INFECTION AND IMMUNITY, July 1979, p. 357-3650019-9567/79/07-0357/09$2.00/0

Galactomannan Antigenemia in Invasive AspergillosisE. REISS'* AND PAUL F. LEHMANNt

Mycology Division, Center for Disease Control, Atlanta, Georgia 30333,1 and Immunology Division,

Department of Pathology, University of Cambridge, Cambridge CB1 2QQ EnglandReceived for publication 18 April 1979

Galactomannan (GM) extracted from mycelia of Aspergillus fumigatus withcold dilute alkali reacted with antiserum specific for an antigen that circulated ininvasive aspergillosis in rabbits and humans. The GM was purified by its affinityfor concanavalin A and was separated from a nonantigenic glucan by gel permea-tion on Sephacryl S-200. The GM molecular weight of between 25,000 to 75,000was smaller than the antigen present in infected rabbit serum which was retainedby an ultrafiltration membrane that had a nominal molecular weight limit of125,000. The ratio of galactose to mannose present in GM was 1:1.17. Theserological activity of GM was stable to boiling but labile to 0.01 N HOl,implicating galactofuranose as an antigenic determinant. Analysis of purified GMby methylation-gas chromatography suggested a structure consisting of a 1 -- 6-linked mannan backbone with oligogalactoside side chains 3 units long, terminat-ing in galactofuranose. The presence of mannose as a side chain component was

also inferred. Another antigen ofA. fumigatus, which did not bind to concanavalinA, was isolated after tandem chromatography on diethylaminoethyl- and car-

boxymethyl-Sephadex and was identified as a galactan. The galactan inhibitedthe immune precipitation of GM with specific antiserum.

Invasive aspergillosis is encountered in someleukemia patients who are receiving intensivechemotherapy and broad spectrum antibiotics.The trend towards heavy immunosuppressionand the success in treating previously lethalopportunistic bacterial infections have led to anincrease in the number of invasive aspergillosiscases. One major cancer treatment center re-

ported 93 cases of invasive aspergillosis in a 5.5-year period (16), as reviewed by Armstrong etal. (1). The demand for timely laboratory diag-nosis of this infection has not been met becauseof the shortcomings of present methodology. Anantemortem serodiagnosis is difficult becausethe absence of precipitins may be linked to hy-pogamma-globulinemia (1). Only a few survivorshave been reported, and treatment with theantifungal agent amphotericin B must be startedearly in the disease if it is to be successful (10).

In an earlier report from this laboratory anti-gen was detected in rabbits that were immuno-suppressed with cortisone and cyclophospha-mide before infection with Aspergillus fumiga-tus began (12). By using serum from an infectedrabbit as the source of circulating A. fumigatusantigen, it was possible to produce, in a secondrabbit, an antiserum that detected the circulat-ing antigen. Using this reference antiserum, the

t Present address: Department of Microbiology, MedicalCollege of Ohio, CS 1008 Toledo, OH 43699.

first demonstration of circulating antigen in a

human patient with invasive aspergillosis was

made. Antisera produced by the conventionalmeans of injecting rabbits with killed mycelium,or with the fungus culture filtrate, were notcapable of detecting circulating A. fumigatusantigen.

Preliminary results (12) showed that an alka-line borohydride extract (CA) of autoclavedwhole mycelium contained a nondialyzable fac-tor capable of being absorbed and precipitatedby the antibody directed against circulating an-

tigen. This antigenic activity was resistant todigestion by pronase, but was susceptible toperiodate oxidation, suggesting the involvementof carbohydrate determinants. The CA extractcomigrated in immunoelectrophoresis with cir-culating antigen from immunosuppressed, in-fected rabbits. With these observations as a

guide, a series of column chromatographic pro-cedures was devised to further purify and char-acterize this antigen.

MATERIALS AND METHODS

Antiserum production. Antiserum to circulatingantigen was produced in rabbits by the method de-

scribed previously (12). Briefly, rabbits were immu-nosuppressed by receiving 200 mg of cyclophospha-mide and 30 mg of cortisone, intravenously, 1 daybefore intravenous injection of 5 x 10' A. fumigatusconidia. Within 6 days, mycelium was detected in the

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358 REISS AND LEHMANN

liver, kidney, brain, and heart of each rabbit, as wellas antigenemia. The serum containing fungal antigenwas removed. Other rabbits were immunized with theserum in FCA to produce a reference antibody. Anti-genemia was detected by counterimmunoelectropho-resis (CIE) (Fig. 1). The circulating antigen was des-ignated AFSA (A. fumigatus serum antigen) and an-tisera, which reacted with it as anti-AFSA. Furtherbatches of anti-AFSA were produced in rabbits, withprecipitin lines used as sources of antigen (21). Aprecipitin line was formed between anti-AFSA andAFSA in the serum of an infected rabbit by using CIE(Fig. 1) (12). These lines were cut out of several gelsand, after washing in cold saline eight times over 2days, they were homogenized and injected intramus-cularly in Freund complete adjuvant. After 3 weeksthe rabbits were given booster injections intradermallyin several sites on their backs.

