INFECTION AND IMMUNITY, Apr. 1977, p. 181-188Copyright © 1977 American Society for Microbiology

Vol. 16, No. 1Printed in U.S.A.

Serial Enzymatic Hydrolysis of Cell Walls ofTwo Serotypes ofYeast-Form Histoplasma capsulatum with a(1-*3)-

Glucanase, f(1->3)-Glucanase, Pronase, and ChitinaseE. REISS

Mycology Division, Center for Disease Control, Atlanta, Georgia 30333

Received for publication 19 October 1976

A serial enzymatic hydrolysis procedure for the partial lysis ofHistoplasmacapsulatum yeast-form cell walls was described, and its application for thedifferentiation oftwo serotypes was evaluated. Cell walls were serially digestedwith a(1- >3)-glucanase and ,B(1- 3)-glucanase of Cladosporium resinae, thenby Pronase, and then by chitinase. The walls of serotype 1, 2, 3 (61.5% digested)were not susceptible to a(1-3)-glucanase, and they contained 30.3% chitin, thusidentifying the strain as chemotype 1 (chem 1). Serotype 1, 4 cells walls (51.6%digested) released 27.3% as glucose after treatment with a(1-33)-glucanase andcontained 7.8% chitin, compatible with chemotype 2 (chem 2). In addition toquantitating the monomeric products of enzymolysis, I recovered soluble nondi-alyzable polysaccharide from the digests of both serotypes.

Various fractions of the Histoplasma capsu-latum cell have been studied for their contribu-tion to the antigenic structure of this fungus.Among these are the cell wall (17, 33), theribosomal fraction (10), and certain secretedglycoproteins, the H and M antigens (4, 14).Since the cell wall matrix appears to be thesubstrate in the specific fluorescent-antibodyand complement fixation tests (18), it was con-sidered worthwhile to further delineate the mu-ral structure (8, 33) by using polysaccharasesand a protease as probes. Only a few reportshave appeared in which H. capsulatum cellwalls were exposed to enzymes of known actionpatterns (8, 31, 33). Although considerable in-formation has been acquired about the chitincontent and protease susceptibility of the wallsstudied, a quantitative analysis ofsoluble prod-ucts and enzyme-resistant wall residues has notbeen achieved. Advances in the characteriza-tion of microbial polysaccharases specific fora(1- 3)-glucans, ,8(1-+3)-glucans, and chitin(29, 34, 35) have made it possible to design amore comprehensive digestion scheme. The pri-mary objectives of this study were to measurethe extent of lysis of cell walls after a multi-stage or serial enzymatic hydrolysis and toquantitate the products formed at each stage.On the chance that depolymerization of thewall would be accompanied by the release oflarge and potentially antigenic soluble frag-ments, care was taken in the recovery of non-dialyzable products. The serological evaluationof soluble extracts and resistant residues, gene-

rated at each stage of serial enzymolysis, is thesubject of a separate report (E. Reiss, S. E.Miller, W. Kaplan, and L. Kaufman, Infect.Immun., in press).

Other factors influencing the selection ofH.capsulatum cultures for enzymatic treatmentwere their serotype and chemotype (8, 17, 19,33). Fluorescent-antibody cross-staining andadsorption studies among a variety of yeast-form isolates ofH. capsulatum revealed a max-imum of four markers and five serotypes (19,20). Adsorption of fluorescein-conjugated anti-globulins to one ofthese serotypes (1,2,3,4) withyeast-form cells of Blastomyces dermatitidisproduced a conjugate specific for members ofthe genus Histoplasma. Some H. capsulatumisolates, considered "nonantigenic," however,did not stain with this specific reagent (20).These isolates were subsequently recognized asserotype 1,4, which cross-reacted with B. der-matitidis. Thus, factors 2 and 3 specific for H.capsulatum were identified by fluorescent-anti-body staining of whole, yeast-form cells.

In the present study, two isolates represent-ing the 1,2,3 and the 1,4 serotypes were com-pared for their susceptibility to enzymes. It washoped that chemical analysis of partially di-gested cell walls might provide clues to thenature of the haptenic groups that determineserotype specificity. Thus far, the antigenicanalysis of cell walls had relied on preliminaryextraction with dilute alkali (17, 33) or organicbase (8, 12). Using dilute alkali, Kanetsunaextracted a(1-3)-glucan from some H. capsu-

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latum yeast-form isolates (17). The occurrenceof alkali-extractable acetic acid-precipitablea(1-33)-glucan identified isolates designated aschemotype 2 (chem 2); those yeast-form isolateslacking a(1-*3)-glucan and having an elevatedchitin content comprised chemotype 1 (chem 1).An enzymatic approach to solubilization of wallmaterial appeared to have merit from thestandpoint of selectively cleaving glycosidiclinkages under mild conditions with respect topH.

