INlCrION AND IMMUNrrY, June 1975, p. 1187-1194Copyright i 1975 American Society for Microbiology

Vol. 11, No. 6Printed in U.S.A.

Immunochemical Studies on the Human Pathogen Sporothrixschenckii: Effects of Chemical and Enzymatic Modification of

the Antigenic Compounds upon Immediate and DelayedReactions

H. SHIMONAKA,* T. NOGUCHI, K. KAWAI, I. HASEGAWA, Y. NOZAWA, AND Y. ITO

Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan

Received for publication 6 November 1974

The rhamnose-containing polysaccharide-peptide compound derived from thecells of the pathogenic fungus Sporothrix schenckii has been shown to contain87.1% carbohydrate and 12.5% peptide and to give rise to both immediate- anddelayed-type reactions in sensitized guinea pigs. The capacity to induceimmediate-type reaction, passive cutaneous anaphylaxis, was completely lost bydegradation of the carbohydrate moiety by periodate, whereas the ability toinduce the delayed-type reactions of migration inhibition (in vitro) and thesporotrichin reaction (in vivo) were only slightly affected by periodate treatment.On the other hand, delayed "reactivities" to the compound were considerablyreduced by treatment with papain, whereas the immediate-type reactionremained positive. These results lead to a conclusion that the rhamnose-contain-ing polysaccharide of the polysaccharide-peptide antigenic compound plays animportant role in the immediate-type reaction, whereas the peptide is largelyresponsible for the delayed-type reaction.

The chemistry of numerous fungal polysac-charides and glycoproteins has been studiedintensively and some comprehensive reviewshave appeared (5, 17); however, there is littleinformation concerning the chemistry of humanpathogenic fungi (1, 3, 4, 11). Sporothrixschenckii is the agent of sporotrichosis whichusually causes a subcutaneous infection in man;a definite diagnosis of this mycotic disease hasbeen chiefly made by means of routine mycolog-ical procedures. In addition, some immunologi-cal diagnoses by skin tests or serological tech-niques have also been available. The recogni-tion of the importance of the hypersensitivityreaction in sporotrichosis was first due to thework of Widal in 1910 (30). Because the increas-ing incidence of sporotrichosis is recognized(31), there is urgent need for a more reliableantigen for immunological tests. To date, thecrude antigen from concentrated culture fil-trates of S. schenckii has been commonly usedfor the serological diagnosis of patients havingsporotrichosis (14), and little attention has beengiven to the relationship between the chemicalcomposition and the immunological activity ofantigenic compounds existing in the cells of S.schenckii (2, 15, 20).Our previous paper (19) has reported the

isolation and purification of immunologically

active polysaccharide-peptide compounds fromS. schenckii which have been shown to induceboth immediate and delayed hypersensitivityreactions (20). In the present study, attentionwas focused on elucidating the relationshipbetween the chemical composition of the anti-gen and immediate and delayed hypersensitivereactivities in experimental sporotrichosis. Forthis purpose, modifications of polysaccharidesor peptides moieties of the antigenic compoundderived from S. schenckii cells were performed;the immunological activities of the modifiedantigens were examined in sensitized guineapigs using several criteria: the precipitin test,the migration inhibition test, the sporotrichintest, and passive cutaneous anaphylaxis.

MATERIALS AND METHODSOrganisms. A strain of S. schenckii was cultured

at 27 C in Sabouraud broth for 4 weeks on a surfaceculture. The fungal mats were separated from theculture medium by filtration and washed repeatedlywith distifled water.

Extraction and purification of antigenicsubstances. The antigenic substances in this studywere extracted according to the method previouslydescribed (19) (Fig. 1). Antigenic substances wereextracted with 45% phenol (29), and a crude polysac-charide fraction was further purified by chromatogra-phy on a Sephadex G-100 column. The main fraction

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passing through the column in the void volume wasdesignated CP.

Modif'ication of the CP fraction. For digestion ofthe peptide moiety in the CP fraction, the CP andpapain were dissolved in acetate buffer (pH 5.7)containing 5 mM cysteine-hydrochloride and 1 mMethylenediaminetetraacetic acid to give 0.5 and 0.01%concentrations, respectively, and incubated at 37 Cfor 48 h. Gel filtration on a Sephadex G-75 columnwas then performed to remove small degraded pep-tides and enzyme (24).

