JOURNAL OF BACTERIOLOGY, Apr. 1972, p. 208-218Copyright 0 1972 American Society for Microbiology

Vol. 110, No. 1Printed in U.SA.

Biochemical Studies on the ThermalDimorphism of Paracoccidioides brasiliensislFUMINORI KANETSUNA, LUIS M. CARBONELL, ICHIRO AZUMA, AND YUICHI YAMAMURA

Center of Microbiology and Cell Biology, Instituto Venezolano de Investigaciones Cientificas, Apartado 1827,Caracas, Venezuela, and The Third Department of Internal Medicine, School of Medicine, Osaka University,

Osaka, Japan

Received for publication 5 January 1972

The biochemical and morphological changes of the yeastlike (Y) form to themycelial (M) form of Paracoccidioides brasiliensis were examined. The mainpolysaccharide of hexoses of the Y-form cell wall was a-glucan, whereas thepolysaccharides of the M-form cell wall were ,-glucan and galactomannan. Thea-glucan of the Y form contained mainly a-(1 - 3)-glycosidic linkage. The ,-glucan of the M form contained mainly ,B-(1 3)-glycosidic linkage with a fewbranches at C-6 position. The incorporation of "4C-glucose into the cell wallglucans showed that synthesis of a-glucan decreased rapidly after the tempera-ture of the culture was changed from 37 to 20 C. The synthesis of ,B-glucan wasaugmented at an early stage of the morphological change. The M-form cell wallcontained 12 times more disulfide linkage than the Y form. The cell-free ex-tracts of the whole cell of the Y form had five times more protein disulfide re-ductase activity than the M form, whereas extracts of the M form containedfive to eight times more f,-glucanase activity than the Y form. From these re-

sults, a hypothesis for the production of the M form from the Y form is pro-

posed.

Paracoccidioides brasiliensis shows thermaldimorphism: a yeastlike (Y) form at 37 C anda mycelial (M) form at 20 C (27). The morpho-logical changes of the fungus were studied ex-tensively by Carbonell et al. (11-14), using theelectron microscope. However, the biochemicalmechanism of the morphological changes ofthe fungus is not yet clear, although Nickersonproposed the formation of the M form fromthe Y form as a result of the selective inhibi-tion of cell division without simultaneousgrowth inhibition (27, 31).Both forms of P. brasiliensis contain en-

zymes of glycolysis, the hexose monophosphateshunt, and the citric acid cycle, suggestingthat they may utilize glucose through the samemetabolic pathways (20). The main cell wallconstituents of both forms are lipids, chitin,glucans, and proteins (23), and the main cellwall glucan of the Y form is a-glucan whereasthe M-form cell wall contains larger amountsof ,B-glucan (21). This change of cell wall glu-cans may play an important role in the di-morphism of the fungus. However, more de-

'This report was presented in part at the First PanAmerican Symposium on Paracoccidioidomycosis, Medel-lin, Colombia, 25-27 October 1971.

tailed information on the cell wall componentsand several enzymatic activities which mayconcem the cell wall are required for under-standing the dimorphism.

In this paper, chemical structure of a- andf,-glucans of the cell walls, the time course ofthe thermally induced change of glucans, andseveral enzymatic activities of cell-free ex-tracts of both forms are described. A hypoth-esis for the thermal dimorphism of P. brasi-liensis is also proposed.

MATERIALS AND METHODSThe Y and M forms of P. brasiliensis (strain 7193,

Instituto Nacional de Tuberculosis, Caracas) wereobtained as described previously (20). The cell wallsof both forms were prepared by the combined use ofa French press and a sonic oscillator as describedpreviously (21, 23).

Fractionation of cell walls. A schematic repre-sentation of the various treatments and agents ap-plied to the cell wall of both forms is shown in Fig.1.Y-form cell wall. The Y-form cell wall (4 g) was

suspended in 200 ml of 1 N NaOH and stirred for 1hr at 20 C. After centrifugation at 8,000 x g for 10min, the precipitate was treated with 100 ml of 1 NNaOH three times as described above. The alkali-insoluble residue was washed with water, ethyl alco-

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Yeastlike formCell wall (4.0 g)

1 N NaOH

IExtract

Neutralization

Insoluble residue

(Fr.Y-1; 1.97 g)

Precipitate

(Fr. Y-2; 1.51 g)

Extract

Neutralization

Mycelial formCell wall (5.0 g)

1 N NaOH

Insoluble residue

(Fr.M-1; 3.50 g)0.5 M Acetic acid (90C)

Supernatant

(Fr.M-3; 0.70 g)

Precipitate

(Fr. M-2; 0.016 g)

Insoluble residue

Chitinase

Insoluble residue

1 N NaOH

Insoluble residue

(Fr. M-6; 0.10 g)

Extract

Neutralization

Supernatant Precipitate(Fr. M-5; 0.18 g) (Fr. M-4; 0.81 g)

FIG. 1. Fractionation of the cell walls of the yeastlike and mycelial forms of Paracoccidioides brasiliensis.The yield of each fraction is given in parentheses.

hol, and ether, successively (fraction Y-1). From thecombined alkaline extracts, glucan was precipitatedby neutralization with acetic acid and collected bycentrifuging at 12,000 x g for 10 min. Purification ofthe glucan by precipitation from the alkaline solu-tion (50 ml) by neutralization with acetic acid was

repeated two more times. The precipitated glucanwas washed with water, ethyl alcohol, and ether,successively (fraction Y-2). The supernatant solu-tions obtained after removal of glucan from the neu-tralized alkaline extracts were concentrated by ly-ophilization, dialyzed against water, and again ly-ophilized (fraction Y-3).