MATERIALS AND METHODS

Organisms and conditions of growth. H. capsula-tum A811 (serotype 1,2,3) and H. capsulatum 105(serotype 1,4), obtained from the Mycology Divi-sion's culture collection, were the test cultures. Bothconverted readily to the yeast form at 370C, andbatches were grown on a medium of the followingcomposition, in grams per liter: D-glucose, 10; caseinhydrolyzate (Casitone, Difco), 10; yeast extract(Difco), 3; K2HPO4, 1.34; KH2PO4, 1.67; L-cysteineHCl, 0.5; and MgSO4 7H2O, 0.1; final pH, 6.5. Nineliters was dispensed in 30 1-liter flasks, seeded with5-ml amounts of a 50-h starter culture, and incu-bated for 6 days at 370C on gyratory shakers at 150cycles/min. Yields of 200 to 250 g of wet packedyeast-form cells were recovered from each batchafter centrifugation (10,400 x g, 20 min).

Preparation of cell walls. Yeast-form cells were

washed twice with water and diluted to give a ratioof packed cells to water of 1:2.5 (vol/vol). A 32.5-mlcell suspension and 42.5 g of 0.5-mm-diameter glassbeads were combined in 60-ml screw-cap bottles anddisrupted for 2.25 min in a Braun cell homogenizer(Bronwill Scientific Inc., Rochester, N.Y.). The ho-mogenate was decanted, and walls were cleaned ofcytoplasm and membranes by repeated centrifuga-tion in 0.1% sodium dodecyl sulfate solution andwater, as previously described (37). The yields oflyophilized walls were 4.4 g for serotype 1,2,3 and 9.1g for serotype 1,4.

Preparation of glucanases and chitinase. Thesource of the constitutive extracellular complex ofa(l- 3)- and /3(1-*3)-glucanases was an isolate ofCladosporium resinae var. avellaneum QM7998, ob-tained from Elwyn T. Reese of the U.S. Army Na-tick Development Center. The medium of Mandelset al. (24) was used for growth and enzyme produc-tion, modified by providing mannitol, 5 g/liter, as

the carbon source (34). After growth for 14 days at300C, 8 liters of culture was centrifuged at 10,400 x g

for 15 min. The supernatant was adjusted to pH 5,filtered through celite, and concentrated under N2pressure in the cold to 1 liter in an Amicon thin-channel filtration apparatus (TCF 10, Amicon Corp.,Lexington, Mass.) over a PM10 membrane. The re-

tentate then received 2 volumes of cold acetone, andthe resulting precipitate was resuspended in citratebuffer (0.05 M, pH 4.5), lyophilized, and stored at-400C.Serratia marcescens QM1466, obtained from E. T.

Reese, was the source of microbial chitinase. The

basal medium for enzyme induetion was modifiedfrom Monreal and Reese (28) and contained in gramsper liter: yeast extract, 0.5; (NH4)2HP04, 2.75; andMgSO4 7H20, 0.3; adjusted to a final pH of 7.5 withKH2PO4. To this base 0.33% (wt/vol) king crab chi-tin, bleached and ground to 20 mesh, was added. A500-ml, 18-h starter culture of the bacterium inbrain-heart infusion broth was sedimented (10,400x g, 20 min), resuspended in 0.025 M potassiumphosphate buffer, pH 7.6, seeded into 2 liters ofbasalsalts medium with chitin, and incubated at 300C at120 cycles/min. Chitinase was detectable after 5days, and production peaked on the 8th day. Afterthe culture was centrifuged, the supernatant wasflash evaporated at 300C to 160 ml.Enzyme purification. The separation of a(1l-3)-

glucanase from f(1-*3)-glucanase (EC 3.2.1.6) pres-ent in the C. resinae culture filtrate was effected bycolumn chromatography on diethylaminoethyl-cel-lulose (DE-32, Whatman). Since operating condi-tions were different from those Reese et al. (34)suggested, the following details are provided. Allsteps were carried out at 4 to 90C. An acetone powdersample (1.5 g) that contained 214 mg of protein wasdialyzed versus starting buffer (0.01 M potassiumphosphate, pH 6.8). The column, a 60-ml syringe,was packed with 28 ml of DE-32, and, after beingequilibrated, it was charged with the sample in 50ml and eluted at 50 ml/h. Fractions of 8 ml werecollected with a Racetrack fractionator (Gilson Med-ical Electronics, Middleton, Wis.) and screened withan ultraviolet flow analyzer (Uvicord II, LKB In-struments). At fraction 23 the salt concentrationwas increased to 0.05 M NaCl, and fractions 58through 130 were eluted with a linear gradient of0.05 to 0.5 M NaCl. Portions were sampled for activ-ity, and those containing a(1-*3)-glucanase, that is,fractions 31 through 50, were pooled and lyophilized.Fractions 73 through 110, which contained ,3(1- 3)-glucanase, were first diafiltered over a PM10 mem-brane with 0.025 M citrate buffer, pH 4.5, containing0.025 M NaCl, and the retentate was lyophilized.

