Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Glycobiology vol. 13 no. 11 pp. 743±747, 2003 DOI: 10.1093/glycob/cwg069 Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis Oussama Ahrazem2, Alicia Prieto2, Gioconda San-Blas3, Juan Antonio Leal2, Jes us Jimenez-Barbero2, and Manuel 1,4 Bernabe 2 Centro de Investigaciones Biol ogicas, CSIC, Vel azquez 144, 28006-Madrid, Spain; 3Centro de MicrobiologõÂa y BiologõÂa Celular, IVIC, P.O. Box 21827, Caracas 1020A, Venezuela; and 4Instituto de QuõÂmica Organica, CSIC, Juan de la Cierva 3, 28006-Madrid, Spain Received on January 13, 2003; revised on March 13, 2003; accepted on March 13, 2003 Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of ! 6)-[a-Galf -(1 ! 6)a-Manp-(1 ! 2)]-a-Manp-(1 ! . The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of ! 6)-[-a-Manp-(1 ! 2)]-a-Manp-(1 ! , alternating with a trisaccharide repeating block of ! 6)-[bGalf -(1 ! 6)-a-Manp-(1 ! 2)]-a-Manp-(1 ! . Key words: cell wall polysaccharides/fungi/NMR spectroscopy/Paracoccidioides brasiliensis Introduction Paracoccidioides brasiliensis is a pathogenic dimorphic fungus, which at environmental temperatures (20±26 C) grows in a mycelial form while transforming to a yeast phase at the temperature of the mammalian host (36±37 C). It is endemic to the region between the south of Mexico and the north of Argentina, causing paracoccidioidomycosis, the most widespread systemic mycosis in this geographical region (San-Blas et al., 2002), which invades lungs and afterward disseminates to almost any organ. If not treated, it is almost always fatal. Different aspects of the pathobiology and dimorphism of P. brasiliensis have been reviewed (SanBlas et al., 2002; Borges-Walmsley et al., 2002; Franco, 1987; San-Blas, 1982; San-Blas and San-Blas, 1977). 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 1 To whom correspondence should be addressed; e-mail: [email protected] metabolites whose presence would modulate host±parasite 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 (2±8%), are the glycosidic moieties of peptidopolysaccharides. 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; Latge et al., 1991; De Ruiter et al., 1991) and are probably involved in cell±cell and/or cell±host 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. Results Polysaccharide F1SS from the mycelial phase The analyses of the crude alkali-extractable water-soluble cell wall polysaccharidic material from the mycelial phase of P. brasiliensis gave glucose (10%), galactose (30%), and mannose (60%). After purification through Sepharose CL6B, the main component (polysaccharide F1SS) consisted of mannose (70%) and galactose (30%), as shown by gasliquid chromatography (GLC) of the alditol acetates. The absolute configuration was shown to be D for both sugars (Gerwig et al., 1979). Methylation analysis allowed to deduce the linkage types summarized in Table I. Both polysaccharides are polydisperse, with an average molecular mass around 70 kDa as determined by gel permeation chromatography on Sepharose CL6B. Table I. Linkage types (%) deduced from methylation analyses of the polysaccharides F1SS of the mycelial and yeast phases of P. brasiliensis Linkage type Manp-(1 ! Mycelial Yeast 2.7 21.3 27.3 12.6 ! 2)-Manp-(1 ! 9.3 10.1 ! 6)-Manp-(1 ! 28.7 20.7 ! 2,6)-Manp-(1 ! 31.7 35.0 Galf-(1 ! Glycobiology vol. 13 no. 11 # Oxford University Press 2003; all rights reserved. 743 O. Ahrazem et al. The 1 H±nuclear magnetic resonance (NMR) spectrum contained three anomeric signals at 5.08, 5.04, and 5.02 ppm (Figure 1a) in the proportion 1:1:1, as deduced from integration. The corresponding residues were labeled A±C, from low to high field. A and C appeared as slightly broad singlets (J1,2 2 Hz), and B exhibited a clear doublet, J1,2 4.4 Hz, that could be indicative of a galactofuranose with a-configuration (see also the 13 C d information later and compare with b-configuration, J1,2 2 Hz) (Cyr and Perlin, 1979). The 13 C-NMR spectrum (Figure 1b) showed 18 singlets, 3 of them anomeric, at 103.0, 101.2, and 99.0 ppm. A series of 2D homo-NMR (double quantum filtered homonuclear correlated spectroscopy [DQCOSY], total correlation spectroscopy [TOCSY]) and hetero- (heteronuclear multiplequantum coherence [HMQC], heteronuclear single quantum coherence [HSQC]-TOCSY) NMR