M-form cell wall. The M-form cell wall (5 g) wastreated with 1 N NaOH as described above. From thecombined extracts, however, only 16 mg of glucan(fraction M-2) was precipitated by neutralizationwith acetic acid, leaving large amounts of polysac-charides in the supernatant solution. The superna-tant solutions were concentrated by lyophilization,dialyzed against water, and again lyophilized (frac-tion M-3). The alkali-insoluble residue (fraction M-1) was then extracted four times with 100 ml of 0.5 Macetic acid at 90 C for 6-hr periods (2, 25, 26), and asmall amount (about 18 mg) of polysaccharide wasfound in the dialyzed extracts. Since preliminary

Supernatant

(Fr.Y-3; 0.06 g)

Extract

Extract

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treatment of the acetic acid-treated cell wall with 1N NaOH (at 20 or 75 C) or with dimethyl sulfoxide(26) did not extract glucan, 2.65 g of the acetic acid-treated cell wall was treated with chitinase (1mg/ml) in 200 ml of 0.05 M acetate buffer, pH 5.0, at37 C for 1 week. Toluene (1 ml) was added to thereaction mixture to prevent bacterial growth. Thistreatment hydrolyzed about 90% of chitin in the cellwall. About 20% of glucan was also hydrolyzed byglucanases in the commercial chitinase preparation.After the treatment with chitinase, the insoluble res-idue (1.65 g) was treated three times with 50 ml of 1N NaOH at 20 C for 2-hr periods. From the com-bined extracts, glucan was precipitated by neutrali-zation with acetic acid and purified as describedabove (fraction M-4). The supernatant solutions ob-tained after removal of glucan from the neutralizedalkaline extracts were concentrated by lyophiliza-tion, dialyzed against water, and lyophilized (frac-tion M-5). The final alkali-insoluble residue waswashed with water, ethyl alcohol, and ether, succes-sively (fraction M-6).Methylation of glucans and analysis of methyl-

ated glucose. The methylation of glucans was per-formed essentially by the method of Sandford andConrad (32). Since the alkali-soluble a-glucan (frac-tion Y-2) of the Y form is insoluble in dimethyl sulf-oxide, 200 mg of the glucan of the Y form was sus-pended in 20 ml of dimethyl sulfoxide at 40 C undernitrogen gas. After the addition of 3 ml of methylsul-finyl anion prepared by the method of Sandford andConrad (32), the reaction mixture was incubatedwith continuous stirring for 5 to 7 hr at 40 C. Then,after the temperature was lowered to 20 C, 1 ml ofmethyl iodide was added dropwise, and the stirringwas continued overnight. The reaction mixture wasdialyzed against running tap water for 24 hr andevaporated until dry at 40 C under reduced pressurein a rotary evaporator. The above procedures wererepeated four times more, and finally the methylatedglucan was extracted with chloroform, and afterbeing washed with water the chloroform extract wasevaporated until dry. Yield, 140 mg; [a](5 = +2550(C, 0.33) in chloroform.The methylation of the alkali-soluble ,-glucan

(fraction M-4) (200 mg) of the M form was performedsimilarly, except that temperature was 20 C insteadof 40 C, because the glucan of the M form is solublein dimethyl sulfoxide. The procedures were repeatedthree times. Yield, 217 mg; [a]"5 0 (C, 1.0) inchloroform.

The methylated glucans of the Y and M forms didnot show a significant absorption band in the 3,500cm- I regions, indicating that most of the hydroxylgroup was substituted by the methoxyl group.To the methylated glucan (10 mg) we added 0.1

ml of 72% sulfuric acid, and the suspension was keptfor 1 hr at 4 C. Then, after the addition of 0.8 ml ofwater, hydrolysis was continued for 7 hr at 100 C.The reaction mixture was passed through a smallcolumn (6 by 0.6 cm) of Amberlite IR4B (carbonateform) to remove sulfonium ions and concentrated toabout 1 ml. The methylated glucose was reducedwith sodium borohydride (10 mg) at 20 C for 2 hr,and, after treatment with a small amount of Dowex

50 (H+ form), boric acid was removed by codistilla-tion with methyl alcohol. The dried residue wastreated with 2 ml of a mixture of acetic anhydrideand pyridine (1:1) at 100 C for 1 hr. The reactionmixture was diluted with water, concentrated untildry, and extracted with chloroform. The partiallymethylated glucitol acetates dissolved in chloroformwere analyzed on a glass column (200 by 0.4 cm)containing 3% of nitril silicone-polyester copolymer(ECNSS-M) on Chromosorb-W (100-120 mesh) at180 C at a gas flow rate of 60 ml of nitrogen per min,using a gas chromatograph GC-4BPF (ShimadzuSeisakusho Ltd., Kyoto, Japan) (8).The methylated glucan (10 mg) was also methano-

lyzed by treatment with 1 ml of 5% methanolic hy-drogen chloride in a sealed tube at 100 C for 24 hr.The methanolysate was neutralized with silver car-bonate, filtered, and concentrated. The syrupy res-idue dissolved in methyl alcohol was examined on aglass column (200 by 0.4 cm) containing 15% 1,4-butanediole succinate polyester on Shimalite-W (60-80 mesh) at 185 C at a gas flow rate of 75 ml of ni-trogen per min in a gas chromatograph GC-4BPF (1).To isolate tri-O-methyl-D-glucose, 40 mg of meth-

ylated a- or ,3-glucan was hydrolyzed with H2S04 asdescribed above. After removal of sulfonium ions,the concentrated hydrolysate was chromatographedon Toyo filter paper no. 51A (Toyo Kagaku Sangyo,Osaka, Japan) using ascending development for 24hr in n-butyl alcohol-ethyl alcohol-water (4:1:5, v/v,upper phase). The main spot (RF 0.78) was elutedwith 50% methyl alcohol, and the crystalline methyl-ated glucose was obtained by concentration. Yield:26 mg from a-glucan and 29 mg from f3-glucan.Incorporation of l 4C-glucose into glucans.

