Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Ilha do Fundão, 21941-970, Rio de Janeiro, RJ, Brazil1
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK2
Instituto Biomédico, Universidade do Rio de Janeiro (UNIRIO), Rio de Janeiro, Brazil3
Disciplina de Biologia Celular, Universidade Federal de São Paulo, São Paulo, Brazil4
Author for correspondence: Eliana Barreto Bergter. Tel: +55 21 590 3093. Fax: +55 21 5608344. e-mail: immgbel{at}microbio.ufrj.br
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: fungal pathogen, mycelium, peptidopolysaccharide, 1H- and 13C-nuclear magnetic resonance
Abbreviations: COSY, correlated spectroscopy; DEPT, distortionless enhancement by polarization transfer; PAS, periodic acid/Schiff; TSP, trimethylsilylpropionic acid sodium salt
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Very few studies on the antigenic composition and the immune response elicited during infection by P. boydii have been reported. Soluble antigens in culture filtrates were isolated and separated by anion-exchange chromatography; they consisted of 7096% carbohydrate and 416% protein (Lupan & Cazin, 1976 ). No detailed chemical composition of these antigens has been established. Here we report the characterization of a peptidorhamnomannan from mycelial forms of P. boydii, using GC, methylation analysis, 1H- and 13C-NMR spectroscopy, ELISA and immunofluorescence techniques, and comparison with Sporothrix schenckii, another pathogenic fungus that also synthesizes rhamnose-containing antigenic structures (Travassos et al., 1973
; Mendonça et al., 1976
; Lopes-Alves et al., 1994
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extraction and fractionation of peptidopolysaccharides.
Crude extracts were obtained by extraction of the mycelium with hot phosphate buffer (50 mM, pH 7·2), at 100 °C for 2 h, and were then fractionated by Cetavlon precipitation, according to Lloyd (1970) . The mother liquors from the first Cetavlon precipitation were adjusted to pH 8·8 with sodium borate and the resulting precipitate recovered by centrifugation to give the major fraction. This fraction, after uncoupling Cetavlon from the complex with acetic acid, was dialysed and lyophilized.
Fractionation of the peptidopolysaccharides (fraction B) on DE-52.
The pH 8·8 Cetavlon-precipitated material was dissolved in 0·02 M sodium phosphate buffer, pH 6·1, and applied to a DE-52 column (Whatman, 9·0x2 cm). A discontinous gradient of 0·1 M NaCl in 0·02 M sodium phosphate buffer to 1 M NaCl was used to fractionate the sample. Eluted fractions were monitored by A280 for protein, and colorimetrically (A490) for carbohydrate (Dubois et al., 1956 ).
SDS-PAGE.
Peptidopolysaccharides were solubilized at 100 °C for 5 min in 0·06 M Tris/HCl, pH 6·8, 2% (w/v) SDS, 10% (v/v) glycerol and 0·025% (w/v) bromophenol blue under reducing conditions by adding 5% (w/v) 2-mercaptoethanol immediately before use (Laemmli, 1970 ). Electrophoresis was performed in a 12% polyacrylamide separating gel using a Bio Rad Mini-Protean II electrophoresis system. Gels were stained for carbohydrate with the periodic acid/Schiff (PAS) reagent and for protein with Coomassie blue.
Analytical procedures.
Total carbohydrate was determined by the phenol/sulfuric acid method (Dubois et al., 1956 ), protein by the Lowry method, phosphate by the procedure of Ames (1966)
, and hexosamine by the method of Belcher et al. (1954)
.
Component monosaccharides.
Peptidopolysaccharides were hydrolysed with 3 M trifluoroacetic acid at 100 °C for 3 h; the resulting monosaccharides were analysed (a) by HPTLC in n-butanol/acetone/water (5:4:1, by vol.) (Ovodov et al., 1967 ) and (b) as their alditol acetates, which were characterized and quantified by GC-MS (Sawardeker et al., 1965
).
Methylation analysis.
Peptidopolysaccharides were methylated by the lithium methyl sulfinyl carbanion method (Parente et al., 1985 ). The partially methylated derivatives were converted to the corresponding alditol acetates and were analysed by GC coupled to electron-impact mass spectrometry. The partially methylated alditol acetates were separated on a fused silica capillary column of OV-225 (30x0·25 mm, i.d.), programmed from 50 to 220 °C at a rate of 50 °C min-1, then hold.
1H- and 13C-NMR spectroscopy.