experiments allowed the assignment of all the proton and carbon signals of the three residues (see Table II). Comparison of the chemical shifts values with those reported by Bock and Pedersen (1983) permitted us to deduce the glycosylation positions. Fig. 1. (a) 1 H-NMR (500 MHz) and (b) 13 C-NMR (125 MHz) spectra in D2O at 40 C for the cell wall F1SS polysaccharide from the mycelial form of P. brasiliensis. The anomeric protons have been labeled A±C. Thus, A is 2,6-di-O-substituted mannopyranose; B, terminal galactofuranose; and C, 6-O-substituted mannopyranose, which is in accordance with the methylation results. Concerning the anomeric configuration of the mannose residues, a carbon-coupled HMQC experiment allowed the measurement of their 1 JC-1,H-1 values, giving 173 Hz for units A and C, which are indicative of a-configurations for the mannose residues (Bock and Pedersen, 1974). 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 H1A/C-6A0 , H-1B/C-6C, and H-1C/C-2A could be observed. We denote a second unit of A as A0 . 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. The small quantities (510%) of terminal and 2-Osubstituted mannopyranoses observed in the methylation analyses were not detected in the NMR spectra. Therefore, to further investigate those minor components, we selectively hydrolyzed the galactofuranose residues, taking advantage of the lability of the glycosylic linkages of the furanoid rings, as compared with those of the pyranoid rings. Treatment of the polysaccharide with 0.05 M sulfuric acid gave a new polysaccharide composed exclusively of mannose. Methylation analysis gave terminal mannopyranose (34%); 2-O-substituted, 3-O-substituted, and 6-O-substituted mannopyranoses (23.5%, 1.1%, and 9%, respectively); and 2,6-di-O-substituted mannopyranose Table II. 1 H- and 13 C-NMR chemical shifts (d) for the alkali-extractable water-soluble cell wall polysaccharide F1SS isolated from the mycelial form of P. brasiliensis Proton or Carbon Units A 1 H C B H C C H C 1 5.08 99.0 5.04 101.2 5.02 103.0 4.01 80.0 4.15 77.4 4.08 70.9 3 3.94 71.4 4.22 75.2 3.83 71.3 4 3.87 67.1 3.84 82.1 3.81 67.3 5 6a 3.80 72.2 3.79 72.8 3.91 72.6 13 Fig. 2. (a) H-NMR (300 MHz) and (b) C-NMR (75 MHz) spectra in D2O at 40 C for the partially hydrolyzed mycelial form of P. brasiliensis. 744 2 Numbers in boldface type represent glycosylation sites. 4.04 6b 3.74 66.4 3.72 3.63 63.4 4.05 67.0 3.66 Cell wall polysaccharides in phases of P. brasiliensis Table III. 1 H- and 13 C-NMR chemical shifts (d) for the alkali-extractable water-soluble cell wall polysaccharide F1SS isolated from the yeast form of P. brasiliensis Proton or Carbon Units 1 D H E H C C F H C Fig. 3. (a) 1 H-NMR (500 MHz) and (b) 13 C-NMR (125 MHz) spectra in D2O at 40 C for the cell wall F1SS polysaccharide from the yeast form of P. brasiliensis. G H H H C C (32.4%). The 1 H-NMR spectrum (Figure 2) suggests the presence of a small proportion of short chains of (1 ! 2) and (1 ! 3) linked mannopyranoses and also small amounts of unbranched mannopyranoses in the main chain, as observed in several yeasts (see, for instance, Vinogradov et al., 1998). Polysaccharide F1SS from the yeast phase The analysis of the alkali-extractable water-soluble yeast cell wall polysaccharidic material from the yeast form gave mannose (67.9%), galactose (22.1%), and glucose (9.9%). After purification through Sepharose, the main component (polysaccharide F1SS) gave mannose (77%) and galactose (23%). The absolute configuration was shown to be D for both sugars. Methylation analysis led to the results gathered in Table I. The 1 H-NMR spectrum (Figure 3a) showed a severely crowded anomeric region, from which very little information could be inferred. The 13 C-NMR spectrum (Figure 3b) contained four main and one small anomeric singlets. The signal at 108.7 ppm suggests a b-galactofuranose moiety, and those around 103 ppm are characteristic of mannopyranose units. The small singlet appeared at identical position to that of the a-galactofuranose unit (101.2 ppm) found in the spectrum of the mycelial phase. The 2D 1 H-13 C correlation (HMQC) spectrum contained six anomeric crosspeaks, which were labelled D±I. 