About 16 mg (dry weight) of the Y form was inocu-lated into each of several 500-ml Erlenmeyer flaskswhich contained 100 ml of the liquid medium com-posed of 2% glucose, 1% glycine, and 0.2% yeast ex-tract (Difco) (20). All the flasks were placed on a re-ciprocating shaker (3-cm amplitude and 120 strokesper min) at 37 C. After 24 hr of culture, uniformlylabeled "4C-glucose was added to four flasks (38.8MCi per flask) and again after 24 hr of culture at 37C; 5 ml of formaldehyde (35%) was added to theflasks containing "4C-glucose to kill the fungus. Theremaining flasks were transferred to another shaker(3-cm amplitude and 80 strokes per min), and cul-turing was continued at 20 C. At 48, 72, 120, 192,and 288 hr after inoculation, "4C-glucose was addedto four flasks, and culture was continued for 24 hr at20 C as described above.The fungus was collected by centrifugation,

washed with water thoroughly, lyophilized, andweighed. The dried fungus was treated with 30 ml ofether five times. Carrier a-glucan (30 mg) of the Yform was added to the defatted fungus, and treat-ment of the fungus with 20 ml of 1 N NaOH at 20 Cfor 2 hr was repeated three times. The alkali extractswere neutralized with acetic acid, and the a-glucanwas collected by centrifugation. The a-glucan waspurified by reprecipitation from alkaline solution (10ml) by neutralization, followed by washing with wa-ter, ethyl alcohol, and ether, successively. The a-glucan was hydrolyzed with 1 ml of 1 N HCl at 105 C

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for 5 hr. After removal of HCI by repeated evapora-tions in vacuo over NaOH pellets, the residues weredissolved in 1 ml of water, and 0.5 ml of the hydroly-sate was used to determine radioactivity in the a-glucan fraction. The remaining hydrolysate was usedfor paper chromatography.The alkali-insoluble residue of the fungus was

washed with water and treated with Diazyme (2 mgper ml) in 30 ml of 0.05 M acetate buffer, pH 5.0, at37 C for 7 hr to remove any remaining glycogen. Theinsoluble residue was washed with water (30 ml) fivetimes and incubated with a fB-1, 3-D-glucanase prepa-ration (2.4 mg of protein) in 10 ml of 0.05 M acetatebuffer, pH 5.0, at 37 C for 24 hr to solubilize ,-glucan. Five drops of toluene were added to the reac-tion mixture to prevent bacterial growth. The super-natant solution was obtained by centrifugation at12,000 x g for 10 min, and the precipitate waswashed with 10 ml of water two times. The superna-tant fractions and washings were combined and fil-tered. The filtrate was concentrated to 1 ml by ly-ophilization, and 0.5 ml was used to determine ra-dioactivity in the j-glucan fraction.The radioactivity of the a- and jB-glucan fractions

(0.5 ml) was determined in a Packard Tri-Carbliquid scintillation spectrometer, model 3320, with10 ml of solvent [10 g of 2, 5-diphenyloxazole (PPO)and 30 mg of p-bis-2-(5-phenyloxazolyl)-benzene(POPOP) per liter of p-dioxanel. The identificationof a radioactive compound such as glucose was per-formed by paper chromatography. After 24 hr of de-scending development in n-butyl alcohol-pyridine-water (30:20:15), the paper was cut into 1-cm seg-ments, and radioactivity was determined in 5 ml ofthe solvent (4 g of PPO and 50 mg of POPOP perliter of toluene) (36).

Disulfide linkage of the cell walls. The cellwalls (10 to 20 mg) were suspended in 1 ml of 8 Murea and incubated at 37 C for 1.5, 4, and 5 hr withor without 50 mg of sodium borohydride. After thedestruction of borohydride by dropwise addition of 1N HCI, the resulting sulfhydryl groups in the cellwalls were determined by the method of Ellman (16)as follows. To the slightly acidified solution (2.5 ml),1.0 ml of 0.2 M sodium phosphate buffer (pH 8) and0.05 ml of 5,5'-dithiobis(2-nitro-benzoic acid)(DTNB) reagent (4 mg of DTNB per ml of 0.05 Mphosphate buffer, pH 7) were added. After centrifu-gation at 12,000 x g for 20 min, the optical densityof the clear supernatant solution was determined at412 nm, and extinction coefficient e M = 13,600 wasused to estimate the sulfhydryl group.The cystine-cysteine content of the cell walls was

also determined by conversion of cystine and cys-teine to cysteic acid by dimethyl sulfoxide, followedby automatic amino acid analysis, according to themethod of Spencer and Wold (34).

Preparation of cell-free extracts and enzymeassays. Whole cells of the Y and M forms werewashed with 0.05 M potassium phosphate buffer, pH7.0, and ground with about two volumes of glasspowder (5 gm, Heat-Systems Company, Nelville,N.Y.) for 10 min. After suspension of the resultingpaste in 0.05 M phosphate buffer, pH 7.0, cell-freeextracts were obtained by centrifugation at 500 x g

for 10 min.Protein disulfide reductase (EC 1.6.4.4) activity

was determined as follows. Ammonium sulfate (13.3g) was added to the cell-free extract (25 ml), and theprecipitate was collected by centrifugation at 12,000x g for 20 min. The precipitate was dissolved in 5ml of water and dialyzed against 2 liters of 0.005 Mphosphate buffer, pH 7, overnight. The reaction mix-ture contained 3 mg of ovalbumin, a dialyzed en-zyme preparation (5-9 mg of protein), and 0.4 Mmoleof reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleo-tide phosphate (NADPH) in a total volume of 2 mlof 0.05 M phosphate buffer, pH 7.0, in a Thunbergtube. After evacuation, the reaction mixture wasincubated at 37 C for 1 hr, and 1.0 ml of 0.2 M so-dium phosphate buffer, pH 8.0, and 0.05 ml ofDTNB reagent were added to the tube. After centrif-ugation at 12,000 x g for 20 min, the resulting sulf-hydryl group was estimated from the optical densityat 412 nm of a clear supernatant solution as de-scribed above. The tubes containing the heated en-zyme preparation (100 C, 10 min) were used as con-trol.

