Centro de Investigaciones Biológicas, c/Velázquez 144.28006-Madrid, Spain1
Instituto de Química Orgánica, Grupo de Carbohidratos, c/Juan de la Cierva 3.28006-Madrid, Spain2
Author for correspondence: J. Antonio Leal. Tel: +34 91 561 18 00. Fax: +34 91 562 75 18. e-mail: aleal{at}cib.csic.es
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ABSTRACT |
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Keywords: Paecilomyces, cell wall, galactomannan, chemotaxonomy, fungal antibodies
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INTRODUCTION |
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These genera are economically important since some species are responsible for food contamination and spoilage (King et al., 1969 ; Engel & Teuber, 1991
), can infect immunodeficient humans (Byrd et al., 1992
) or are pathogenic for invertebrate animals such as the silk-worm (Bombyx mori) (Aoki & Yanase, 1970
).
Taxonomy based only on the morphology of these genera is sometimes unclear. The use of biochemical and molecular techniques is needed in order to give some insight into the classification of related groups of fungi (Taylor et al., 1990 ; Berbee et al., 1995
). The fungal cell wall is mainly composed of polysaccharides, which have proved to be useful chemotaxonomic and antigenic markers for identification purposes (Bartnicki-García, 1970
; Weijman et al., 1982
; Weijman & Golubev, 1987
; Weijman & Van der Walt, 1989
; Kamphuis et al., 1992
; Leal, 1994
; Leal & Bernabé, 1998
).
The finding that fungal polysaccharides have immunological properties, and that some of them are almost genus-specific (Notermans & Soentoro, 1986 ), enhanced interest in the elucidation of the structure of these molecules since they might be used as chemotaxonomic characters. Most studies on serological properties of fungal polysaccharides deal with exopolysaccharides (Notermans & Soentoro, 1986
; Notermans et al., 1987
; De Ruiter et al., 1992
) and cell wall glycoproteins (Gailliez & Poulain, 1988
; Sridhara et al., 1990
; Hearn, 1991
). An antigenic water-soluble galactomannan isolated from cell walls of Paecilomyces fumosoroseus and Paecilomyces farinosus (section Isarioidea) has been studied in our laboratory (Domenech et al., 1996
). In the present research we report on the structural and immunological characterization of polysaccharides purified from cell walls of species of section Paecilomyces and discuss their use as chemotaxonomic characters for the delimitation of the section.
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METHODS |
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Wall material preparation and fractionation.
Wall material was obtained as reported by Gómez-Miranda et al. (1990) . Cell wall material (8 g) was repeatedly extracted with 1 M NaOH (300 ml) at 20 °C. After centrifugation, the supernatants were combined and an equal volume of 96% ethanol was added. The precipitate was collected by centrifugation, dialysed against running tap water and then freeze-dried. This polysaccharidic material (500 mg) was suspended in distilled water (100 ml), stirred at room temperature for 2 h and centrifuged. The precipitate was extracted again as described above and the supernatant solutions were combined and freeze-dried (F1S).
Isolation of polysaccharides from F1S.
Fraction F1S (190 mg) was dissolved in 3 ml distilled water and centrifuged at 13000 g for 15 min to eliminate insoluble material. The supernatant was added to a column (90x3 cm) of Sepharose CL-6B and eluted with distilled water (flow rate 21·4 ml h-1). Fractions of 2·5 ml were collected and monitored for carbohydrate by the phenol-sulphuric acid method (Dubois et al., 1956 ). Appropriate fractions were combined, concentrated to a small volume (15 ml) and freeze-dried.
Isolation of the mannan core.
A sample of each polysaccharide (100 mg) was treated with 20 ml 0·05 M H2SO4 at 100 °C for 6 h. The degraded polysaccharide was dialysed against distilled water (exclusion limit 3000 Da), concentrated to a small volume (10 ml) and freeze-dried.
Chemical analyses.
Monosaccharides were released by Saeman hydrolysis (Adams, 1965 ) and analysed by GLC as their corresponding alditol acetates as previously described (Gómez-Miranda et al., 1981
). Methylation analysis and GLC-MS of the derivatives were performed as described by Domenech et al. (1996)
. Permethylated samples were subjected to reductive cleavage as described by Gray (1987)
in order to identify the ring size (pyranose or furanose) of the sugar residues. The partially methylated anhydroalditol acetates obtained were analysed by GLC-MS using a fused silica SPB-1 column and a temperature range of 150 to 200 °C (3 min initial hold and ramp rate 3 °C min-1). Absolute configuration was determined according to Gerwig et al. (1978)
by GLC-MS of the tetra-O-TMSi-(+)-2-butylglycosides obtained. Protein was determined by the Lowry method.
