides. Arch. Microbiol., 173, 296302.[CrossRef][ISI][Medline]
Almeida, I.C., Neville, D.C., Mehlert, A., Treumann, A., Ferguson, M.A., Previato, J.O., and Travassos, L.R. (1996) Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis. Glycobiology, 6, 507515.[Abstract]
Azuma, I., Kanetsuna, F., Tanaka, Y., Yamamura, Y., and Carbonell, L.M. (1974) Chemical and immunological properties of galactomannans obtained from Histoplasma duboisii, Histoplasma capsulatum, Paracoccidioides brasiliensis and Blastomyces dermatitidis. Mycopathol. Mycol. Appl., 54, 111125.[ISI][Medline]
Bernabé, M., Ahrazem, O., Prieto, A., and Leal, J.A. (2002) Evolution of polysaccharides F1SS and proposal of their utilisation as antigens for rapid detection of fungal contaminants. EJEAFChe, 1(1).
Bock, K. and Pedersen, C. (1974) A study of 13C-H coupling constants in hexopyranoses. J. Chem. Soc. Perkin Trans., 2, 293297.
Bock, K. and Pedersen, C. (1983) Carbon-13 nuclear magnetic resonance spectroscopy of monosaccharides. Adv. Carbohydr. Chem. Biochem., 41, 2766.[ISI]
Borges-Walmsley, M.I., Chen, D., Shu, X., and Walmsley, A.R. (2002) The pathobiology of Paracoccidioides brasiliensis. Trends Microbiol., 10, 8087.[CrossRef][ISI][Medline]
Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[CrossRef][ISI]
Cyr, N. and Perlin, A.S. (1979) The conformations of furanosides. A 13C nuclear magnetic resonance study. Can. J. Chem., 57, 25042511.[ISI]
De Ruiter, G.A., Smid, P., Van der Lugt, A.W., Van Boom, J.H., Notermans, S.H.W., and Rombouts, F.M. (1991) Immunogenic extracellular polysaccharides of Mucorales. In: Latgé, J.P. and Boucias, D. (Eds), Fungal cell wall and immune response. Springer Verlag, Berlin, pp. 169180.
Domenech, J., Barasoaín, I., Prieto, A., Gómez-Miranda, B., Bernabé, M., and Leal, J.A. (1996) An antigenic water-soluble glucogalactomannan extracted from cell walls of Paecilomyces fumosoroseus and Paecilomyces farinosus. Microbiology, 142, 34973503.[Abstract]
Domenech, J., Prieto, A., Barasoaín, I., Gómez-Miranda, B., Bernabé, M., and Leal, J.A. (1999) Galactomannans from the cell walls of species of Paecilomyces sect. Paecilomyces and their teleomorphs as immunotaxonomic markers. Microbiology, 145, 27892796.
Franco, M.F. (1987) Hostparasite relationships in paracoccidioidomycosis. J. Med. Vet. Mycol., 25, 518.[ISI][Medline]
Gerwig, G.J., Kamerling, J.P., and Vliegenthart, J.F.G. (1979) Determination of the absolute configuration of mono-saccharides in complex carbohydrates by capillary GLC. Carbohydr. Res., 77, 1017.[CrossRef][Medline]
Kanetsuna, F. and Carbonell, L.M. (1970) Cell wall glucans of the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol., 101, 675680.[Medline]
Kanetsuna, F., Carbonell, L.M., Moreno, R.E., and Rodríguez, J. (1969) Cell wall composition of the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol., 97, 10361041.[ISI][Medline]
Laine, R.A., Esselman, W.J., and Sweeley, C.C. (1972) Gas-liquid chromatography of carbohydrates. Meth. Enzymol., 28, 159167.
Latgé, J.P., Debeaupuis, J.P., Moutaouakil, M., Diaquin, M., Sarfati, J., Prévost, M.C., Wieruszeski, J.M., Leroy, Y., and Fournet, B. (1991) Galactomannan and the circulating antigens of Aspergillus fumigatus. In: Latgé, J.P. and Boucias, D. (Eds), Fungal cell wall and immune response. Springer-Verlag, Berlin, pp. 143155.
Leal, J.A. and Bernabé, M. (1998) Taxonomic applications of polysaccharides. In: Frisvad, J.C., Bridge, P.D., and Arora, D.K. (Eds), Chemical fungal taxonomy. Marcel Dekker, New York, pp. 153181.
Leal, J.A., Prieto, A., Ahrazem, O., Pereyra, T., and Bernabé, M. (2001) Cell wall polysaccharides: characters for fungal taxonomy and evolution. Rec. Res. Devel. Microbiol., 5, 235248.
Levery, S.B., Toledo, M.S., Straus, A.H., and Takahashi, H.K. (1998) Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen. Biochemistry, 37, 87648775.[CrossRef][ISI][Medline]
Mendonça, L., Gorin, P.A., Lloyd, K.O., and Travassos, L.R. (1976) Polymorphism of Sporothrix schenckii surface polysaccharides as a function of morphological differentiation. Biochemistry, 15, 24232431.[ISI][Medline]
Mischnick, P. and De Ruiter, G.A. (1994) Application of reductive cleavage in the structural investigation of the antigenic polysaccharides of Aspergillus fumigatus and Penicillium digitatum with respect to the determination of the ring size of the galactose moieties. Carbohydr. Pol., 23, 512.[CrossRef][ISI]
Nickerson, W.J. (1948) Enzymatic control of cell division in micro-organisms. Nature, 162, 241245.[ISI]
Notermans, S., Veeneman, G.H., Van Zuylen, C.W.E.M., Hoogerhout, P., and Van Boom, J.H. (1988) (15)-Linked ß-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species. Mol. Immunol., 25, 975979.[CrossRef][ISI][Medline]
Prieto, A., Leal, J.A., Gómez-Miranda, B., Ahrazem, O., Jiménez-Barbero, J., and Bernabé, M. (2001) Structure of a cell wall polysaccharide isolated from Hypocrea gelatinosa. Carbonhydr. Res., 333, 173178.
San-Blas, G. (1982) The cell wall of fungal human pathogens: its possible role in host-parasite relationships. Mycopathologia, 79, 159184.[ISI][Medline]
San-Blas, G. and San-Blas, F. (1977) Paracoccidioides brasiliensis: cell wall structure and virulence. A review. Mycopathologia, 62, 7786.[ISI][Medline]
San Blas, G. and San Blas, F. (1994) Biochemistry of Paracoccidioides brasiliensis dimorphism. In: Franco, M., Lacaz, C., Restrepo-Moreno, A., and Del Negro, A., (Eds), Paracoccidioidomycosis. CRC Press, Boca Raton, Florida, pp. 4966.
San-Blas, G., Niño-Vega, G., and Iturriaga, T. (2002) Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med. Mycol., 40, 225242.[ISI][Medline]
Toledo, M.S., Levery, S.B., Straus, A.H., Suzuki, E., Momany, M., Glushka, J., Moulton, J.M., and Takahashi, H.K. (1999) Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus. Biochemistry, 38, 72947306.[CrossRef][ISI][Medline]
Vinogradov, E., Petersen, B., and Bock, K. (1998) Structural analysis of the intact polysaccharide mannan from Saccharomyces cerevisiae yeast using 1H and 13C NMR spectroscopy at 750 MHz. Carbohydr. Res., 307, 177183.[CrossRef][ISI][Medline]
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