For degradation of the polysaccharide moiety in theCP fraction, the CP was partially degraded with 16mM sodium periodate (16) at 4 C for 100 h.

Chemical constituents of antigenic compounds.Total sugar content was determined by the anthronemethod, and qualitative analysis of monosaccharideswas done by gas-liquid chromatography (22). Qualita-tive and quantitative determinations of amino sugarwere carried out according to the previous paper (21).For amino acid analysis, the samples were hydrolyzedat 105 C in sealed ampoules, and the hydrolysateswere analyzed using an amino acid autoanalyzer(Hitachi model KLA-3). The amounts of total nitro-gen and phosphorus were determined by the Kjeldahl-Nessler and Gomori methods, respectively.

Immunological activities. (i) Preparation ofantiserum to CP. Male, albino rabbits (about 3 kg)were inoculated every 7 days for 5 weeks with amixture of 1.0 ml of Freund complete adjuvant and1.0 ml of 0.9% antigen (CP) in 0.85% NaCl solutionadministered intramuscularly in each thigh. Theywere bled 1 week after the last injection, and anti-CPserum was frozen until use.

(ii) Immunoelectrophoresis. The immunoelectro-phoretic technique in agar was essentially as de-scribed by Grabar (10).

(iii) Passive cutaneous anaphylaxis. Themethod was performed principally by Ovary's method(25). Normal, albino guinea pigs, 300 to 400 g, wereinjected intracutaneously with 0.05 ml of dilutedanti-CP serum. At the same time, both the diluent0.85% NaCl solution (which was used to dissolve theantigen) and nonsensitized rabbit serum were in-jected as controls. After 5 h the animals were injectedintravenously with a mixture of 0.25 mg of antigenand 1% Evans blue dissolved in 1.0 ml of saline. Thediameter of the blue staining around the injection sitewas measured 30 min later.

(iv) Migration inhibition test. Male, albinoguinea pigs, 300 to 400 g, were sensitized with 5 mg ofdead whole cells emulsified (1:1, vol/vol) in Freundcomplete adjuvant injected into the thigh muscleseach week for 2 weeks. The procedure employed wasprincipally the capillary tube method of George andVaughan (9) with minor modifications. To obtainperitoneal exudate, the sensitized (5 weeks aftersensitization) and nonsensitized guinea pigs wereinjected intraperitoneally with 20 ml of liquid paraf-fin. Four days later exudate cells were harvested bywashing the peritoneal cavity with chilled Hanksbalanced salt solution. The peritoneal exudate cellsuspension was sucked into sterile capillaries, and thecapillaries were affixed with a high-vacuum grease

(Dow Corning Co.) in tissue culture chambers. Thechambers were filled with Eagle medium containing20% normal heat-inactivated calf serum and 0.006%kanamycin with or without antigens (64 gg of antigenper 1.2 ml of nutrient medium). The chambers wereincubated at 37 C for 24 h, and the rate of migration ofcells from the capillaries was determined by theformula of Carpenter (7). Migration indexes of 80% orless were recorded as positive (6).

(v) Delayed skin test (sporotrichin test). Male,albino guinea pigs, 300 to 400 g, were inoculatedintraperitoneally with 10 mg of whole cells suspendedin 1 ml of 0.85% NaCl solution. Skin tests wereperformed 6 weeks after a single sensitizing dose ofantigen, using 0.1 mg of antigen in 0.1 ml of saline.Readings were made at 24 h, and the degrees oferythema and induration were recorded in millimetersas the product of the two diameters of the test sitereactions measured at right angles to one another.Palpable induration of 7 mm or greater was consid-ered to be a positive reaction according to Edwards(8). Controls consisted of reactions in sensitizedguinea pigs injected with the various antigens.

RESULTS

Figure 1 shows the extraction procedure forisolating antigens derived from the cells of S.schenckii. The crude polysaccharide fractionwas obtained from the water layer; the elutionpattern obtained by Sephadex G-100 gel filtra-tion during subsequent purification of the crudepolysaccharide fraction is presented in Fig. 2. Itwas found that the main CP fraction assayedwith anthrone coincided with the peptide curvemonitored by absorbance at 275 nm. Thischromatographic result suggests that the CPfraction is a polysaccharide-rich peptide com-pound. Actually, the CP contained 87.1% carbo-hydrate and 12.5% peptide (Table 1). The yieldof the CP was about 0.6% of the startingmaterial of S. schenckii dried mycelia. Gas-liq-uid chromatographic analysis of sugar compo-nents showed that the CP was composed ofrhamnose, mannose, and galactose, and theirmolar proportion was 2.1:5.6:1.0. The results ofchemical analysis suggest that the CP is arhamnose-containing polysaccharide peptidecompound. As for amino acid composition, allfractions contained large quantities of asparticacid, threonine, serine, glutamic acid, glycine,and alanine (Table 2).To investigate the effect of alteration in the

chemical components of the CP fraction upontheir capacity to elicit the hypersensitivityreactions, papain and periodate treatmentswere performed (Fig. 1). The papain-treatedfraction was designated as CP-I and the perio-date-treated fraction as CP-II. Analytical dataon the modified fractions (CP-I and CP-II) are

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Defatted mycelia

extracted with 45% phenol

centrifuged

Water layer

dialyzed against distilled water

lyophilized

Crude polysaccharide fraction

eluted with 0.85% NaCl throughSephadex G-100 column

lyophilized

Purified polysaccharide fraction (CP)(yield = 0.6% of mycelia)

Phenol layer

(proteinous material)discarded

Breakdown of protein moiety

digested with 0.01% papain in 50mM acetate buffer (pH 5.7)containing 5 mM cysteine-HCland 1 mM EDTA at 37 C for48 h

eluted with distilled waterthrough Sephadex G-75 column

dialyzed against distilled water

Degradation of carbohydrate moiety

incubated in 16 mM NaIO4at 4 C for 100 h

dialyzed against distilledwater

lyophilized

CP-II (yield = 53% of the CP)

lyophilized

CP-I (yield = 39% of the CP)

FIG. 1. Procedure for isolation, purification, and modification of antigenic compounds (CP, CP-I, CP-If)from S. schenckii cells. EDTA, Ethylenediaminetetraacetic acid.

given in Tables 1 and 2. Little difference of thesugar component was observed between the CPfraction and the enzyme-treated fraction, CP-I.However, the proteolytic digestion caused a 50%reduction in the nitrogen content of the CPfraction. In contrast, treatment with periodate,which disrupted the glycol group of sugar,drastically affected the sugar moiety of the CP.Any carbohydrate component was not detecta-ble by anthrone reaction and gas-liquid chroma-tography.

For immunological assay the antigens of CP,CP-I, and CP-II were used. Figure 3 shows theresults of immunoelectrophoretic analyses usingthe anti-CP rabbit serum. A few strong precipi-tin arcs were obtained at the negative electrodewith CP and CP-I, whereas CP-II failed toproduce a precipitin arc.

Passive cutaneous anaphylactic activities areshown in Table 3. No bluing spot was observedin any of the controls. The dimension of theinjected area was greater than 100 mm2 with CPand CP-I. In contrast, the activity was com-pletely abolished by periodate.The migration patterns are shown in Fig. 4.

The results of the migration inhibition test areshown in Table 4 and are recorded as themigration indexes of 24-h packed cell prepara-tions made from the peritoneal exudates ofsensitized guinea pigs. The migration was sig-nificantly inhibited by the addition of the CP(average migration index of 10 sensitized guineapigs = 69.7%) and the periodate-treated CP-II(70.5%). However, papain-treated CP-I showeda weak inhibition of the migration (88.6%). Theaverage migration index in six nonsensitized

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0 50 100 1

- E620(a.-)_ o- EVs

I\\\I \I

I5 0

FIG. 2. Fractionation on Sephadex G-100 column (2.8 by 40 cm) with 300 mg of the crude polysaccharidefraction in 0.85% NaCI solution. The 5-mi fractions were pooled. -, Absorbance at 620 nm; -----, absorbanceat 275 nm.

guinea pigs was 93 to 99% to each antigen.For delayed skin tests, intradermal injection

of the antigens was carried out in four guineapigs sensitized with dead S. schenckii cells.Control sites were injected with 0.85% NaClsolution; all antigens used were injected intofour nonsensitized guinea pigs as another con-

trol. Sites injected with 0.85% NaCl showedreactions of less than 4.6 by 4.3 mm of erythemaand no induration in the sensitized guinea pigs.The mean diameter of induration for CP was12.1 by 11.5 mm, and the delayed skin reactiv-

ity of papain-treated CP-I was reduced to lessthan 20%o of the original CP (Table 5). On theother hand, little change was observed in theactivity of the periodate-treated CP-II. It wasfound that there was close correlation betweenthe in vitro and in vivo manifestations of thedelayed-type reactions.