Glucanase activities were determined as follows.The cell-free extracts were dialyzed overnightagainst 2 liters of water. The reaction mixture con-tained a substrate (5-6 mg) and an extract (3-5 mgof protein) in a total volume of 3.4 ml of 0.05 M ace-tate buffer, pH 5.0, incubated at 37 C for 0, 3, and 5hr. As substrates, laminarin and the ,B-glucan of theM form were used for ,B-glucanase activity and thea-glucan of the Y form was used for a-glucanase ac-tivity. At the indicated time, 1.0 ml of the reactionmixture was put into 3 ml of water, heated at 100 Cfor 10 min, and filtered. The reducing power of thefiltrates was determined by Somogyi's method (33),with glucose used as a reference. The reaction mix-ture without substrate was used as control.

Chitinase activity was estimated by a proceduresimilar to that described for glucanases, except thatchitin was used instead of glucans. Incubation wascontinued for 10 and 24 hr, and the amino sugar inthe filtrates was estimated (7).

Analytical procedures. Hexoses, amino sugars,amino acids, total nitrogen, and phosphorus wereestimated as described previously (21, 23). Themolar ratio of sugars was determined by the methodof Wilson (37). Protein was determined by themethod of Lowry et al. (24), using bovine serum al-bumin as standard. 6-1,3-D-Glucanase [,B-1,3(4)-glucan glucanohydrolase, EC 3.2.1.6] of Basidiomy-cete QM 806 was prepared as described previously(21). One milligram of the enzyme preparation hy-drolyzed 25 mg of laminarin per hr under the condi-tions described before. Digestion of glucans withsnail digestive juice was performed as described pre-viously (23), and the liberated glucose was estimatedby Somogyi's method (33) or by glucose oxidase.

Materials. ECNSS-M on Chromosob-W and 1,4-butanediole succinate polyester on Shimalite-Wwere obtained from Shimadzu Seisakusho Ltd.,Kyoto, Japan; laminarin (ex Laminaria hyperborea),from Koch-Light Laboratories, Ltd., England;DTNB, from K & K Laboratories, Inc., Plainview,

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N.Y.; uniformly labeled "4C-D-glucose, from Interna-tional Chemical and Nuclear Corp., Irvine, Calif.;chitinase (EC 3.2.1.14), from Calbiochem, Los Ange-les, Calif.; digestive juice of Helix pomatia, fromIndustrie Biologique Franqaise, Gennevilliers,France; Diazyme [glucoamylase (EC 3.2.1.3)], fromMiles Chemical Co., Elkhart, Ind.; glucose oxidase(EC 1.1.3.4) and ovalbumin (grade V), from SigmaChemical Co., St. Louis, Mo.

RESULTS

Fractionation of cell walls. The chemicalcomposition of the cell wall preparations,which is similar to the cell wall compositionreported previously (23), is shown in Table 1.On paper chromatograms, the Y-form cell wallhydrolysate revealed only glucose, whereas theM form contained glucose, galactose, andmannose in a molar ratio of 1: 0.3:0.6. Thechemical composition of the various fractionsof the cell walls is also shown in Table 1. Al-most all of the glucan which was extractedwith alkali from the Y-form cell wall was pre-

cipitated by neutralization of the extracts,leaving very small amounts of polysaccharidecomposed mainly of galactose and mannose(molar ratios, glucose: galactose: mannose = 1:12.3:16.5) in the supernatant solution (fractionY-3). About 14% of the cell wall glucans of theY form remained in the alkali-insoluble res-

idue (fraction Y-1).Treatment of the M-form cell wall with al-

kali extracted a small amount of glucan whichwas obtained as an alkali-soluble glucan of theM form. This glucan (fraction M-2) was notanalyzed further. The polysaccharide com-

posed mainly of galactose and mannose(glucose: galactose: mannose = 1:9.4: 19.8) was

found in the supernatant solution (fraction M-3) after neutralization of the alkali extracts.Sevag's method (35) was applied repeatedlv

(10 times) to remove proteins from the super-

natant solution (680 mg of dry matter con-taining 427 mg of hexose as mannose), butonly 38.2% of polysaccharide was recovered inan aqueous phase. However, since the aqueousphase still contained proteins or peptides, esti-mated to be as much as 14% from the nitrogencontent (2.31%), the polysaccharide fraction(160 mg of hexose as mannose in 20 ml of 0.01N HCl) was passed through a Dowex 50 column(2.2 by 13 cm, H+ form in water) and the non-

adsorbed fraction was collected. After neutrali-zation with NaOH, the nonadsorbed fractionwas concentrated by lyophilization, dialyzedagainst water, and again lyophilized. This pu-rified polysaccharide fraction (144 mg, [a]D =

+700 in water) contained 97.2% hexose as man-nose determined by the anthrone reaction,0.17% nitrogen, and 0.21% phosphorus. Themolar ratio of glucose, galactose, and mannosewas 1:8.5:20.6. The polysaccharide was pre-cipitated by Fehling's solution (19), and themolar ratio of glucose, galactose, and mannoseof the precipitate ([a] 5 = +750 in water) was0: 1: 2.2, which strongly indicates that the poly-saccharide is a galactomannan.Treatment of the alkali-insoluble residue