Chitinase (EC 3.2.1.14). The concentrated culturefiltrate (160 ml), induced on crabshell chitin, wasadjusted to pH 6.8 and 0.05 M with citrate-phosphatebuffer and clarified by centrifugation. Solid(NH4)2S04 was added to the supernatant, and theprecipitate formed at 20% saturation was sedi-mented at 20,000 x g for 20 min. After the superna-tant had been adjusted to 70% saturation, the sus-pension was held in an ice bath overnight and thencentrifuged at 20,000 x g for 1 h. The residue thenwas dissolved in citrate-phosphate buffer (0.025 M,pH 6.8) and diafiltered versus this buffer over aPM10 membrane; the retentate was lyophilized andstored at -40°C.Enzyme assays. The substrate for the chitinase

assay was the chitodextrins produced from crabshellchitin flakes by overnight solubilization in cold con-centrated H3PO4. After being filtered through glasswool, the chitodextrins were added to cold water,titrated to neutrality with 2 N NaOH, washed ex-haustively with water, and lyophilized. The diges-tion cocktail contained, per milliliter: chitodextrins,2.5 mg; gelatin (Difco), 100 jig, as stabilizer; cyclo-

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heximide, 50 jig; and chloramphenicol, 25 ,g; dis-solved in 0.05 M citric acid-K2HPO4 buffer, pH 6.6.At zero time 1 ml of enzyme was added and incu-bated for 5 h at 370C, when the reaction was termi-nated by boiling for 10 min. The product, N-acetyl-D-glucosamine (glcNAc), was detected in the superna-tant according to the Morgan-Elson procedure (16),and units of activity were expressed as nanomoles ofglcNAc/milliliter per minute.The substrate for a(1-*3)-glucanase was the in-

soluble sporophore layer of the -racket fungus Poly-porus betulinus, purified by removal of 3(13)-glucan(34), a gift of E. T. Reese; for the .3(1-33)-glucanaseassay, the standard was laminarin (K & K Labora-tories). In other respects, the digestion conditionswere identical and contained, per milliliter: sub-strate, 5 mg; gelatin, 100 ,ug; CaCl2, 55 ,ug; cyclohex-imide, 50 ug; and chloramphenicol, 25 ug; in a 0.05M citric acid-NaOH buffer, pH 4.5. A 1-ml samplewas added to 1 ml of digestion solution, incubatedfor 1 h with /8(1-*3)-glucanase or 4 h with a(1-3)-glucanase, and then terminated by boiling. Glucosewas detected enzymatically in the supernatantswith Glucostat reagent.The protease, Pronase CB (Calbiochem), was in-

cubated with cell wall samples for varying times at370C in a 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.5, containing, per milli-liter: CaCl2, 555 ,ug; cycloheximide, 50 ug; andchloramphenicol, 25 .g. The amino acid so liberatedwas estimated with ninhydrin.

Other polysaccharides used as reference stan-dards were soluble starch (Nutritional BiochemicalsCo.); pustulan, a 83(1--6)-glucan from Polyporus pa-pullosa (Calbiochem); and mycodextran, alternatinga(1-3) and a(1-4) linkages, from Aspergillus ja-ponicus (a gift of E. T. Reese).

Enzymatic hydrolysis of cell walls. Cell walls ofserotype 1,4 were dried to constant weight over P205in vacuo, and a starting weight of 500 mg was resus-pended in 17.5 ml of buffer with the aid of a motor-driven tissue homogenizer (Tri-R Instruments,Rockville Centre, N.Y.). Then a(1-*3)-glucanasecontaining 1 mg of protein (specific activity, 33.1nmol of glucose/min per mg of protein) was added,and the suspension was digested at 370C for 6 h in areciprocating water bath shaker at 90 cycles/min.Completeness of the reaction was judged by testingsamples of the digest for the release of glucose. Afterthe digest was centrifuged (13,500 x g, 15 min),the pellet was washed with 10 ml of buffer, resus-pended in 15 ml of fresh buffered enzyme, and fur-ther digested for 18 h. The soluble fraction againwas recovered, and fresh buffered enzyme was addedfor a total contact time of 46 h. The a(1-*3)-glucan-ase-resistant cell wall residue was washed once withglucanase buffer and three times with water andthen lyophilized. Supernatants from each digestionperiod were passed through filters (Swinnex 25,0.45-,um pore size; Millipore Corp., Bedford, Mass.),dialyzed overnight in the cold versus water, lyophi-lized, and stored at -40°C.