1H- (500 MHz) and 13C- (125 MHz) NMR spectra were recorded using a Varian Unit 500 spectrometer. Samples (10 mg) were dissolved in 0·75 ml D2O (99·96%) with trimethylsilylpropionic acid sodium salt (TSP) as internal reference. 13C chemical shifts were determined taking as reference the rhamnose methyl signal at 18·4 p.p.m. (Gorin, 1981 ). The DEPT (distortionless enhancement by polarization transfer) spectrum was recorded using the manufacturers software. A double-quantum-filtered, gradient-assisted, magnitude-mode COSY spectrum was recorded using the pulse sequence GMQCOSY supplied by the spectrometer manufacturer.
Immunoreactivity of P. boydii peptidorhamnomannan.
Rabbit sera against whole P. boydii hyphae and Sporothrix schenckii mycelium phase (strain 1099.18) were used in this study. They were analysed by a direct ELISA method (Voller et al., 1976 ). Wells of flat-bottomed polyvinyl microtitre plates (Hemobag) were coated with 100 µl of a 10 µg ml-1 solution of P. boydii peptidopolysaccharide. They were subsequently treated with PBS-Tween 20 (0·1%) containing 2% BSA as blocking agent; this and all subsequent incubations were at 37 °C for 1 h, The hyperimmune rabbit serum was used at a dilution of 1/200 to 1/1638400, and antibody binding was also measured using goat anti-rabbit IgG conjugated to horseradish peroxidase. The chromogen used was o-phenylenediamine, added with H2O2. Other steps were carried out as described by Haido et al. (1998)
.
Immunofluorescence.
Indirect immunofluorescence analysis was carried out according to Haido et al. (1998) . Fixed mycelia and conidia (2% formaldehyde in methanol) from P. boydii were treated with rabbit anti-P. boydii serum at a dilution of 1:50 and incubated for 60 min at 37 °C in a moist chamber. A pre-immune serum was used as control. After washing, fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG diluted 1:50 in PBS (10 mM, pH 7·2) was added. The slides were incubated for 60 min, washed and examined in a fluorescence microscope. For the inhibition tests, the rabbit anti-P. boydii serum diluted 1:50 was pre-incubated for 60 min at 37 °C with an equal volume of 50 and 100 µg ml-1 peptidorhamnomannan from both P. boydii and S. schenckii (Lima & Lopes-Bezerra, 1997
; Penha & Lopes-Bezerra, 2000
) and with a peptidogalactomannan from Aspergillus fumigatus strain 2109 (Haido et al., 1998
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
NMR spectroscopy
Well-resolved signals in the 13C-NMR spectrum of P. boydii are listed in Table 3, with values for similar compounds taken from Travassos et al. (1974)
for comparison.
|
|
1H-NMR spectroscopy of the peptidopolysaccharide (Fig. 4) reveals a strong spectrum originating from carbohydrate, with broad, weaker signals from the peptide. Distinctive signals arising from rhamnose H-6 methyl protons are seen at about 1·3 p.p.m.
|
|
Immunological reactivity of purified peptidorhamnomannan (fraction III)
Fraction III was tested in ELISA using hyperimmune rabbit antisera against mycelial forms of both P. boydii and S. schenckii. P. boydii fraction III reacted strongly with the homologous antiserum whereas a much lower reactivity was obtained with the anti-S. schenckii serum (Fig. 6).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The family of peptidopolysaccharides from P. boydii which was fractionated by DE-52 column chromatography migrated in SDS-PAGE gels as a broad band in the 5080 kDa range, stained by both PAS and Coomassie blue. No other protein band appeared in the gel, which suggested that a defined group of closely related peptidopolysaccharides had been isolated. The degree of heterogeneity among these glycoconjugates was determined by the carbohydrate content and carbohydrate/protein ratios of the different fractions analysed in this work. Methylation analysis suggests that -L-Rhap(1
3)-
-L-Rhap disaccharides are present in side chains, possibly linked (1
3) to (1
6)-linked mannose residues. Mannose was also present as non-reducing end units, 2-O-, 3-O-, 6-O- and 2,4-di-O-substituted units and a few 2,6- and 3,4-di-O-substituted units. The relatively high proportion of 2-O-substituted mannosyl units could be due to their inclusion in side chains with and without rhamnose, or the presence of O-linked mannosyl oligosaccharides in the glycoconjugate.
Methylation analysis identified several structures for which prominent signals would be expected in the 13C-NMR spectrum. Reasonable correspondence can be established between chemical shifts found in this study and literature values for several carbon signals in the anomeric and other well-resolved regions of the spectrum (Table 3). Unequivocal assignment of all NMR spectra awaits study of purified carbohydrate species from this rhamnomannan.