(fraction M-1) of the M-form cell wall with ace-tic acid extracted small amounts (about 18 mg)of polysaccharides containing glucose, galac-tose, and mannose in a molar ratio of 1:1.9:3.2. Subsequent treatment of the acetic acid-treated cell wall with chitinase hydrolyzed a

part of the glucan. After chitinase treatment,glucan (fraction M-4) was easily extracted withalkali at 20 C and precipitated by neutraliza-tion, leaving a small amount of polysaccharide(glucose: galactose: mannose = 1:0.4:0.8) inthe supernatant solution (fraction M-5). Thefinal alkali-insoluble residue (fraction M-6)contained a small amount of glucan. The re-

covery of polysaccharides was about 70% in the

TABLE 1. Chemical composition of various fractions of the cell walls of yeastlike and mycelial forms ofParacoccidioides brasiliensis

Fraction

Component Yeastlike form Mycelial form

Cell Y-i Y-2 y-3 Cell M-1 M-3 M-4 M-5 M-6wall wall

Total phosphorus (%) ... 0.15 0.11 0.00 0.10 0.47 0.10 0.11 0.02 0.16 0.21Total nitrogen (%) 4.......25 5.25 0.00 6.10 5.21 5.10 4.05 0.06 5.54 7.03Hexosea (%) . 4............4. 13.4 100.1 29.8 37.5 42.8 35.1 99.0 22.7 1.2Amino sugara (%) ....... 32.2 70.8 0.0 1.0 18.0 25.1 0.5 0.3 0.8 74.5Amino acida(%).......... 3.0 0.0 31.7 20.6 18.2 21.4 0.6 30.2 8.3

aThe amounts of hexose, amino sugar, and amino acids are estimated as glucose, glucosamine hydro-chloride, and alanine, respectively.

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case of the M form, contrasting with 101% inthe Y form. This may be due to the partialhydrolysis of polysaccharides in a step of theacetic acid treatment.

These results suggest that (i) the f,-1, 6-glucan which can be extracted with hot aceticacid (25) does not exist in an appreciableamount in the M form, and (ii) the solubility ofthe M-form glucan in alkali depends on theprevious treatment of the cell wall.Structure of glucans. Table 1 shows the

chemical composition of the main cell wallglucans (fractions Y-2 and M-4) which wereused for the methylation experiments. Thealkali-soluble glucan (fraction Y-2) of the Yform showed high dextrorotation ( [a]2 =

+ 2330 in 1 N NaOH) and might be pure a-

glucan since the snail digestive juice and thef3-1, 3-glucanase preparation did not liberateglucose from the glucan. The infrared spec-

trum of the Y-form glucan was identical to thatof the a-1,3-glucan (21). The M-form glucan(fraction M-4) was also practically pure f3-glu-can, as seen from the following facts. The spe-

cific optical rotation of the glucan was low([a]D5 = +7O in 1 N NaOH), and its infraredspectrum showed an absorption band at 885cm-', due to f,-glycosidic linkages, and no

adsorption bands at 840 and 815 cm-' whichdo appear in a-glucan (21). Snail digestivejuice liberated 96% of glucose estimated bySomogyi's method. ,B-1,3-Glucanase also solu-bilized the M-form glucan completely, al-though only 48% of hydrolyzed glucan was esti-mated as free glucose by glucose oxidase.The a-glucan of the Y form mainly contains

(1 - 3)-glycosidic linkage (Fig. 2, Table 2).There is also a small amount of (1 4)- or (1- 6)-linkage as shown by peak III in Fig. 2.Due to the small amount of peak III, furtheranalysis was not performed. Although the pos-sibility of an artifact of incomplete methyla-tion or demethylation during hydrolysis is notexcluded, there may be few branches at the C-2, C-3, or C-6 position.

,W-Glucan of the M form contains mainly (1- 3)-glycosidic linkage, and there are a fewbranches at the C-6 position. Since partial acidhydrolysis of ,B-glucan did not show the exist-ence of cellobiose (21), peak III in Fig. 2 mayshow the existence of a small amount of (16)-linkage in the f3-glucan.To eliminate the possibility of the existence

of 1,2, 5-tri-O-acetyl-3, 4,6-tri-0-methyl glu-citol in peak II, which is derived from (1 2)-

glycosidic linkage, methyl glucosides were alsoanalyzed by gas-liquid chromatography. Twomain peaks were obtained corresponding to a-

and f,-methyl glucosides of 2,4,6-tri-O-

methyl-D-glucose. There were no peaks ofmethyl glucosides of 3,4,6-tri-O-methyl glu-cose, indicating that there is no appreciableamount of 3,4,6-tri-O-methyl glucose in thehydrolysates of methylated a- and ,B-glucans.

Further identification of the (1 - 3)-glyco-sidic linkage in the a- and f3-glucans was per-formed by the isolation of 2,4,6-tri-O-methylglucose by preparative paper chromatography.The resulting crystalline methyl glucose frommethylated a- and ,3-glucans showed the sameproperties: melting point and mixed meltingpoint, 115 to 117 C; [a] 25 = +91° (5 min)+ 76° (1 day) (C, 0.575 in water) in the a-glucan; and [a]25 = +93O (5 min)- +75° (1day) (C, 0.605 in water) in the ,B-glucan. Aftertreatment of the tri-O-methyl glucose (5 mg)with 40 mg of aniline in 0.5 ml of ethyl alcoholfor 4 hr in a boiling water bath, the crystallineaniline derivative (N-phenyl-2,4,6-0-methyl-D-glucosylamine) was obtained. It had a melt-ing point of 164 to 165 C.

I n II IV V VI

0 5 10 IS5

FIG. 2. Gas-liquid chromatography of the methyl-ated glucans of cell walls of the yeastlike and my-celial forms of Paracoccidioides brasiliensis. Solidline, a-glucan of yeastlike form; dotted line, ,3-glucan of mycelial form. (1) 1,5-di-0-acetyl-2,3,4,6-tetra-0-methyl- D-glucitol; (II) 1 ,3,5-tri-0-ace-tyl-2,4,6-tri- 0-methyl- D-glucitol; (III) 1 ,5,6-tri-0-acetyl-2,3,4-tri-0-methyl- D-glucitol or 1,4,5-tri-O-acetyl-2, 3, 6-tri-0-methyl- -glucitol; (IV) 1,3,4, 5-tetra-0-acetyl-2, 6-di-0-methyl-D-glucitol (?); (V)1 , 2, 3, 5-tetra-0-acetyl-4, 6-di-0-methyl-D-glucitol(?); (VI) 1,3,5,6-tetra-0-acetyl-2,4-di-0-methyl-D-glucitol.

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TABLE 2. Hydrolysis products from methylated a-and ,8-glucans of Paracoccidioides brasiliensis

Molar ratios

Peak" Methyl glucose a- tGlu- Glu-can can

I 2,3,4,6-tetra-O-methyl- 1.0 1.0D-glucose

II 2,4, 6-tri-O-methyl- 105.9 30.3D-glucose

III 2,3,6- or 2,3,4-tri-O-methyl- 6.2 1.9D-glucose

IV 2,6-di-O-methyl-D-glucose (?) 0.8 0V 4,6-di-O-methyl-D-glucose (?) 0.6 0VI 2, 4-di-O-methyl-D-glucose 0.8 0.9a From Fig. 2.

f,-Glucan in the Y-form cell wall. Thetreatment of the Y-form cell wall with snaildigestive juice or j3-1, 3-glucanase liberatedabout 10% of total glucan, suggesting the exist-ence of about 4% ,8-glucan in the Y-form cellwall. The alkali-insoluble residue of the Y-form cell wall contained 13.4% glucose; due tothis fact about 14.5% of the cell wall glucanswas not extracted with alkali, and this alkali-insoluble glucan was susceptible to snail diges-tive juice and fl-1,3-glucanase. These resultssuggest that there may be about 4 to 5% ,8-glucan in the Y-form cell wall.Incorporation of 14C-D-glucose into glu-

cans. The results of the incorporation of "4C-glucose into cell wall glucans showed the timecourse of the change of the cell wall glucanswith thermally induced morphological change(Fig. 3). After changing the cultivation temper-ature from 37 to 20 C, synthesis of a-glucandecreased rapidly while the synthesis of ,3-glucan was augmented at an early stage of themorphological change. The ratio of the ra-dioactivities of the a-glucan fraction to the ,B-glucan fraction in the Y form was 7.2. Thisratio dropped to 0.52, 0.25, and 0.08 after2, 4, and 7 days, respectively, of conversionfrom the Y form to the M form. At 20 C ,B-glucan was synthesized predominantly.

Disulfide linkage in the cell walls. Disul-fide linkage in the cell walls was estimated bytwo methods. The treatment of cell walls withborohydride, followed by the determination ofthe produced sulfhydryl group, showed thatthe Y- and M-form cell walls contained 0.29and 1.92 ,moles of sulfhydryl group per 100mg, respectively. The nontreated cell walls ofboth forms did not show the existence of freesulfhydryl groups, indicating that all the sulf-hydryl groups formed disulfide linkages in thecell wall preparations.

On the other hand, the cystine-cysteine con-tent of the Y- and M-form cell walls, whichwas determined by the conversion of cystineand cysteine to cysteic acid by dimethyl sulf-oxide followed by amino acid analysis, was0.25 and 3.40 umoles of half-cystine per 100mg, respectively. The discrepancy in theamount of the sulfhydryl group of the M formbetween the two methods may result from theincomplete reduction of the disulfide linkageby sodium borohydride due to the more com-pact nature of the M-form cell wall, althoughlonger treatment of the cell wall with borohy-dride did not increase significantly the amountof the sulfhydryl group.

ZNE-CQ%'ZI

'10

:3Chc:3

LA..

0

I:~

0

Time (days)

FIG. 3. Time course of the change of incorporationof 'IC-glucose into a- and j,-glucans of Paracoc-cidioides brasiliensis due to shift of the temperatureof culture from 37 to 20 C. At the time indicated byarrows, "'C-glucose was added to culture mediumand incubated for 24 hr. The amount of the fungusgrown during 24 hr was calculated from the growthcurve. Symbols: 0, growth curve; 0, counts per min-ute of a-glucan fraction per milligram of fungusgrown during 24 hr; A, counts per minute of j,-glucan fraction per milligram of fungus grown during24 hr; x, ratio of radioactivities of a-glucan fractionto pl-glucan fraction.

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Enzymatic activities of cell-free extracts.Protein disulfide reductase activity was muchhigher in the Y form. When NADPH was usedas a coenzyme, 27.1 and 4.9 nmoles of the sulf-hydryl group were produced per hr from disul-fide linkages by 1 mg of protein of the cell-freeextracts of the Y- and M-forms, respectively.NADH had a lower effect: 18.6 nmoles in theY form and 3.9 nmoles in the M form. Withoutcoenzyme, the Y and M forms produced 6.9and 1.6 nmoles of the sulfhydryl group, respec-tively.

f-Glucanase activity was much higher in theM form than in the Y form. With laminarinused as substrate, the Y and M forms liberated34 and 277 nmoles of glucose equivalent permg per hr, respectively. When the f-glucan ofthe M form was used as substrate, only 7.8 and39.4 nmoles of glucose equivalent were liber-ated per mg per hr by the Y and M forms, re-spectively.

Activities of a-glucanase and chitinase werenot found in either form.

DISCUSSIONSeveral attempts have been made to explain

dimorphism (31). For example, Nickerson pro-posed the formation of the M form from the Yform as the selective inhibition of cell divisionwithout simultaneous growth inhibition (27).However, it seems to be difficult to learn thedetailed mechanism of the morphologicalchanges of dimorphic fungi from the aboveassumption.

In the case of P. brasiliensis, the moststriking change in thermal dimorphism is thechange of the glycosidic linkage in cell wallglucans. The main cell wall glucan of the Yform has long chains of a-(l - 3)-linked glu-cose, as suggested from the high ratios of2,4, 6-tri-O-methyl glucose to 2,3,4, 6-tetra-O-methyl glucose and dimethyl glucose, in con-trast to the a-1,3-glucan of Polyporus betu-linus which has 12 moles of 2,4,6-tri-O-methylglucose and 4 moles of 4,6-di-O-methyl glu-cose per mole of tetra-O-methyl glucose (15).The a-glucan of the Y form contains also smallamounts of (1 - 4)- or (1 - 6)-glycosidiclinkage. The existence of an a-(1 - 4) linkagein a-1, 3-glucan of Aspergillus niger has beenreported (18, 29).The occurrence of a--1, 3-glucan has been

demonstrated in Aspergillus (18, 29), Schizo-saccharomyces (3), Polyporus (3, 15), Crypto-coccus (3), and Blastomyces (22), and a-1,3-glucan may be distributed more widely infungal cell walls than has been suspected.The fi-glucan of the M-form cell wall con-

tains mainly 03-1,3-glycosidic linkage, and

there are a few branches at the C-6 position.Small amounts of the (1 - 6)-glycosidiclinkage were also found in f3-glucan. The f3-glucan used in the present study may be some-what degraded by the acetic acid treatment,since the f3-glucan is weakly positive in theperiodic acid-Schiff reaction, in contrast to p-glucans which are isolated from chitinase-treated M-form cell walls without acetic acidtreatment and are positive in the periodicacid-Schiff reaction, as described in a previousreport (21). A 0-1,3- and ,8-1,6-linked glucanis distributed widely in fungal cell walls (5).Recently, Bacon et al. (2) studied in detail thesolubility in alkali of cell wall p-glucan of Sac-charomyces cerevisiae. They found that var-ious chemical, physical, and enzymatic treat-ments of the cell wall affected the solubility ofglucans in alkali. In the case of the M form ofP. brasiliensis, without previous treatmentwith chitinase, no appreciable amount ofglucan is extracted with alkali. However, afterchitinase treatment a part of f,-glucan is easilyextracted with 1 N NaOH at 20 C. These re-sults indicate that the solubility of f3-glucandepends on the previous treatment of the cellwalls. The nonreproducible results of alkali-soluble and alkali-insoluble glucans of the Mform reported previously (21, 23) may be ex-plained partially by these facts. In contrastwith previous results (21, 23) in the presentstudy, almost all f3-glucan of the M form isextracted with alkali. This may be due to theeffect of the acetic acid treatment of the cellwall as reported for the Saccharomyces cellwall glucans (2).

It may be confusing to divide j3-glucans intoalkali-soluble and alkali-insoluble glucanswhich were described in previous papers (21,23). Therefore, we will use only the term f3-glucan hereafter.

In previous papers (21, 23), almost all theglucan of the Y-form cell wall was identified asa-glucan. This may have been due to hydrol-ysis of ,B-glucan in the Y-form cell wall by 3-glucanases in the commercial chitinase prepa-ration which was used before alkali treatmentof the cell wall. The isolation and identifica-tion of fp-glucan of the Y form were not con-tinued because chitinase preparation free of ,B-glucanases was not available. However, suscep-tibility of the glucan in the alkali-insolubleresidue of the Y-form cell wall to snail diges-tive juice and f3-1, 3-glucanase strongly sug-gests the existence of about 4 to 5% ,B-glucanin the Y-form cell wall, because the a-glucan isnot hydrolyzed by these enzyme preparations(23).

The localization and function of ,B-glucan in

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the Y-form cell wall is unknown. However, it isinteresting to note that the cell walls of Sac-charomyces contain about 1 to 2% chitin lo-cated at budding sites (4, 10) and that the Y-form cell wall of Blastomyces dermatitidiscontains about 2 to 3% ,8-glucan (22). We mayassume that f3-glucan in the Y-form cell wallsof P. brasiliensis and B. dermatitidis has a re-lationship with budding as does chitin in Sac-charomyces.The incorporation of "4C-glucose into glu-

cans shows that the synthesis of a-glucan de-creases rapidly when the fungus is transferedfrom 37 to 20 C, accompanying the concomi-tant increase of the synthesis of ,3-glucan. Asfor the synthesis of a-glucan in the M-formcell wall, the results are not constant. Amongfive preparations of the M-form cell wall, threepreparations did not contain any appreciableamount of a-glucan, and in the other two prep-arations only about 17% (21) and 60% (23), re-spectively, of cell wall glucans were of the a-type. In the case of B. dermatitidis, about 60%of the glucans of the M form are a-type (22).The factors responsible for the amount of a-glucan in the M-form cell wall are unknown.Since the existence of a-glucan is not neces-sary for the production of the M form, the cellwall of hyphae may not contain a-glucan;therefore some segments may have a differentcomposition. For example, the occurrence ofchlamydospores (intercalary or terminal) (13)or arthrospores (30) may be one of these fac-tors, because the cell wall of a chlamydosporeis much thicker than that of hyphae, sug-gesting a different chemical composition be-tween them.The existence of galactose and mannose in