Cell walls of either serotype 1,4 or serotype 1,2,3were dried in vacuo and reconstituted in 17.5 ml ofbuffer with the aid of a tissue homogenizer. Then

j8(1- 3)-glucanase containing 0.77 mg of protein(specific activity, 25 umol of glucose/min per mg ofprotein) was added, and the mixture was gentlyshaken at 370C. After 5 h, the supernatant wasrecovered by centrifugation (13,500 x g, 15 min),fresh buffered enzyme was added, and digestion wascontinued for a total time of 24 h. Supernatants werefiltered, dialyzed, and then lyophilized, and the,8(1- 3)-glucanase-resistant walls were washed andlyophilized.

Cell walls resistant to fl(1-3)-glucanase were re-suspended in 15 ml of buffered enzyme solution con-taining 3 mg of Pronase and incubated for 10 h at370C with reciprocal shaking and then centrifugedat 13,000 x g for 20 min. The residue was washedwith buffer containing disodium ethylenediamine-tetraacetic acid (Na2 EDTA, 10-2 M) to inactivatethe enzyme (30) and three times with water, andthen it was lyophilized. The supernatant and wash-ings were sampled for liberated amino acids; thenthey were pooled and dialyzed first versus Na2EDTA (10-3 M) and then overnight versus waterbefore being lyophilized.

Pronase-resistant walls were resuspended in 12.5ml of buffered chitinase solution containing 4 mg ofenzyme (total activity, 55.5 nmol of glcNAc/minwith respect to a chitodextrin standard). The incu-bation at 370C was interrupted at 6 h. Fresh bufferedenzyme was added, and digestion was continued for18 h, when sampling indicated that the reaction wasnot yet complete. An additional 10 h of incubationwith fresh enzyme was required. The total contacttime was 34 h. The suspension was sedimented bycentrifugation at 13,000 x g for 20 min; the superna-tants were filtered and dialyzed versus water; andthe residues were washed once with buffer and threetimes with water. The dialyzed supernatants andthe washed chitinase-resistant cell walls were thenlyophilized and stored at -40°C.

Chemical analyses. Glucose was determined en-zymatically by the semimicro version of the Gluco-stat test (Glucostat Special Reagent, WorthingtonBiochemicals Co., Freehold, N.J.), according to themanufacturer's directions. Amino sugar was esti-mated in a modification of the Morgan-Elson reac-tion (16) with respect to glcNAc. Total carbohydratewas measured with phenol-sulforic acid and totalprotein with Folin-phenol with respect to bovineserum albumin fraction V (Miles Laboratories,Kankakee, Ill.). Amino acid liberated by Pronasewas detected with ninhydrin (30).

RESULTSYields and specific activities of polysac-

charases (Fig. 1). The a(1-*3)-glucanase re-tained activity after exposure to pH 6.8, con-trary to a previous report of its lability to pHabove 5 (34). Perhaps working at a cold temper-ature, 90C, moderated the effect of pH. Thetotal activity after purification obtained from1.5 g of acetone powder containing 214 mg ofprotein was 1.08 ,umol of glucose/min, with aspecific activity of 99.4 nmol of glucose/min per

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Ec0

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0.1

500

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20 40 60 80 100 120FRACTION NUMBER

FIG. 1. Elution profile of Cladosporium resinae culture filtrate on a diethylaminoethyl-cellulose column.Symbols: (-----) Ultraviolet flow analyzer logarithmic recording; (0) /3(1 -> 3)-glucanase activity; (a) a(l-3)-glucanase activity; (A) activity measured against cell walls ofH. capsulatum chem 2 (fractions 30 through 60)and against chem 1 walls (fractions 78 through 126).

mg of protein. The production of (3(1-*3)-glu-canase was more abundant than that ofa(1-33)-glucanase, and the irregular elutionprofile on DE-32 suggested the presence of iso-zymes, which has been described in the case ofother f8(1- 3)-glucanase producers (22). A morecompact zone of elution occurred when a lineargradient of 0.1 to 0.5 M NaCl was used. A totalactivity of 503 Amol of glucose/min per 111 mgof protein was recovered. The a(1-*3)-glucan-ase gave negative reactions on ,B(1-+3)- and,8(1-*6)-glucans or on the mycodextran refer-ence polysaccharide; however, a(1->4)-glucan-ase was detected (13 nmol of glucose/min permg of protein). Unexpectedly, it was found thatchitinase was produced constitutively and coe-luted with (3(1- 3)-glucanase during columnchromatography. The chitinase activity was di-minished by mass adsorption on insoluble chi-todextrins (16). A sequence of four 30-min ad-sorptions at ice bath temperature removed 86%of the chitinase activity. The specific activity of/3(1-*3)-glucanase after this step and molecularfiltration over a PM10 membrane was 25 umolof glucose/min per mg of protein. Reese verifiedthe finding that a constitutive chitinase was

coproduced by C. resinae on a separate batch. Itwas found that the total activity was three-fourths as potent as the inducible Serratia chi-tinase. Acetone precipitation was found to be a