NMR spectroscopy.
The polysaccharides (approx. 20 mg) obtained after column chromatography were dissolved in D2O (1 ml) followed by centrifugation (10000 g for 20 min) and lyophilization of the supernatant. The process was repeated twice and the final sample was dissolved in D2O (0·7 ml, 99·98% D). 1H- and 13C-NMR spectra were recorded at 40 °C on a Varian INOVA 300 spectrometer. Proton chemical shifts refer to residual HDO at 4·61 p.p.m. Carbon chemical shifts refer to internal acetone at
31·07 p.p.m.
Antibody production and serology.
Antibodies were obtained by immunization of rabbits with F1S-B polysaccharide (the major fraction isolated by gel permeation chromatography of F1S on Sepharose CL-6B) of P. variotii CBS 990.73A, P. fumosoroseus CBS 375.70 and Eupenicillium crustaceum CBS 635.70. The protocols for immunization and titration by ELISA were as previously described (Domenech et al., 1996 ) except that the ELISA plates were coated with different concentrations of polysaccharide (40400 g ml-1) depending on the antigens. The specificity of the antisera obtained was tested by inhibition studies (Domenech et al., 1996
). The polysaccharides isolated from all species tested in this work and the water-soluble polysaccharide from E. crustaceum (Leal et al., 1993
) were used as inhibitors. Staining of P. variotii CBS 990.73A was carried out by immunofluorescence under the same conditions as reported by Domenech et al. (1996)
, but in this case it was not necessary to adsorb the secondary antibody with mycelium since nonspecific staining was not observed.
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RESULTS |
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Structural studies of the polysaccharides
The water-soluble polysaccharides F1S-B were studied using a combination of chemical and spectroanalytical techniques. The methylation results (Table 1) demonstrated that all the polysaccharides contained almost identical substituted galactofuranose and mannopyranose residues, although in different proportions. Three of them had also very small amounts (2%) of terminal glucopyranose. All the 1H-NMR spectra (Fig. 1a
) showed broad uneven signals coexisting with small signals, indicative of irregular polysaccharides. In addition, the 13C-NMR spectra (Fig. 1b
) contained sharp and broad signals, suggesting the presence of a low mobility backbone with side chains of higher flexibility (Rath et al., 1995
; Prieto et al., 1997
). The chemical shifts of the sharp signals had values over 105 p.p.m., indicative of ß-galactofuranose units, while the broad signals were assigned to the mannopyranose residues. These values are in agreement with the existence of ß-galactofuranose chains linked to a mannan core (Prieto et al., 1997
). Some of the polysaccharides were analysed by comparison with known polysaccharides. The signals for the galactofuranose residues of P. variotii and B. fulva were found to be closely related to those of Penicillium expansum (Parra et al., 1994
) since (1
5)-ß- and (1
6)-ß- linkages appeared in a 3:1 proportion. F1S-B of Ta. leycettanus and Ta. byssochlamydoides had outer chains of ß-Galf O-5-substituted, giving signals comparable to those found for the (1
5)-ß-galactan isolated from E. crustaceum (Leal et al., 1993
).
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The peaks observed in the 1H- and 13C-NMR spectra (Fig. 1) are consistent with these residues (Leal et al., 1993
; Parra et al., 1994
; Prieto et al., 1997
). Signals corresponding to the mannan residues appeared poorly resolved in most of the cases. Hence, taking advantage of the lability of the glycosidic linkages of the furanoid rings as compared with those of the pyranoid rings, we further investigated them by selectively hydrolysing the galactofuranan moieties of all the polysaccharides to leave only the mannan skeleton.
The methylation results demonstrated that all the mannan cores also had identical residues (Table 2), although in different proportions. The 1H-NMR spectra of the mannans obtained are depicted in Fig. 2
, the mannan from B. fulva and Th. crustaceus (not shown) being very similar to that of P. variotii.
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Immunological studies
The titre of the sera after the fifth injection reached 1/8000 for P. variotii and P. fumosoroseus, and 1/6000 for E. crustaceum. The results of inhibition studies are shown in Fig. 3. F1S-B polysaccharides of all isolates of P. variotii inhibited the serum anti-F1SB of P. variotii CBS 990.73A (Fig. 3b
) at low concentrations (3·269·3 g ml-1), but the polysaccharide F1S-B from the remaining species of section Paecilomyces studied did not block the immunological reaction (results not shown).
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The hyphae of P. variotii CBS 990.73A reacted with polyclonal antibodies raised against fraction F1S-B of this micro-organism as demonstrated by immunofluorescence (Fig. 4).
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DISCUSSION |
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Polyclonal antibodies raised against F1S-B polysaccharide from P. variotii CBS 990.73A were highly specific, since the binding to their specific antigen was only inhibited by different isolates of the same species (Fig. 3b). Polysaccharides with a structure similar to that of the antigen used, such as those from other species of section Paecilomyces, did not inhibit the immunochemical reaction. This could be related to the differences in the length of the side chains and the irregularity of the polysaccharides F1S-B of this group. Inhibition experiments were also performed using serum anti-F1S-B from E. crustaceum, which is a linear (1
5)-ß-galactofuranan (Leal et al., 1993
), in order to check if these antibodies recognized the galactofuranan side chains of the polysaccharides studied here. In this case, the polysaccharides F1S-B from Ta. byssochlamydoides, P. variotii, Th. crustaceus and B. fulva inhibited the binding of the antiserum to its specific antigen at concentrations that varied from 9 to 300 µg ml-1. F1S-B fractions from these species have a higher content of galactofuranose than those from Ta. leycettanus and B. nivea, which reacted only at concentrations higher than 300 µg ml-1. This may indicate that the (1
5)-ß-galactofuranose residues are immunodominant as in the extracellular polysaccharides of Penicillium and Aspergillus (Notermans et al., 1988
). On the other hand, the polysaccharides of fungi from section Paecilomyces did not inhibit the binding of the serum anti-F1SB of P. fumosoroseus to its specific antigen, a (1
6)-
-mannan substituted at O-4 by one residue of ß-galactopyranose or
-glucopyranose.
Mycelium of P. variotii reacted specifically with serum anti-F1S-B of the same species (Fig. 4), indicating the accessibility of the epitope in the hyphae. This would allow the detection of fungal contamination in food and biological samples.
The structure of the polysaccharides F1S-B from fungi of section Paecilomyces presented in this work differs from the one described for P. fumosoroseus and P. farinosus from section Isarioidea (Domenech et al., 1996 ) indicating the heterophyletic character of the genus Paecilomyces. The availaible data support the division of the genus into two sections. The characteristic polysaccharides from both sections are different from those described for Penicillium species. The existing classification of the genus was proposed by Samson (1974)
on the basis of the morphology and the structure of the phialides and conidiophores. More recent studies using molecular techniques, such as the cladistic analysis of PCR-based DNA markers, clearly showed that morphological characteristics of asexual structures are insufficient to yield a cohesive taxonomy of Paecilomyces, and that classifications based on morphological criteria alone had problems in distinguishing among species of closely related genera (Tigano-Milani et al., 1995
). It is interesting to note that F1S-B polysaccharides from the two species of Talaromyces included in this work (Ta. leycettanus and Ta. byssochlamydoides) are clearly distinct from the complex glucogalactans with small amounts of mannose found in species of the same genus which are closely related to Penicillium (Prieto et al., 1995
). Our data reinforce the results of rDNA analysis (Taylor et al., 1990
) showing that Ta. leycettanus and Ta. byssochlamydoides are closely related to Byssochlamys and Thermoascus and phylogenetically separated from other Talaromyces, and agree with the proposal of Pitt (1993)
that Talaromyces species with Paecilomyces anamorphs should be transferred to Byssochlamys.
As has been shown in other fungal taxa, polysaccharides seem to be useful characters for delimiting genera and establishing relationships among anamorphs and teleomorphs (Leal et al., 1992 , 1997
; Domenech et al., 1994
, 1996
; Leal, 1994
; Jiménez-Barbero et al., 1995
; Prieto et al., 1995
, 1997
; Leal & Bernabé, 1998
).
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ACKNOWLEDGEMENTS |
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Received 8 February 1999;
revised 10 June 1999;
accepted 18 June 1999.