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. The configuration of the anomeric centers was deduced from a carbon-coupled HMQC experiment, which allowed the measurement of the 1 JC-1,H-1 values, giving 174.4, 174.3, 174.4, 175.9, 172.9, and 173.0, for units D to I, respectively, which, in accordance with the values of the chemical shifts, I H C 2 5.11 99.2 5.08 99.2 5.06 108.7 5.04 101.2 5.04 103.1 5.02 103.0 3 4.03 79.5a 4.03 79.9 a 4.14 81.8 4.15 77.5 4.07 70.9 4.07 70.8 3.94b 71.5 3.93b 71.5 4.07 77.8 4.22 75.2 3.81 71.5 3.83 71.5 4 5 3.85 67.7c 6a ~.78 72.2 3.87 ~.81 c ~3.78 67.4 4.01 84.0 3.83 82.2 3.68 67.7 3.81 67.4 3.84 71.8 3.78 72.7 3.76 74.1 6b 4.02d 3.69 66.6e 4.03d 3.72 66.4e 3.73 3.66 63.8 3.73 3.64 63.4 3.88 3.77 62.0 4.03 3.67 67.4 Numbers in boldface type represent glycosylation sites. a,b,c,d,e These values may have to be interchanged within each respective row. are demonstrative of b-configuration for the furanose unit G and a for the rest of the residues (Bock and Pedersen, 1974; Cyr and Perlin, 1979). To discriminate among the various possibilities of arrangement of the different fragments, we recorded a long-range proton±carbon correlation HMBC experiment that, in addition to trivial cross-peaks, showed signals for H-1D/C-6E (D0 ), H-1E/C-6D (E0 ), 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 D0 and E0 . 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. In addition, a small proportion of short chains of (1 ! 2) linked mannopyranoses is deduced from the methylation analysis, analogously to that observed in the mycelium phase. Discussion P. brasiliensis grows as filamentous fungus when cultivated at 23 C and is easily converted to the yeast phase by simply changing the temperature to 37 C. This process is 745 O. Ahrazem et al. reversible, as demonstrated by Nickerson (1948), who also showed that this reversibility was exclusively due to the temperature factor and was independent from other changes in the culture medium. 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 a-(1 ! 3) glucan was found in the yeast phase, whereas a b-(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 BernabeÂ, 1998) revealed that the mycelium from these microorganisms contained either a-(1 ! 3) or b-(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 a-(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 a-glucan working as a protection mechanism of the fungus against host defences (San-Blas, 1982). Structural variations have also been observed by MendoncËa 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 b-galactofuranose domains of polysaccharides from several pathogenic fungi (Latge et al., 1991; Notermans et al., 1988; Azuma et al., 1974) is known, yet little or nothing is known of the role of a-galactofuranoses because these residues have only been described in cell wall polysaccharides from a few species belonging to Onygenales (Bernabe et al., 2002). Material and methods Strains and growth conditions P. brasiliensis strain IVIC Pb73 (ATCC 32071) was grown in peptone yeast glucose medium and incubated at either 746 23 C (mycelium form) or 37 C (yeast form) with continuous shaking. 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 0.25 mm ID 0.2 mm film thickness) with a temperature program (210 C to 240 C, initial time 3 min, ramp rate 15 C min ÿ1 , 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 (1±5 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 1 H- and 13 C-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 (1 H, 300 MHz). Proton chemical shifts refer to residual HDO at d 4.61 ppm. Carbon chemical shifts refer to internal acetone at d 31.07 ppm. The polysaccharides F1SS (~20 mg) were dissolved in D2O (1 ml) followed by centrifugation (10,000 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. Acknowledgments The authors thank J. L opez and Lic. B. Moreno for technical assistance. This work was supported by Grant BQU2000-1501-C02-01 from Direcci on General de Investigaci on CientõÂfica y Tecnica. Cell wall polysaccharides in phases of P. brasiliensis Abbreviations DQCOSY, double quantum filtered homonuclear correlated spectroscopy; GLC, gas-liquid chromatography; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple-quantum coherence; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; TFA, trifluoroacetic acid; TOCSY, total correlation spectroscopy References Ahrazem, O., G omez-Miranda, B., Prieto, A., BarasoaõÂn, I., Bernabe, M., and Leal, J.A. (2000a) An acidic water-soluble cell wall polysaccharide: a chemotaxonomic marker for Fusarium and Gibberella. Mycol. Res., 104, 603±610. Ahrazem, O., G omez-Miranda, B., Prieto, A., Bernabe, M., and Leal, J.A. (2000b) Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides. Arch. Microbiol., 173, 296±302. 