means for the facile separation of a(1-*3)-glu-

canase, 8l(1-*3)-glucanase, and chitinase. Onevolume of cold acetone precipitated 94% of the,8(1--3)-glucanase, whereas 2 volumes of coldacetone precipitated chitinase but not a- or (3-glucanase (E. T. Reese, personal communica-tion).The 8(1--3)-glucanase did not have activity

for the a(1-*3)-glucan reference material. How-ever, activity was detected on mycodextran(17.7 nmol of glucose/min per mg of protein), ona(1--4)-glucan (134 nmol of glucose/min per mgof protein), and on pustulan (58.3 nmol of glu-cose/min per mg of protein). These coproducedenzymes were present at levels of less than 1%of the major 83(1-*3)-glucanase activity.The kinetics of chitinase induction by Serra-

tia were previously found to depend on particlesize of the substrate (29). In the present work,20-mesh chitin flakes were so large that a longlatent period occurred before enzyme was re-leased into the culture filtrate. The coproduc-tion of small amounts of the red prodigiosinpigment was also noted. In this regard molecu-lar filtration over a PM10 membrane was help-ful because some pigment was removed in thefiltrate. The total activity recovered in the pre-cipitate formed at 70% saturation with(NH4)2SO4 was 10.3 Amol of glcNAc/min, witha specific activity of 13.9 nmol of glcNAc/minper mg ofpowder after molecular filtration. Thediafiltered chitinase preparation was tested

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SERIAL ENZYMOLYSIS OF H. CAPSULATUM 185VOL. 16, 1977

and found to be nonreactive upon the a(l-3)-,a(1-*4)-, /3(1- >3)-, or f8(1--6)-glucans or myco-

dextran.Serial enzymatic hydrolysis. The cell walls

of each of two serotypes ofH. capsulatum wereserially subjected to digestion with a(1- 3)-glu-canase, /8(1-3)-glucanase, Pronase, and thenchitinase, a process referred to below as serialenzymolysis. Serotype 1,2,3 walls were not sus-ceptible to a(1-*3)-glucanase, and, in addition,the amino sugar released by chitinaseamounted to 30.3% of the wall dry weight.These two findings were sufficient grounds foridentifying the isolate as chem 1 (8, 17, 33)(Table 1). In contrast, the walls of serotype 1,4contained a(1--3)-glucan, 27.3% released byenzyme, and a low chitin content (7.8%), indi-cating it was a chem 2 culture (Table 2). Theamount of glucose released by 18(1--.3)-glucan-ase from chem 1 (18.1%) was greater than thatsolubilized from chem 2 (11.3%). Pronase ex-tracted amino acids to the extent of 8.3% from

chem 1 and 7.0% from chem 2. At each stage ofextraction, carbohydrate in nondialyzable formwas recovered. Since an objective of the studywas to probe the mural antigenic structurewith enzymes, the soluble nondialyzable frag-ments were considered to be presumptive anti-gens, and their activity is reported separately(Reiss et al., in press). Most soluble nondialyza-ble material could be extracted from chem 1walls by f8(1-*3)-glucanase, whereas chem 2walls yielded polysaccharide predominantly atthe a(1-3)-glucanase stage and also upon

,8(1--3)-glucanase treatment. Lesser amountsof nondialyzable carbohydrate-containing ma-terial was recovered from Pronase and chiti-nase digests. The total extent to which wallswere solubilized by serial enzymolysis was61.5% for chem 1 and 56.1% for chem 2. Thiswas judged on the basis of monomers releasedand the amount of cell wall core recovered afterthe solubilization procedure. The saccharidenature of the enzyme-resistant core is reported

TABLE 1. Serial hydrolysis ofyeast-form cell walls ofHistoplasma capsulatum (chemotype 1) with /3(1 -3)-glucanase, Pronase, and chitinase

f3(1- 3)-Glucanase Pronase ChitinaseDetermination Amt Amt Percent Amt Percent

(mg) Percent (mg) (mg)Starting weight 400 100 220 100 155 100Monomer released 72.3a 18.1 18.2b 8.3 89.5c 57.7Extract, nondialyzabled 28.8 7.2 3.4 1.6 6.7 4.3Total extracted 101 25.3 21.6 9.9 96.2 62.1Resistant residue 239 59.8 179 81.4 59.5 38.4Not accounted for 60 15 19 8.6 0 0

Cell wall extractede (%) 25.3 5.9 30.3a 1)-Glucose.b Amino acid.c N-acetylglucosamine.d Total carbohydrate.e Monomer + polysaccharide as percent of original wall dry weight.