the M-form cell wall was reported previously,although these sugars are not detected in theY-form cell wall (23). The present study showsthe existence of very small amounts of galac-tose and mannose, estimated to be about 1 to2% of the amount of glucose, in the Y form cellwall also. Such a small amount of galactoseand mannose could not be detected on paperchromatograms. The polysaccharide composedmainly of galactose and mannose is extractedwith alkali and is soluble in water. The poly-saccharide may be a galactomannan, since theprecipitation of the polysaccharide with Fehl-ing's solution does not change significantly themolar ratio of galactose to mannose. The gal-actomannan may be important for the inter-pretation of the immunological properties ofthe fungus as demonstrated in other fungi (17).The M-form cell wall has more of a disulfide

linkage than the Y form. Although the oxida-tion of the sulfhydryl group must occur artifi-

cially during the preparation of the cell walls,we assume that the M-form cell wall containsmuch more protein and disulfide linkage thanthe Y-form cell wall. This assumption mayexplain the intimate coexistence of proteinswith f,-glucan in the M-form cell wall as re-ported previously (21, 23).As for the several enzymatic activities con-

cemed with the cell wall, the ones pertainingto the cell wall itself must be determined.However, since at present we have no reliablemethods for the determination of cell wallenzymes of P. brasiliensis, the cell-free ex-tracts were used in the determination of pro-tein disulfide reductase, glucanases, and chiti-nase. We assume that the enzymes in the cell-free extracts may have some interaction withthe cell wall components.

Nickerson et al. (28, 31) demonstrated theimportant role played by protein disulfidereductase as a division enzyme in the di-morphism of Candida albicans; i.e., higher ac-tivity in the normal yeast than in the division-less filamentous mutant. In a strain of S. cer-evisiae which shows ovoid and elongated formsdepending on the culture conditions, similarresults have been reported (9). Since it is diffi-cult to use cell wall proteins as a substrate inthe determination of protein disulfide reduc-tase, ovalbumin was used in the present studyas reported in the determination of the en-zyme activity of bakers' yeast (9). The cell-freeextract of the Y form had a much higher ac-tivity than that of the M form in P. brasilien-sis, as in the case of C. albicans (28) and S.cerevisiae (9).From the above-mentioned data we propose

a hypothesis for the thermal dimorphism of P.brasiliensis as shown in Fig. 4. This hypothesisis based on the assumption that a sphericalform is produced by the simultaneous syn-thesis of the entire cell wall and that a cylin-drical form is produced by the synthesis of theapical region only, as demonstrated in the caseof Mucor (6). The Y-form cell wall contains a-1, 3-glucan in the outer layer, chitin in theinner layer (12), and small amounts of ,8-glu-can, proteins, and lipids. Although we have nodata on the localization of ,B-glucan, we as-sume that the j3-glucan may exist as islets inthe cell wall as does chitin in the cell wall of S.cerevisiae (4, 10). By some mechanism, there isa loss of rigidity around the ,B-glucan, and theweakened part is blown out as a bud. f3-Glu-canase and protein disulfide reductase mayplay a role in the formation of the bud. At 37C, at the budding site and also in all parts ofthe cell wall of the daughter cell, a-glucan andchitin are synthesized much more actively

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DIMORPHISM IN P. BRASILIENSIS

Chitin= Proteins

#- Glucon-- a- GlucOn

FIG. 4. Proposed hypothesis for the productionbrasiliensis. Explanation is given in the text.

than j3-glucan, resulting in the formation of aspherical form. Proteins may have no rigiditydue to the small amount of disulfide linkageand also due to the active protein disulfidereductase. The chitin strengthens the producedspherical form.On the other hand, at 20 C, the synthesis of

a-glucan decreases at the budding sites and ,B-glucan fibers grow continuously by the linkageof the glucose unit, one by one, to the nonre-ducing terminal of ,-glucan, forming an apicalgrowth. Proteins may have some degree of ri-gidity because of the high amount of disulfidelinkage and the low activity of protein disul-fide reductase and prevent a balloon-likegrowth of ,B-glucan. Chitin also may partici-pate in the production of the M form, because,B-glucan is not extracted with alkali withoutprevious hydrolysis of chitin by chitinase. Inother words, fibers of proteins and chitin maybe interwoven with fibers of ,B-glucan in theM-form cell wall, in contrast to the Y-formcell wall in which a-glucan and chitin formtwo layers (12). Electron microscope studyreveals that in the conversion from the Y formto the M form, the outer layer (a-glucan layer)of the Y form does not continue to the cell wallof the M form, and in general there are no dis-tinct layers in the M-form cell wall (11, 14).By the above mechanism, all Y-form cells

have the capacity to produce the M form asobserved in the conversion from the Y form to

of yeastlike and mycelial forms of Paracoccidioides

the M form (11). On the other hand, the con-version from the M form to the Y form occursin only small parts of the M form (11). Al-though there may not be sufficient biochemicaldata to present a hypothesis for the conversionfrom the M form to the Y form, the informa-tion for the synthesis of a-glucan-synthesizingenzymes, protein disulfide reductase, etc., maynot be distributed evenly throughout the hy-phae, but localized only in some segments.With the transference of the cells from 20 C to37 C, the synthesis of ,B-glucan decreases, andthe cell wall of some segments may be soft-ened by the combined action of ,B-glucanaseand protein disulfide reductase. Simultane-ously, synthesis of a-glucan may occur in allparts of the cell wall of the segments replacing,B-glucan, resulting in the Y form.

ACKNOWLEDGMENT

We thank Akira Misaki for valuable suggestions con-cerning the analysis of the methylated glucans.

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3. Bacon, J. S. D., D. Jones, V. C. Farmer, and D. M.

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