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, 507±515. 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, 111±125. Bernabe, 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 13 C-H coupling constants in hexopyranoses. J. Chem. Soc. Perkin Trans., 2, 293±297. Bock, K. and Pedersen, C. (1983) Carbon-13 nuclear magnetic resonance spectroscopy of monosaccharides. Adv. Carbohydr. Chem. Biochem., 41, 27±66. Borges-Walmsley, M.I., Chen, D., Shu, X., and Walmsley, A.R. (2002) The pathobiology of Paracoccidioides brasiliensis. Trends Microbiol., 10, 80±87. Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209±217. Cyr, N. and Perlin, A.S. (1979) The conformations of furanosides. A 13 C nuclear magnetic resonance study. Can. J. Chem., 57, 2504±2511. 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: Latge, J.P. and Boucias, D. (Eds), Fungal cell wall and immune response. Springer Verlag, Berlin, pp. 169±180. Domenech, J., BarasoaõÂn, I., Prieto, A., G omez-Miranda, B., Bernabe, M., and Leal, J.A. (1996) An antigenic water-soluble glucogalactomannan extracted from cell walls of Paecilomyces fumosoroseus and Paecilomyces farinosus. Microbiology, 142, 3497±3503. Domenech, J., Prieto, A., BarasoaõÂn, I., G omez-Miranda, B., Bernabe, 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, 2789±2796. Franco, M.F. (1987) Host±parasite relationships in paracoccidioidomycosis. J. Med. Vet. Mycol., 25, 5±18. 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, 10±17. Kanetsuna, F. and Carbonell, L.M. (1970) Cell wall glucans of the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol., 101, 675±680. Kanetsuna, F., Carbonell, L.M., Moreno, R.E., and RodrõÂguez, J. (1969) Cell wall composition of the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol., 97, 1036±1041. Laine, R.A., Esselman, W.J., and Sweeley, C.C. (1972) Gas-liquid chromatography of carbohydrates. Meth. Enzymol., 28, 159±167. Latge, J.P., Debeaupuis, J.P., Moutaouakil, M., Diaquin, M., Sarfati, J., Prevost, M.C., Wieruszeski, J.M., Leroy, Y., and Fournet, B. (1991) Galactomannan and the circulating antigens of Aspergillus fumigatus. In: LatgeÂ, J.P. and Boucias, D. (Eds), Fungal cell wall and immune response. Springer-Verlag, Berlin, pp. 143±155. Leal, J.A. and Bernabe, 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. 153±181. Leal, J.A., Prieto, A., Ahrazem, O., Pereyra, T., and Bernabe, M. (2001) Cell wall polysaccharides: characters for fungal taxonomy and evolution. Rec. Res. Devel. Microbiol., 5, 235±248. 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, 8764±8775. MendoncËa, 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, 2423± 2431. 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, 5±12. Nickerson, W.J. (1948) Enzymatic control of cell division in microorganisms. Nature, 162, 241±245. Notermans, S., Veeneman, G.H., Van Zuylen, C.W.E.M., Hoogerhout, P., and Van Boom, J.H. (1988) (1 ! 5)-Linked b-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species. Mol. Immunol., 25, 975±979. Prieto, A., Leal, J.A., G omez-Miranda, B., Ahrazem, O., JimenezBarbero, J., and Bernabe, M. (2001) Structure of a cell wall polysaccharide isolated from Hypocrea gelatinosa. Carbonhydr. Res., 333, 173±178. San-Blas, G. (1982) The cell wall of fungal human pathogens: its possible role in host-parasite relationships. Mycopathologia, 79, 159±184. San-Blas, G. and San-Blas, F. (1977) Paracoccidioides brasiliensis: cell wall structure and virulence. A review. Mycopathologia, 62, 77±86. 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. 49±66. San-Blas, G., Ni~ no-Vega, G., and Iturriaga, T. (2002) Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med. Mycol., 40, 225±242. 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, 7294±7306. Vinogradov, E., Petersen, B., and Bock, K. (1998) Structural analysis of the intact polysaccharide mannan from Saccharomyces cerevisiae yeast using 1 H and 13 C NMR spectroscopy at 750 MHz. Carbohydr. Res., 307, 177±183. 747