TABLE 2. Serial hydrolysis ofyeast-form cell walls of Histoplasma capsulatum (chemotype 2) witha(1 -33)-glucanase, (8(1 -.3)-glucanase, Pronase, and chitinase

a(1-.3)-Glu- ,B(1- 3)-Glu- Pronase Chitinasecanase canase

DeterminationAmt Per- Amt Per- Amt Per- Amt Per-(mg) cent (mg) cent (mg) cent (mg) cent

Starting weight 500 100 260 100 130 100 80 100Monomer released 137a 27.3 52.1a 20.0 9.1b 7.0 24c 30.0Extract, nondialyzabled 19.2 3.8 5.1 2.0 1.5 1.2 3.1 3.9Total extracted 156 31.2 57.2 22.0 10.6 8.2 27.1 33.9Resistant residue 281 56.2 147 56.5 94.0 72.3 51.5 64.4Not accounted for 63.3 12.7 55.8 21.4 25.4 19.5 1.4 1.8

Cell wall extractede (%) 31.1 12.4 4.8 7.8a-e See Table 1.

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separately (Reiss et al., in press). The cell wallcore resistant to serial enzymolysis comprised18.7% of the initial dry weight of chem 1 and14.8% ofchem 2, and the total amount of mate-rial not accounted for as monomer, nondialyza-ble polysaccharide, and resistant residue was19.8% for chem 1 and 33.6% for chem 2. Al-though losses during manipulation of 10% ateach stage are not atypical, dialyzable oligosac-charides may have been produced, but this pos-sibility was not investigated.

DISCUSSION

The objective of the present work was to se-lectively cleave glycosidic bonds in the cellwall, thus causing release of sugars, and torecover any polysaccharide fragments liberatedas a result of wall lysis. Therefore, enzymeswith action patterns specific for linkagesknown to occur in yeast-form cell walls of H.capsulatum had to be chosen (8, 17, 33). Incontrast to the walls of baker's yeast or Can-dida albicans (5, 6, 21, 23, 26, 27, 32, 39), thehistoplasmal wall contained larger amounts ofchitin (12 to 39%) (8, 38), and some isolatescontained a substantial a(1-*3)-glucan compo-nent (up to 41.7% [17]). H. capsulatum containsgalactomannan, which is serologically active inimmunodiffusion (17, 36), in addition to,8(1-)3)-glucan.

Since a commercial preparation of chitinasealso contained potent /8(1-)3)-glucanase activ-ity (31), it was considered advisable to prepareand characterize a(1-*+3)-glucanase, f(1- 3)-glucanase, and chitinase. Although other mi-crobial sources of a(1- 3)-glucanases (9, 11, 13)and f8(1-)3)-glucanase (25, 35) were known, thepresence of an inducer was required to obtainappreciable yields, whereas C. resinae was aconstitutive producer of these two activitieswhen the carbon source for growth was manni-tol. a- and 18-glucanases were separated (34),and the problem of concurrent chitinase secre-tion was minimized by mass adsorption. Thepattern of endo- or exo-splitting action has notbeen established for the Cladosporium glucan-ases. The serial manner in which these en-zymes were presented to the yeast-form cellwalls was suggested by Hunsley and Burnett(15). Those workers found that preliminarytreatment with f(1- 3)-glucanase and proteasepromoted chitinase digestion of Neurosporawalls. The extent of lysis of histoplasmal wallsafter serial enzyme extraction (61.5%, chem 1;51.6%, chem 2) was sufficient to justify thistechnique. These values for total lysis were thesummation of the monosaccharide or amino

acid and soluble, nondialyzable polysacchariderecovered at each stage. The data indicate thatthe yeast-form serotype of the two culturesstudied could be determined on grounds of en-zymatic susceptibility. In particular, the actionof a(1-+3)-glucanase monitored colorimetri-cally with Glucostat reagent was presumptiveevidence for chem 2. Further verification wasprovided by the extent of release of glcNAc bychitinase (7.8%); for a typical chem 2 strain,Kanetsuna et al. (17) determined this to be11.5% of the wall dry weight.The first evidence 'for chemical differen-

tiation of yeast-form isolates of H. capsulatumwas indicated by significant variations in chitincontent. Pine and Boone (33) found that isolatesreactive with fluorescein-labeled H. capsula-tum antibodies had a higher chitin content andlower alkali susceptibility than "nonreactive"isolates. Later Domer (8) designated certainyeast-form isolates as chemotype 1 based on achitin content of 37.6% and low solubility in theorganic base, ethylenediamine. Chemotype 2status was accorded to two isolates with low-ered chitin content, 12.3%, which were 48.2%solubilized by ethylenediamine. Chemotypeswere distinguished without knowledge of theserotypic nature of the isolates involved. Thepresent data suggest that serotype and chemo-type may be covariant properties, since the1,2,3 serotype was compatible with chem 1 be-cause of its high chitin content and absence ofa(1-33)-glucan, and serotype 1,4 was deter-mined to be chem 2 on the basis of the presenceof a(1-*3)-glucan and low chitin content. Ex-tension of serotyping and chemotyping to otherisolates ofH. capsulatum is warranted to sub-stantiate this relationship.The serial enzymolysis procedure should be

applicable to study of the cell wall compositionof other zoopathogenic fungi that are classifiedas ascomycetes or Fungi Imperfecti due to thecommon theme of glucans and chitin withinthis group (3). The aim of such an approachwould be to recover fragments of high molecu-lar weight that may be released during partialenzymolysis. In addition to the common glucan-chitin theme, those haptenic variations respon-sible for species specificity may be less subjectto denaturation within the pH and temperaturelimits of enzymatic digestion.The chemical structure of the yeast-form

walls that resisted enzymolysis is also worthpursuing, since in an analogous system themannan core ofbaker's yeast resistant to exo-a-mannanase was found to be enriched in phos-phomannan linkages which, when exposed tophosphodiesterase, rendered the core suscepti-

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SERIAL ENZYMOLYSIS OF H. CAPSULATUM 187

ble to further digestion (7). The serological ac-

tivity and saccharide composition of soluble ex-

tracts and resistant residue products of serialenzymolysis are described separately with re-

spect to the two serotypes of H. capsulatumstudied above (Reiss et al., in press).

ACKNOWLEDGMENTI thank Elwyn T. Reese for his valuable guidance and for

providing polysaccharolytic enzyme-producing microorga-nisms.

LITERATURE CITED

1. Alexander, M. 1971. Microbial ecology, p. 357-385. Aca-demic Press Inc., New York.

2. Bacon, J. S. D. 1973. The contribution of 3-glucanasesto the lysis of fungal cell walls, p. 61-74. In J. R.Villaneuva, I. Garcia-Acha, S. Gascon, and F. Uru-buru (ed.), Yeast, mould, and plant protoplasts. Pro-ceedings of the Third International Symposium on

Yeast Protoplasts, Salamanca. Academic Press Inc.,New York.

3. Bartnicki-Garcia, S. 1968. Cell wall chemistry, mor-

phogenesis, and taxonomy of fungi. Annu. Rev. Mi-crobiol. 22:87-108.

4. Bradley, G., L. Pine, M. W. Reeves, and C. W. Moss.1974. Purification, composition, and serological char-acterization of histoplasmin-H and M antigens. In-fect. Immun. 9:870-880.

5. Cabib, E., and B. Bowers. 1971. Chitin and yeast bud-ding. Localization of chitin in yeast bud scars. J. Biol.Chem. 246:152-159.

6. Chattaway, F. W., M. R. Holmes, and A. J. E. Barlow.1968. Cell wall composition of the mycelial and blas-tospore forms ofCandida albicans. J. Gen. Microbiol.51:367-376.

7. Colonna, W. J., and J. 0. Lampen. 1974. Structure ofthe mannan from Saccharomyces strain FH4C, a mu-

tant constitutive for invertase biosynthesis. I. Signif-icance of phosphate to the structure and refractori-ness of the molecule. Biochemistry 13:2741-2748.

8. Domer, J. 1971. Monosaccharide and chitin content ofcell walls of Histoplasma capsulatum and Blasto-myces dermatitidis. J. Bacteriol. 107:870-877.

9. Ebisu, S., K. Kato, S. Kotani, and A. Misaki. Isolationand purification of Flavobacterium a-1,3-glucanase-hydrolyzing, insoluble, sticky glucan ofStreptococcusmutans. J. Bacteriol. 124:1489-1501.

10. Feit, C., and R. P. Tewari. 1974. Immunogenicity ofribosomal preparations from yeast cells of Histo-plasma capsulatum. Infect. mnmun. 10:1091-1097.

11. Fleet, G. H., and H. J. Phaff. 1974. Glucanases inSchizosaccharomyces. Isolation and properties of thecell wall-associated (3(1- 3) glucanases. J. Biol.Chem. 249:1717-1728.

12. Garcia, J. P., and D. H. Howard. 1971. Characteriza-tion of antigens from the yeast phase ofHistoplasmacapsulatum. Infect. Immun. 4:116-125.

13. Hasegawa, S., and J. H. Nordin. 1969. Enzymes thathydrolyze fungal cell wall polysaccharides. I. Purifi-cation and properties of an endo-a(D-(1-.3) glucanasefrom Trichoderma viride. J. Biol. Chem. 244:5460-5470.

14. Heiner, D. C. 1958. Diganosis of histoplasmosis usingprecipitin reactions in agar gel. Pediatrics 22:616-627.

15. Hunsley, D., and J. H. Burnett. 1970. The ultrastruc-tural architecture of the walls of some hyphal fungi.J. Gen. Microbiol. 62:203-218.

16. Jeuniaux, C. 1966. Chitinases, p. 644-650. In E. F.

Neufeld and V. Ginsburg (ed.), Methods in enzymol-ogy, vol. 8. Academic Press Inc., New York.

17. Kanetsuna, F., L. M. Carbonell, F. Gil, and I. Azuma.1974. Chemical and ultrastructural studies on the cellwalls of the yeastlike and mycelial forms of Histo-plasma capsulatum. Mycopathol. Mycol. Appl. 54:1-13.