By comparison with the rhamnomannan extracted with hot dilute alkali from S. schenckii, Ceratocystis stenoceras and C. ulmi (Travassos et al., 1973 ; Gorin & Spencer, 1970
) it was evident that the rhamnomannan of P. boydii was significantly different. The dirhamnosyl side chains -
-L-Rhap(1
2)-
-L-Rhap present in S. schenckii mycelial polysaccharides obtained at 25 °C (Mendonça et al., 1976
) were not found in the P. boydii rhamnomannan, which has
-(1
3)-linked rhamnosyl disaccharide.
The structural differences in the rhamnomannan of P. boydii as compared with a similar polysaccharide from S. schenckii, shown by methylation analysis, were confirmed by 13C-NMR spectroscopy. A consistent difference was the absence of signals at 96·8 p.p.m. and 80·3 p.p.m. corresponding respectively to the C-1 of 2-O-substituted -L-Rhap residues of
-L-Rhap-(1
2)-
-Rhap and C-2 of 2-O-substituted
-L-Rhap units (Travassos et al., 1974
).
The 1H-NMR spectrum (Fig. 4), and the COSY spectrum (Fig. 5
), with a limited number of clearly defined cross-peaks, is indicative of regularity in the carbohydrate structure; the peptide signals are too broad and too weak to give visible cross-peaks. Technical difficulties arise in obtaining a complete structural analysis by NMR of the whole peptidopolysaccharide, which has a high molecular mass and some degree of heterogeneity. However, studies of purified carbohydrate chains are currently being undertaken to determine their structures in detail.
Preliminary results on the nature of the antigenic determinants in P. boydii peptidorhamnomannan showed that fraction III contains antigenic determinants recognizable by anti-P. boydii antibodies, but that do not cross-react with anti-S.schenckii antibodies raised against whole cells of this fungus. This result can be explained on the basis of the immunodominance of the carbohydrate epitopes in the peptidorhamnomannans of both species. Lloyd & Travassos (1975) showed that anti-S. schenckii antibodies reacted with ß-L-Rhap-(1
3)-
-D-Manp but Lopes-Alves et al. (1994)
later showed that a much stronger reactivity was obtained with the O-linked chains containing 2-O- and 4-O-linked rhamnosyl units linked to
-D-glucuronic acid. The very weak cross-reactivity between P. boydii and S. schenckii depends therefore on the low-titre anti-
-L-Rhap(1
3)
-D-Manp antibodies, since the
-L-Rhap-(1
3)-
-L-Rhap(1
3)-L-Rhap-(1
3)-
-D-Manp structure, with or without additional 2-O-linked mannopyranosyl units, appears to be unique to P. boydii.
By immunofluorescence we also showed that the rabbit antiserum against P. boydii mycelium strongly reacted with hyphae and conidia of this fungus, a reaction specifically inhibited by fraction III peptidorhamnomannan. A very weak inhibition was observed with the peptidorhamnomannan from S. schenckii (Penha & Lopes-Bezerra, 2000 ) or a peptidogalactomannan from A. fumigatus (Haido et al., 1998
).
To our knowledge, this is the first characterization of a chemically defined antigen from the mycelial forms of P. boydii. A major aim of this study was to determine whether this antigen could be useful for diagnostic purposes, mainly in mixed allergic bronchopulmonary fungal disease due to P. boydii and Aspergillus (Lake et al., 1990 ). The chemical characterization of fungal antigens is important to allow a rational interpretation of the cross-reactivity between pathogenic species, thus helping to select immunodominant components that can be of diagnostic use. The present work provides evidence for the expression of antigens which are similar but do not cross-react with S. schenckii peptidopolysaccharides and differ from the major Aspergillus glycoconjugate. Further studies are currently under way to determine the fine structure of the P. boydii antigen.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Belcher, R. A., Nutten, A. J. & Sambrook, C. M. (1954). The determination of glucosamine. Analyst 79, 201-208.
Berenguer, J., Diaz-Mediavilla, J., Urra, D. & Muñoz, P. (1989). Central nervous system infection caused by P. boydii: case report and review. Rev Infect Dis 11, 890-896.[Medline]
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Swith, E. (1956). Colorimetric method for determination of sugar and related substances. Anal Chem 28, 350-356.
Gorin, P. A. J. (1981). Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv Carbohydr Chem Biochem 38, 13-104.
Gorin, P. A. J. & Spencer, J. F. T. (1970). Structures of the L-rhamno D-mannan from Ceratocystis ulmi and the D-gluco-D-mannan from Ceratocystis brunnea. Carbohydr Res 13, 339-349.