DISCUSSIONMany efforts have been made to isolate and

identify antigenic substances from fungi and toclarify the relationship between the chemical

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nature and immunological activity of fungalantigens. Barker et al. reported that a peptidegalactomannan from Trichophyton menta-grophytes is responsible for the immediate anddelayed hypersensitivity reactions (4). Thepure galactomannan of Trichophyton has beenisolated by Blank and co-workers (11, 26) andwas shown to have activities in the comple-ment fixation and precipitin tests; no capacity

TABLE 1. Chemical composition of whole cells andantigenic compounds extracted from S. schenckii cells

Whole Extracted antigenicComposition cell compound

(%) CP Cp-I cp-_a

Total carbohydrateb 48.1 87.1 91.6 0Rhamnosec 11.7 21.1 21.6 0Mannosec 14.4 55.9 58.6 0Galacto4ec 4.7 10.0 11.3 0Glucosec 17.3 Trace Trace 0

Total nitrogend 6.7 2.0 0.9 4.1Peptidee 35.2 12.5 5.6 25.6Glucosaminel 6.1 Trace Trace Trace

Phosphorusg 0.6 0.1 Trace Trace

a Contains decomposed substances due to perioda-tion of polysaccharide moiety.

b By the anthrone method.c Gas-liquid chromatography.d Kjeldahl-Nessler's method.'Nitrogen content (x6.25).'Elson-Morgan's mothod.£ Gormori's method.

to elicit the delayed skin test was found. Re-cently, it has been reported (23) that proteolyticenzyme treatment of antigenic polysaccharide-peptide complexes extracted from mycelia of T.mentagrophytes caused a loss of the ability ofthe extract to elicit a delayed reaction to skintests in humans with naturally acquired tricho-phytosis. The immunological properties of pro-tein and polysaccharide antigens of other fungi,Aspergillus (3), Coccidioides, and Histoplasma(1), were also investigated.

TABLE 2. Amino acid composition of antigeniccompounds extracted from S. schenckii cells

Antigenic compound (10-' mol)Amino acid

CP CP-I CP-II

Aspartic acid 7 6 12Threonine 20 10 36Serine 18 8 38Glutamic acid 4 4 14Proline 1 Trace 1Glycine 9 5 21Alanine 14 6 26Valine 11 3 18Isoleucine 3 1 4Leucine 2 2 5Tyrosine 1 Trace 1Phenylalanine Trace Trace TraceLysine 3 2 5Histidine 2 Trace 2Arginine 1 Trace Trace

+

CP

CP-I

CP-l

FIG. 3. Immunoelectrophoregrams of polysaccharide-peptide compounds. The antigen solutions (2 mg/ml)were placed in the wells (arrow). Rabbit anti-CP serum was added to the center trough after electrophoresis ofthe antigens. Diffusion was at 15 C for 2 days.

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As for S. schenckii, the immunologicallyactive substances were isolated from the cells orculture filtrates, and the serological investiga-tions were performed by using these antigens (2,12, 13, 15, 18-20, 27, 28; S. McMillen and E. R.Laverty, Bacteriol. Proc., p. 115, 1969). Aoki etal. (2), Ishizaki (12), and Lloyd and Bitoon (15)evaluated the use of Sporothrix antigen forclinical diagnosis. Aoki et al. found that the

TABLE 3. Passive cutaneous anaphylaxis producedwith antigenic compounds extracted from S.

schenckii cells

Fraction Dimension (mm2)

CP... 157CP-I .......................... 118CP-II........... 0

Control... 0

Ca

polysaccharide extracted from S. schenckii cellsdisrupted with the French press was a rhamno-mannan compound (1:1); it was effective foreliciting the sporotrichin reaction and showedcross-reaction with Aspergillus antigen. Ishizakiisolated antigens from the culture filtrate of S.schenckii, the major fraction of which contained15% peptide and rhamnose, mannose, and ga-lactose (4:5:1). This fraction proved to be im-munologically active by precipitin reaction inagar gel and by the sporotrichin test. Recently,a serologically active peptido-rhamnomannanwas isolated from the cells and culture mediumof S. schenckii by Lloyd and Bitoon (15). Theyalso showed that the sugar composition of thepolysaccharide included mannose and rham-nose (5:3) and that 18% peptide was present.Our previous studies have shown that the waterlayer after 45% phenol extraction from S.schenckii cells comprises mixtures of polysac-

b

I

FIG. 4. Migration patterns of peritoneal exudate cells from guinea pigs sensitized with S. schenckii cells. (a)Control: migration pattern in the absence of antigen; (b) CP; (c) CP-I; (d) CP-II.

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TABLE 4. Migration inhibition of peritoneal exudatecells induced with antigenic compounds from S.

schenckii cells

Sensitized Migration index (%)guinea pig CP CP-I CP-II

1 79 91 842 69 94 553 85 94 844 67 93 885 78 90 796 72 75 567 51 73 608 64 98 629 74 97 7910 58 81 58

Mean : CIa 69.7 ± 7.2 88.6 ± 6.4 70.5 i 9.4

a CI, Confidence interval0.05).

(significance level, a =

TABLE 5. Comparison of delayed skin reactionsinduced with antigenic compounds extracted from S.

schenckii cells

Sensitized Induration/erythema (mm)guinea pig

CP CP-I CP-II

11.5 x 10.9 5.7 x.5.1 9.9 x 9.514.6 x 13.1 7.6 x 7.4 13.7 x 13.2

2 10.4 x 10.2 1.5 x 1.5 7.7 x 7.113.2 x 11.6 5.4 x 4.9 10.0 x 9.113.0 x 12.2 0 x 0 10.9 x 10.516.8 x 15.0 6.0 x 5.2 15.8 x 15.0

4 13.5 X 12.6 2.3 x 2.3 12.9 x 12.219.3 x 17.9 7.3 x 6.5 16.8 x 15.812.1 x 11.5 2.4 x 2.2 10.4 x 9.8

Mean 16.0 x 14.4 6.6 x 6.0 14.1 x 13.3

Controla 2.3 x 2.3 2.3 x 2.1 3.4 x 2.9

aFrom four nonsensitized animals.

charide and peptide (19) which give rise to bothimmediate and delayed hypersensitivity reac-tions in sensitized guinea pigs (20). The presentreport deals with chemical studies of the puri-fied antigenic polysaccharide-peptide com-pounds derived from S. schenckii cells and withthe chemical components responsible for immu-nological activities. Our main antigenic fraction(CP) consisted of 87.1% of a sugar moiety whichcontained rhamnose, mannose, and galactose(2.1:5.6:1.0) and 12.5% of a peptide moiety, sothe CP is the rhamnose-containing polysaccha-ride-peptide compound. Therefore, this fractionis an ideal model for studying involvement ofpolysaccharide and peptide in the immediate-

and delayed-type reactions. It has been clearlyshown that the CP and the papain-treated CP-Iwere found to be active by the test of passivecutaneous anaphylaxis. However, the perio-date-treated CP-II did not give any bluing spot.On the other hand, treatment with papain(CP-I) significantly abolished the delayed-typereactivities, including both inhibition of perito-neal cell migration and the delayed skin test,whereas treatment of periodate (CP-ll) did notabolish the delayed-type reactivities. The re-sults of this study indicated that the polysac-charide moiety could not elicit delayed-typereactions in guinea pigs sensitized with S.schenckii cells, but provoked the immediate-type reaction, and also that the peptide moietyelicited only the delayed-type reaction. Theseexperimental results are supported by the pre-cipitin activities in agar gel, which are related toa humoral antibody, since the enzyme-digestedfraction (CP-I) showed the same precipitin arcsas those with the undigested fraction (CP).Similar results have been found with the otherfungi (1, 3, 4).

In these experiments peptide moiety couldnot be completely removed by papain digestionso that the delayed-type reaction was not com-pletely abolished. It might be possible that asmall proportion of the polysaccharide is con-cemed with the mediation of the delayed-typereaction. Until the two main components of themolecule can be completely separated, it willnot be certain whether only the peptide isconcerned in the delayed-type reaction or not.Further studies on purification of the antigenmay resolve this question.

ACKNOWLEDGMENTThis work was supported in part by a research grant (no.

877104) from the Japan Ministry of Education.

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