18. Kaufman, L. 1974. Serodiagnosis of fungus diseases, p.557-568. In E. H. Lennette, E. H. Spaulding, and J.P. Truant (ed.), Manual of clinical microbiology, 2nded. American Society for Microbiology, Washington,D.C.

19. Kaufman, L., and S. Blumer. 1966. Occurrence of sero-types amongHistoplasma capsulatum strains. J. Bac-teriol. 91:1434-1439.

20. Kaufman, L., and W. Kaplan. 1961. Preparation of afluorescent antibody specific for the yeast phase ofHistoplasma capsulatum. J. Bacteriol. 82:729-735.

21. Kessler, G., and W. J. Nickerson. 1959. Glucomannan-protein complexes from the cell walls of yeasts. J.Biol. Chem. 234:2281-2285.

22. Kobayashi, Y., H. Tanaka, and N. Ogaswara. 1974.Multiple 3-1,3 glucanases in the lytic enzyme com-plex of Bacillus circulans WL 12. Agric. Biol. Chem.38:959-965.

23. Kopecka, M., H. J. Phaff, and G. H. Fleet. 1974. Dem-onstration of a fibrillar component in the cell wall ofthe yeast Saccharomyces cerevisiae and its chemicalnature. J. Cell Biol. 62:66-76.

24. Mandels, M., F. W. Parrish, and E. T. Reese. 1962.Sophorose as an inducer of cellulase in Trichodermaviride. J. Bacteriol. 83:400-408.

25. Mann, J. W., C. E. Heintz, and J. D. Macmillan. 1972.Yeast spheroplasts formed by cell wall-degrading en-zymes from Oerskovia sp. J. Bacteriol. 111:821-824.

26. Manners, D. J., A. J. Mason, and J. C. Patterson. 1973.The structure of a f8(1- 3)-D-glucan from yeast cellwalls. Biochem. J. 135:19-30.

27. Marriott, M. S. 1975. Isolation and chemical characteri-zation of plasma membranes from the yeast and my-celial forms of Candida albicans. J. Gen. Microbiol.86:115-132.

28. Monreal, J., and E. T. Reese. 1968. Lysis of livingCandida utilis cells by enzymes of Micromonospora.Arch. Biochem. Biophys. 126:960-961.

29. Monreal, J., and E. T. Reese. 1969. The chitinase ofSerratia marcescens. Can. J. Microbiol. 15:689-696.

30. Narahashi, Y. 1970. Pronase, p. 651-664. In G. E. Perl-mann and L. Lorand (ed.), Methods in enzymology,vol. 19. Academic Press Inc., New York.

31. Odds, F. C., 0. Kaufman, D. McLaughlin, C. Calla-way, and S. 0. Blumer. 1974. Effect of chitinasecomplex on the antigenicity and chemistry of yeast-form cell walls and other fractions of Histoplasmacapsulatum and Blastomyces dermatitidis. Sabourau-dia 12:138-149.

32. Phaff, H. J. 1971. Enzymic lysis of yeast cell walls, p.186-193. In A. H. Rose and J. S. Harrison (ed.), Theyeasts, vol. 2. Academic Press Inc., London.

33. Pine, L., and C. J. Boone. 1968. Cell wall compositionand serological reactivity ofHistoplasma capsulatumserotypes and related species. J. Bacteriol. 96:789-798.

34. Reese, E. T., A. Maguire, and F. W. Parrish. 1972. a-1,3-Glucanases and their relationship to mycodex-tranase, p. 735-742. In International FermentationSymposium, 4th, Kyoto.

35. Reese, E. T., and M. Mandels. 1966. ,BGlucanases otherthan cellulase, p. 607-615. In E. F. Neufeld and V.Ginsburg (ed.), Methods in enzymology, vol. 8. Aca-demic Press Inc., New York.

36. Reiss, E., W. 0. Mitchell, S. H. Stone, and H. F.

VOL. 16, 1977

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188 REISS INFECT. IMMUN.

Hasenclever. 1974. Cellular immune activity ofa gal-actomannan-protein complex from mycelia of Histo-plasma capsulatum. Infect. Immun. 10:802-809.

37. Reiss, E., S. H. Stone, and H. F. Hasenclever. 1974.Serological and cellular immune activity of peptido-glucomannan fractions of Candida albicans cellwalls. Infect. Immun. 9:881-890.

38. San-BIas, G., and L. M. Carbonell. 1974. Chemical andultrastructural studies on the cell walls of the yeast-like and mycelial forms of Histoplasma farcimi-nosum. J. Bacteriol. 119:602-611.

39. Sentandreu, R., and D. H. Northcote. 1968. The struc-ture of a glycopeptide isolated from the yeast cellwall. Biochem. J. 109:419-432.

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nloaded from