Haido, R. M. T., Silva, M. H., Ejzemberg, R., Leitao, E. A., Hearn, V. M., Evans, E. G. V. & Barreto Bergter, E. (1998). Analysis of peptidogalactomannan from the mycelial surface of Aspergillus fumigatus. J Med Mycol 36, 313-321.
Jansson, P. E., Kenne, L., Liedgren, H., Lindberg, B. & Longen, J. (1976). A practical guide to the methylation analysis of carbohydrates. Chem Commun Univ Stockholm 8.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lake, F. R., Tribe, A. E., McAleer, R., Frondist, J. & Thompson, P. J. (1990). Mixed allergic bronchopulmonary fungal disease due to Pseudallescheria boydii and Aspergillus. Thorax 45, 489-492.[Abstract]
Lima, O. C. & Lopes-Bezerra, L. M. (1997). Identification of a Concanavalin A-binding antigen of the cell surface of Sporothrix schenckii. J Med Vet Mycol 35, 167-172.[Medline]
Lloyd, K. O. (1970). Isolation, characterization and partial structure of peptidogalactomannan from the yeast form of Cladosporium wernecki. Biochemistry 9, 3446-3453.[Medline]
Lloyd, K. O. & Travassos, L. R. (1975). Immunochemical studies on L-rhamno-D-mannans of Sporothrix schenckii and related fungi by use of rabbit and human antisera. Carbohydr Res 40, 89-97.[Medline]
Lopes-Alves, L., Travassos, L. R., Previato, J. O. & Mendonça-Previato, L. (1994). Novel antigenic determinants from peptidorhamnomannans of Sporothrix schenckii. Glycobiology 4, 281-288.[Abstract]
Lupan, D. M. & Cazin, J.Jr (1976). Serological diagnosis of petrillidiosis (allescheriosis). I. Isolation and characterization of soluble antigens from Allescheria boydii and Monosporium apiospermum. Mycopathologia 58, 31-38.[Medline]
Mendonça, L., Gorin, P. A. J., Lloyd, K. O. & Travassos, L. R. (1976). Polymorphism of S. schenckii surface polysaccharides as a function of morphological differentiation. Biochemistry 15, 2423-2431.[Medline]
Ovodov, Y. S., Evtushenko, E. V., Vaskovsky, V. E., Ovodova, R. G. & Soloveva, T. F. (1967). Thin layer chromatography of carbohydrates. J Chromatogr 26, 111-115.[Medline]
Parente, J. P., Cardon, P., Leroy, Y., Montreuil, J., Fournet, B. & Ricart, J. (1985). A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methyl-sulfinyl-carbanion. Carbohydr Res 141, 41-47.[Medline]
Penha, C. V. L. & Lopes-Bezerra, L. M. (2000). Concanavalin A-binding antigens of Sporothrix schenckii: a serological study. Med Mycol 38, 1-7.
Rippon, J. W. (1998). Pseudallescheriasis. Medical Mycology , 651-680. Philadelphia:W. B. Saunders.
Sawardeker, J. S., Sloneker, J. H. & Jeanes, H. (1965). Quantitative determination of monosaccharides as their acetates by gas-liquid chromatography. Anal Chem 37, 1602-1604.
Spencer, J. T. F. & Gorin, P. A. J. (1971). Systematics of the genera Ceratocystis and Graphium. Proton magnetic resonance spectra of the mannose-containing polysaccharides as an aid in classification. Mycologia 63, 387-402.
Travassos, L. R. & Lloyd, K. (1980). Sporothrix schenckii and related species of Ceratocystis. Microbiol Rev 44, 683-721.
Travassos, L. R., Gorin, P. A. J. & Lloyd, K. O. (1973). Comparison of the rhamnomannans from the human pathogen Sporothrix schenckii with those from the Ceratocystis species. Infect Immun 8, 685-693.[Medline]
Travassos, L. R., Gorin, P. A. J. & Lloyd, K. O. (1974). Discrimination between Sporothrix schenckii and Ceratocystis stenoceras rhamnomannans by proton and carbon-13 magnetic resonance spectroscopy. Infect Immun 9, 674-680.[Medline]
Voller, A., Bidwell, D. & Bartlett, A. (1976). Enzyme-linked immunosorbent assay. In Manual of Clinical Immunology , pp. 506-512. Edited by N. R. Rose & H. M. Freidman. Washington, DC:American Society for Microbiology.
Received 17 October 2000;
accepted 21 February 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |