Cell Wall Composition of the Yeast and Mycelial Forms of Paracoccidioides Brasiliensis

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JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1036-1041 Vol. 97, No. 3 Copyright @ 1969 American Society for Microbiology Printed in U.S.A. Cell Wall Composition of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

FUMINORI KANETSUNA, LUIS M. CARBONELL, RAMON E. MORENO,' AND JOAQUIN RODRIGUEZ Department of Microbiology, Iiistituto Venezolanio de Invesligacionies Cienitificas, Apartado 1827, Caracas, Ventezuela Received for publication 3 December 1968

Isolation and chemical analyses of the cell walls of the yeast (Y form) and mycelial forms (M form) of Paracoccidioides brasiliensis and Blastomyces dermatitidis revealed that their chemical composition is similar and depends on the form. Lipids, chitin, glucans, and proteins are the main constituents of the cell walls of both forms of these fungi. There is no significant difference in the amount of lipids (5 to 10%) and glucans (36 to 47%0) contained by the two forms. In both fungi, the Y form has a larger amount of chitin (37 to 48%) than the M form (7 to 18%c), whereas the M form has a larger amount of proteins (24 to 41 %c) than the Y form (7 to 14%). Several properties of the glucan of P. brasiliensis were studied. Almost all of the glucan in the Y form was soluble in 1 N NaOH, was weakly positive in the periodic acid-Schiff reaction, was not hydrolyzed by snail digestive juice, and had a-glycosidic linkage. Glucans of the M form were divided into alkali-soluble (60 to 65%c) and alkali-insoluble (35 to 40%) types. The alkali-soluble glucan was similar to that of the Y form; the alkali-insoluble glucan was positive in the periodic acid- Schiff reaction and was hydrolyzed by snail digestive juice.

Paracoccidioides brasiliensis and Blastomyces MATERIALS AND METHODS dermatitidis exhibit thermal dimorphism: a yeast P. brasiliensis (strain 7193, Instituto Nacional de form (Y form) at 37 C and a mycelial form (M Tuberculosis, Caracas, Venezuela) and B. derma- form) at room temperature (23). The mecha- titidis (strain BD64, National Communicable Disease nisms by which these fungi change from one Center, Atlanta, Ga.) were used. The Y and M forms form to the other are not yet clear. Both forms of both fungi were obtained as previously described of P. brasiliensis have enzymes of glycolysis, the (18). The fungi were killed in 170 formaldehyde or hexose monophosphate shunt and the citric acid 2.5%G glutaraldehyde, washed with water, and kept in cycle, suggesting that they may utilize glucose a freezer until used. through the same metabolic pathways (18). Cell wall preparation. The frozen fungus was Morphological differences might be due to the crushed from 15 to 25 times with a French press (1.4 chemical composition of the cell wall of each to 2.1 tons per cm2) until almost disrupted. The crushed cells were suspended in approximately 10 form. volumes of water and treated in a sonic oscillator Although Blank (8), by means of X-ray diffrac- (20 kc) for 5 min. The crude cell walls were collected tion studies, demonstrated the presence of chitin by centrifugation at 1,500 X g for 10 min. The sonic in the Y and the M forms of both fungi, and treatment was repeated about 20 times until the super- Kanetsuna (17) found, also in both fungi, that natant solution became clear. When further purifi- glucosamine and glucose are the main constitu- cation was required, the cell walls were suspended in ents of the cell walls of the Y form, detailed in- 85% sucrose and centrifuged at 8,000 X g for 30 to formation on their composition is not yet avail- 60 min. The cell walls were obtained as a sediment, whereas whole cells and cytoplasmic materials passed able. To obtain such information, we studied the Y M P. to the supernatant liquid. After repeating this pro- cell wall compositon of the and forms of cedure several times, the cell walls were washed with brasiliensis, as well as some of the properties of water, dialyzed against several changes of water, and glucan. The cell wall composition of B. dermati- lyophilized. tidis is also described for purposes of comparison. During the preparation procedures, the samples were often examined under light and electron micro-

' Present address: Universidad de Los Andes, Centro de scopes and by the estimation of nucleotides (as Microscopia Electr6nica, Merida, Estado Merida. described below). Nine cell wall preparations of 1036 VOL. 97, 1969 CELL WALL OF PARACOCCIDIOIDES BRASILIENSIS 1037 both fungi were made separately to observe the for 30 min; commercial yeast ribonucleic acid was reproducibility of the methods. used as a reference. Preparation of glucan in P. brasiliensis. Cell walls For sugars, samples were hydrolyzed in sealed of the Y form in the amount of 3.3 g and a 1.0-g tubes with 1 N HCI at 110 C for 5 to 7 hr. A portion of amount of the M form were treated twice separately the hydrolysates was immediately used for reactions with ether and then incubated in 100 ml of 0.3% with anthrone (27) and orcinol (12), with the use of trypsin (EC 3.4.4.4), dissolved in 0.05 M tris(hydroxy- glucose and arabinose, respectively, as standards. methyl)-aminomethane-chloride buffer, pH 8.0, at HCI was removed from the remaining part of the 37 C for 2 hr. After being washed with water, the hydrolysates by repeated evaporations in vacuo over trypsin-treated cell walls were incubated in chitinase silica gel and NaOH pellets. Residues were dissolved (EC 3.2.1.14; 0.6 mg/ml) with 0.05 M acetate buffer in a small amount of water and passed through small (100 ml in the Y form, and 50 ml in the M form), amounts of Dowex-50 (H+ form) and Dowex-1 pH 5.0, at 37 C for 1 week. Ten drops of toluene (OH- form) with water as eluent. Desalted solutions was added to each reaction mixture to prevent bac- were concentrated by lyophilization and used for terial growth. The insoluble materials were collected paper chromatography. by centrifugation at 12,000 X g for 10 min. After For amino sugars and amino acids, samples were this treatment had been repeated two more times, hydrolyzed with 6 N HCI at 110 C for 16 hr. HCI was until amino sugars were no longer liberated, the removed by the above mentioned method, and the insoluble materials were washed with water, ethyl residues were dissolved in water and filtered. The alcohol, and ether, successively. The yield was 1.2 g filtrates were used for the determination of amino of the Y form and 0.47 g of the M form. sugars and amino acids. Amino sugars were de- Treatment of glucan with glucanases. The following termined by the method of Belcher et al. (7) with glucanases were used: glucoamylase (EC 3.2.1.3), glucosamine-HCI as a standard. Amino acids were a-amylase (EC 3.2.1.1), cellulase (EC 3.2.1.4), and analyzed with a JLC-3BC amino acid and nucleic snail digestive juice. Each sample (5 to 7 mg) was acid analyzer (Japan Electron Optics Laboratory Co. incubated with an enzyme (glucoamylase, cellulase, Ltd., Tokyo, Japan) according to the method of or snail digestive juice) in 1.2 ml of 0.05 M acetate Spackman et al. (29), with the use of Aminex A-4 buffer, pH 5.0, at 37 C for 0, 2, 5, 10, and 24 hr. In and A-5 resins (Bio-Rad Laboratories, Richmond, the case of a-amylase, 0.02 M potassium phosphate Calif.) for long and short columns, respectively. buffer, pH 6.9, with 0.0069 M NaCl was used instead Paper chromatography was performed on What- of acetate buffer. At the time indicated above, 0.2 man no. 1 filter paper, with the following solvents: ml of the reaction mixture was placed into 3 ml of (A) n-butyl alcohol-acetic acid-water (4:1:1, v/v), boiling water for 10 min to inactivate the enzymes. ascending; (B) phenol-water (4:1, w/w), ascending; The amount of reducing sugar was then determined by (C) n-butyl alcohol-pyridine-water (6:4:3, v/v), Somogyi's colorimetric method (28) with glucose as a descending; (D) 2 ,4-lutidine-water (65:35, v/v), standard. Glycogen, nigeran, laminarin, and cellulose ascending; (E) ethylacetate-pyridine-n-butyl alcohol- were used in all experiments to determine the sub- n-butyric acid-water (10:10:5:1:5, v/v), descending. strate specificity of the glucanases employed. When the For sugars, multiple development (three times) in enzyme solution had reducing power, it was dialyzed solvent C and two-dimensional chromatography overnight against water before usage. The amount of (first development in solvent B and second develop- enzyme used was that required to give maximal hy- ment in solvent D) were used. For amino sugars, drolysis of a suitable commercial glucan within 2 to solvents A and E were used. Alkaline silver nitrate 5 hr, except for cellulase which needed 24 hr. The (30) and the Elson-Morgan reaction (24) were used results were expressed as a percentage equivalent of for detection of sugars and amino sugars, respec- the glucose liberated from glucans at the 10th hr tively. (glucoamylase, a-amylase, and snail digestive juice) Infrared absorption spectra were taken in a Perkin- or at the 24th hr (cellulase). Elmer 621-Grating infrared spectrophotometer, by Solubility of glucan in dilute alkali. Samples use of the pressed KCI disc technique. suspended in I N NaOH were incubated with stirring, Chemicals. Cellulase (type I), a-amylase (type for 1 to 7 hr at room temperature or at 37 C. After II-A), and yeast ribonucleic acid were obtained from centrifugation at 18,000 X g for 20 min, the pre- the Sigma Chemical Co., St. Louis, Mo.; laminarin, cipitates were washed once with alkali and the from the Seaweed Research Institute, Inverex, supernatant liquids were combined and filtered. The Scotland; nigeran, from the Koch-Light Laboratories, amount of glucan in the filtrates and precipitates Ltd., Colnbrook, England; chitinase, from Calbio- was estimated (27). chem, Los Angeles, Calif.; Diazyme (glucoamylase), Total nitrogen and phosphorus from the Miles Chemical Co., Elkhart, Ind.; digestive Analytical methods. juice of Helix pomatia, from the Industrie Biologique were determined by the methods of Johnson (15) Gennevilliers, Seine, France. and Fiske and SubbaRow (13), respectively. Readily Frangaise, extractable lipids, bound lipids, and ash were de- RESULTS termined by the method of Bartnicki-Garcia and Nickerson (6). Nucleotides were estimated from the Table 1 shows the chemical composition of the ultraviolet spectra of the extracts obtained by treat- cell walls of P. brasiliensis and B. dermatitidis. ing the samples with 1 N perchloric acid at 80 C The chemical composition of the corresponding 1038 KANETSUNA ET AL. J. BACTERIOL. forms of the two fungi was similar. That the pre- (cysteine, tryptophan, etc.) destroyed during acid pared cell wall was practically free from cyto- hydrolysis were not included in Table 2, the M plasmic materials was confirmed by the very small form of both fungi had much larger amounts of amounts of nucleotides and pentoses obtained. proteins than the Y form. Molar ratios of amino This fact was reconfirmed with electron micros- acids differed somewhat, depending on the copy. No significant difference was observed in preparations. In general, however, the M form the lipid and hexose content. Whereas hexose had smaller amounts of lysine and histidine and and amino sugars were the main constituents of larger amounts of proline in relation to a fixed the Y form of both fungi, the M form had small amount of protein than did the Y form. amounts of amino sugar and large amounts of The glucan of the Y form, prepared by treat- amino acids. ing the cell wall with trypsin and chitinase, The sugar constituents of the Y form observed contained 98.5%-o glucose and 2.0% N-acetylglu- by paper chromatography were glucose, glucosa- cosamine. No significant amounts of amino acids mine, and a small amount of an unknown amino were found. Cell walls of the M form treated in sugar. The M form showed, in addition to the the same manner gave insoluble materials which above, small amounts of galactose and mannose. contained 68.3%c glucose, 3.5%c, N-acetylglucosa- Uronic acids were not detected in the naphtho- mine, and 30.0%'c amino acids. Since preliminary resorcinol reaction (14). treatments with Pronase and pepsin failed to In the two forms of P. brasiliensis, the glucosa- remove amino acids, these insoluble materials mine polymer could not be extracted from the were used to examine some properties of the cell walls with 1 N HCI (100 C, 15 min). On the glucans in the M form. other hand, after chitinase treatment the major- Table 3 shows some properties of the glucans ity of the liberated amino sugars were N-acetyl- in both forms of P. brasiliensis. Almost all of the glucosamine (solvent A). These results indicate glucan of the Y form was soluble in 1 N NaOH that chitin is the glucosamine polymer found in at room temperature. It showed high dextrorota- both forms of the fungi. tion and was weakly positive in the periodic acid- The amounts of amino acids were calculated Schiff (PAS) reaction (21). The alkali-soluble from the amino acids shown in Table 2. Although glucan, composed of 100% glucose, was precipi- the amounts of tyrosine, which was overlapped tated by neutralization of the extract. The infra- by a glucosamine peak, and of the amino acids red spectrum (absorption maxima: 815, 840, 925, TABLE 1. Chemical compositioni of the cell walls of Paracoccidioides brasilielnsis and Blastomyces dermatitidis

P. brasiliensis B. dermatitidis Yeast form NIycelia frm foIYeast Component form or Yeast Mycelial form Yeast Mycelial fr

I I~~or la 2 3 4 5 6 7 8 9

c/ C, %c 'IC ,Cl o WC No No Lipid Readily extractable.. 1.5 1.6 1.1 2.9 2.6 1.0 0.9 1.4 2.7 Bound...... I 9.5 6.7 9.6 6.2 7.8 3.8 4.6 8.2 6.2 Hexose as glucose...... 38.4 35.8 37.5 36.8 37.5 39.8 47.1 43.6 43.3 Pentose as arabinose <0.5 <0.5 <0.3 <0.6 <0.2 <0.6 <0.3 <0.5 <0.7 Amino sugar as N-acetyl- glucosamine ...... 43.4 37.2 40.0 7.4 12.6 15.0 48.0 9.2 18.5 Amino acidb ...... 10.1 13.6 9.8 40.8 35.3 35.0 7.1 30.0 23.6 Nucleotide as yeast ribonucleic acid...... '<0.4 <0.5 <0.1 <0.6 <0.1 <0.2 <0.2 <0.1 <0.1 Ash ...... 1.5 3.2 Total phosphorus ...... 0.14 0.17 0.18 0.40 0.61 0.56 0.14 0.23 0.48 Total nitrogen ...... 5.00 4.75 4.21 6.90 6.58 6.50 4.53 5.52 5.10 Sume 104.4 94.9 98.0 94.1 95.8 97.8 107.7 92.4 94.3

a Sample number. Samples 2 and 7 are taken from Kanetsuna (17). b Calculated from Table 2. c Lipid + hexose + amino sugar + amino acid + (ash). VOL . 97, 1969 CELL WALL OF PARACOCCIDIOIDES BRASILIENSIS 1039

1,010, 1,140, 1,350, 1,420, 1,630, 2,900, and 3,350 was soluble in alkali. The alkali-soluble glucan of cm-') showed a-linkage, 840 cm-' (5). Four of the M form had characteristics (precipitability by the glucanases tested did not hydrolyze the glu- neutralization, a-linkage in infrared spectrum, can of the Y form. weakly positive PAS reaction, and nonsuscepti- Glucans of the M form were separated by bility to the glucanases tested) similar to those of treatment with 1 N NaOH at room temperature the Y form, except for lower dextrorotation. for 2 hr. Approximately 60 to 65% of the glucan Approximately 35 to 40% of the M-form

TABLE 2. Amnilto acid compositiont of the cell walls of Paracoccidioides brasilienisis anid Blastomyces dermatitidisa

P. brasiliensis B. dermalilidis

Amino acidb Yeas i Yeast~form Mycelial form form Mycelial form 3 4 5 6 7 8 9

Lysine ...... 9.3 9.1 8.8 8.5 7.7 9.9 6.4 9.3 10.7 Histidine ...... 10.3 12.4 8.8 3.4 3.9 2.1 5.4 4.9 1.8 Arginine ...... 2.7 2.1 1.8 12.7 10.5 10.2 1.4 6.4 3.7 Aspartic acid...... 9.9 10.1 5.8 34.1 28.6 24.0 7.6 21.0 8.1 Threonine ...... 7.9 7.1 8.5 26.1 22.0 23.2 2.4 21.4 19.0 Serine ...... 6.6 7.8 7.7 23.6 23.4 28.8 3.2 17.9 11.8 Glutamic acid...... 9.9 10.1 8.7 38.1 35.6 36.4 5.0 29.1 16.6 Proline ...... 5.0 8.0 5.6 42.8 26.6 40.0 2.7 31.3 28.7 Glycine 12.8 12.8 8.5 53.8 39.5 44.6 4.8 63.0 43.2 Alanine ...... 7.9 11.2 5.1 28.8 29.6 21.9 3.3 16.6 18.0 Valine ...... 4.4 4.1 1.7 18.8 18.1 19.2 1.9 13.0 11.3 Methionine ...... 1.7 3.6 0.7 11.3 4.4 3.0 0.6 1.7 1.3 Isoleucine ...... 3.9 1.8 2.0 14.3 13.9 8.0 1.3 12.5 11.6 Leucine ...... 4.4 5.9 3.6 17.1 21.5 11.2 1.1 14.0 13.4 Phenylalanine ...... 4.5 3.5 2.1 7.5 9.7 11.9 1.2 6.1 7.3

Total (mg/100 mg of cell wall) ...... 10.1 13.6 9.8 40.8 35.3 35.0 7.1 30.0 23.6

a Expressed as micromoles of amino acid per 100 mg of cell wall, except for the total. I Tyrosine was overlapped by glucosamine. c Sample number.

TABLE 3. Some properties of glucants of Paracoccidioides brasiliensis

Mycelial form Propel ty (alkali-soluble)Yeast form Alkali-soluble Alkali-insoluble Percentage of fractionsa 96-98%o 60-65% 40-35% Solubility in hot water Not soluble Not soluble Not soluble [alD in 1 N NaOH +2510 (c, 0.593) +1950 (c, 0.570) Periodic acid-Schiff reaction Weakly positive Weakly positive Positive Infrared absorption spectrum 840 cm-' (a-linkage) 840 cm-' (a-linkage) Susceptibility to glucanasesb Glucoamylase 00%0% 2.1% a-Amylase 0% 0°% 0% Snail digestive juice ...... 0% 4.8% 81.7% Cellulase 0% 0% 15.0%

a Percentage of solubilized or nonsolubilized glucan after treatment with 1 N NaOH at room temperature for 2 hr. I Percentage of liberated glycosidic linkage estimated by increase of reducing power, with glucose as reference. 1040 KANETSUNA ET AL. J. BACTERIOL. glucan was insoluble in alkali. This alkali-in- forms indicate the existence of a-glycosidic link- soluble fraction (40.0% glucose, 5.9% N-acetyl- age. The failure of glucoamylase and a-amylase glucosamine, and 45.3% amino acids) was posi- in hydrolyzing glucans suggests that a-(1 -_4) tive in the PAS reaction, and was hydrolyzed by linkage is not the main linkage. Insolubility in snail digestive juice and cellulase. Since these hot water (16) and the weakly positive PAS reac- crude enzyme preparations could also hydrolyze tion suggest the existence of a glucan mainly a-glucans, the existence of A-linkage in the composed of a-(1 - 3) linkage. The infrared alkali-insoluble glucan was not determined. The spectra of the alkali-soluble glucans of both nature of the glycosidic linkage could not be forms were almost identical to that of a-1,3-glu- determined by infrared spectrum owing to the can of Aspergillus niger (4). The occurrence of impurity of the sample. a-1,3-glucan in fungal cell walls has been reported for Polyporus species (4, 25), A. niger DISCUSSION (16), Cryptococcus, and Schizosaccharomyces It is known that the cell walls of fungi contain (4). various types of polysaccharides, proteins, lipids, It is interesting to note that the M form has and inorganic substances (3), and that, depend- significant amounts of alkali-insoluble glucan(s); ing on environmental conditions, some fungi even after reflux with 1 N NaOH for 15 hr, show dimorphism (26). So far, however, it has approximately 25% of the glucan remained in been difficult to identify a common feature of the insoluble fraction. The alkali-insoluble glucan the cell wall of both the Y and M forms of di- was positive in the PAS reaction and was sus- morphic fungi, although the relationship between ceptible to snail digestive juice and cellulase, cell wall composition and morphology has been contrasting with the alkali-soluble glucan. Since reported in Mucor rouxii (6), Histoplasma cap- the enzyme preparations used could also hydro- sulatum (11, 20), and Candida albicans (10, 19). lyze a-glucans, the nature of the alkali-insoluble The present study revealed that the cell walls glucan is not yet clear. However, the coexistence of the Y form of P. brasiliensis and B. dermatitidis of a- and ,3-glucans in a fungus was reported by have larger amounts of glucosamine and smaller Bacon et al. (4). Furthermore, additional small amounts of amino acids than cell walls of the M amounts of galactose and mannose were observed form. This finding is in agreement with data re- in the cell walls of the M form. These results ported for the cell walls of H. capsulatum (11, 20). indicate the existence of structural differences of The insolubility of the glucosamine polymer in polysaccharides of the two forms, although there hot 1 N HCI and the liberation of N-acetylglu- was no significant difference in the total amount cosamine by chitinase indicate the presence of of neutral sugars. chitin and not of chitosan. Furthermore, the iso- Several attempts have been made to explain lated glucosamine polymer of the Y form of P. dimorphism, but they are too speculative (26). brasiliensis was identified as chitin by X-ray Nickerson (22, 23), proposed the formation of diffraction and infrared spectrum (Moreno et the M form from the Y form as the selective al., Arch. Biochem. Biophys., in press). We ob- inhibition of cell division without simultaneous served no significant difference in the chitin growth inhibition. However, this proposed content of the Y form of the two fungi, although mechanism does not always produce the M form Blank (8) found that the Y form of B. dermati- from the Y form, since there is a possible forma- tidis had three times more chitin than the Y tion of linked, or giant, Y forms. Furthermore, form of P. brasiliensis. the M form of P. brasiliensis and B. dermatitidis Paper chromatography evidenced a very small produces septae (9), suggesting that there is no amount of an unknown amino sugar in both inhibition of cell division. forms. This amino sugar corresponded to the Structural differences as well as rigidity of unusual amino sugar observed in the cell wall of polymers might be of importance in explaining Penicillium notatum (1). Recently, Applegarth dimorphism. Chitin is hard and could maintain and Bozoian (2) described this unusual amino the shape of fungi, but since it is a linear acetyl- sugar as an artifact of acid hydrolysis of N-acetyl- glucosamine polymer it might not be structurally glucosamine. different in the two forms. Although there is a The characteristics of the alkali-soluble glu- difference in the amino acid composition of the cans in both forms of P. brasiliensis differ from two forms, it is not clear whether fungal proteins those of f3-glucans, which are widely distributed are responsible for the rigidity of the cell walls. in fungal cell walls (3). The high dextrorotation However, the more efficient removal of chitin in alkali, and the absorption at 840 cm-' in infra- and proteins by chitinase and trypsin in the Y red spectra, of the alkali-soluble glucans of both form than in the M form of P. brasiliensis may VOL. 97, 1969 CELL WALL OF PARACOCCIDIOIDES BRASILIENSIS 1041

suggest that these components are differently 11. Domer, J. E., J. G. Hamilton, and J. C. Harkin. 1967. Com- arranged in the two forms. parative study of the cell walls of the yeastlike and mycelial phases of Histoplasma capsulatum. J. Bacteriol. 94:466-474. The glucans (or glucans plus proteins in the 12. Fernell, W. R., and H. K. King. 1953. The simultaneous de- M form) have enough rigidity to maintain the termination of pentose and hexose in mixtures of sugars. shape of fungi. After the cell walls of both forms Analyst 78:80-83. of P. brasiliensis were treated with trypsin and 13. Fiske, C. H., and Y. SubbaRow. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400. chitinase, the shape of both forms was still the 14. Hanson, S. W. F., G. T. Mills, and R. T. Williams. 1944. A same. 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