Culture and conditions of growth. A. fumigatus2085 was obtained from the London School of Hygieneand Tropical Medicine, the United Kingdom. It wasoriginally isolated in 1952 from a lung cavity of apatient with Friedlander's pneumonia. The culture ismaintained in the culture collection of the MycologyDivision, Center for Disease Control, as B2570. Forthe production of antigens, a 17-liter glass carboy wasfilled with 15 liters of Czapek-Dox medium (DifcoLaboratories, Detroit, Mich.) supplemented with thefollowing cations: MnCl2.4H20, FeSO4 *7H20, ZnS04.7H20, 10-5 M; CuSO4 . 5H20, 10-6 M; CaCl2.2H20, 10-4M. A 48-h starter culture, grown in two 1-liter flaskseach containing 300 ml was incubated at 30'C and at150 rpm on a gyratory shaker. The carboy was seededwith this growth, and incubation took place at 22 to24°C for 54 h with forced aeration and magnetic stir-ring (Fig. 2). Then, 150 ml of 1% Merthiolate (sodiumethylmercurithiosalicylate, Aldrich Chemicals, Mil-waukee, Wisc.) was added, and the culture was stirredfor 18 h without aeration. The dense growth waspoured over five layers of unbleached cotton muslin ina Buchner funnel, washed with 4 liters of 0.85% NaCland 8 liters of deionized water. The washed fungusmat was peeled away from the filter, squeezed dry,and stored at -40°C.Antigen extraction. The washed mycelium was

suspended in 2 liters of 0.02M citric acid-NaOH buffer(pH 7.0) and autoclaved for 90 min at 121°C and 7.82atmospheres of pressure. After cooling, the suspensionwas centrifuged (8,000 x g, 30 min) and the pellet was

ReferenceArtiserum

Antigenerric.arrNple

FIG. 1. Detection of antigenemia by CIE.

AIR

i~ ZL h

VACUUM LINE

FIG. 2. Carboy device for growth of A. fumigatus.Filtered intake air flows through gas dispersion tubeand circulates through magnetically stirred culture.Two traps are placed in outflow line, leading tovacuum source (design suggested by H. F. Hasen-clever).

resuspended in 1 liter of the same buffer with the aidof a Waring blender. The mycelium was autoclavedagain for 30 min and centrifuged, and the pellet, re-sistant to hot buffer extraction, was resuspended in850 ml of freshly prepared and degassed alkaline bor-ohydride (0.4 N NaOH, 0.1 M NaBH4). The suspensionwas placed in a 1-liter polypropylene bottle, flushedwith nitrogen, then tightly stoppered and extractedwith stirring for 21.5 h in an ice bath. After centrifu-gation (8,000 x g, 30 min) the turbid supernatant wasrecovered and brought to pH 6.5 with acetic acid. Theresulting precipitate was removed by centrifugation(4,000 x g, 20 min), and the supernatant was concen-trated in an ultrafiltration cell (TCF 10, Amicon Corp.,Lexington, Mass.) under positive N2 pressure in thecold using a PM 10 membrance (Amicon) that had anominal molecular weight limit for proteins of 10,000.The retentate, 125 ml, was dialyzed in the cold for 24h versus four changes of 6 liters of deionized water.The material remaining in the dialysis sac was clarifiedby centrifugation (20,000 x g, 20 min), lyophilized, andstored at -40°C over a silica gel dessicant. The prod-uct was designated CA.Binding to lectins. CA at a concentration of 1 mg/

ml in potassium phosphate buffer (0.02 M, pH 7.4)containing 0.14 M NaCl (phosphate-buffered saline)was combined with an equal volume of various lectins,at 2 mg/ml, and incubated at 37°C for 1 h in PBS

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unless otherwise indicated. Then the reaction mixturewas tested in immunodiffusion versus antiserumAFSA. Lectins in buffer and no added CA were oper-ated as controls. The following lectins were tested:concanavalin A (ConA, Miles Laboratories, Inc., Elk-hart, Ind.) in tris(hydroxymethyl)aminomethane-hy-drochloride (0.01 M, pH 7.2, containing 1 mM CaCl2,1 mM MnCl2), Lens culinaris hemagglutinin B (Miles-Yeda), soybean agglutinin (Miles-Yeda), wheat germagglutinin (Miles-Yeda), pokeweed mitogen (SigmaChemical Co., St. Louis, Mo.), and phytohemaggluti-nin-P (Difco Laboratories, Detroit, Mich.).ConA-Sepharose chromatography. A column

(1.2 by 25 cm) packed with ConA-Sepharose (Phar-macia Fine Chemicals, Inc., Piscataway, N.J.) wasequilibrated at 40C with Tris-hydrochloride buffer(0.01 M, pH 7.2) containing MnCl2 (1 mM) and CaCl2(1 mM) (7). The CA sample was dissolved in 3 ml ofTris buffer and applied to the column. The effluentwas monitored at 280 nm with a flow analyzer, (Uvi-cord II, LKB Instruments, Bromma, Sweden), and 5-ml fractions were collected. Material binding to ConAwas eluted with 0.2 M a-methyl mannoside (a-MM).Fractions were screened for protein with the Folinphenol reagent (15) by using a bovine serum albuminfraction V standard. Carbohydrate in the effluent wasdetermined with phenol-sulfuric acid with respect toglucose (9). The eluate was pooled, dialyzed versusdeionized water, and lyophilized.Column chromatography. Small-scale binding in

3-ml plastic syringe columns was carried out to see ifCA antigen would bind to either carboxymetbyl (CM)-Sephadex C-25 or to diethylaminoethyl (DEAE)-Sephadex A-50. The CM columns were operated atpH 3.9, pH 5.5 in acetic acid-sodium acetate (0.05 M);and pH 7.1 in potassium phosphate (0.05 M). TheDEAE column was equilibrated with Tris-hydrochlo-ride buffer (0.05 M, pH 7.1). Columns were chargedwith CA, and the first 20 ml of effluent was reserved.Then 20 ml of buffer containing 0.5 M NaCl was usedfor elution of bound solutes. The pooled effluents andeluates were assayed for serological activity versusanti-AFSA and for protein, carbohydrate, and ribo-nucleic acid (RNA). The orcinol method for determin-ing RNA was used with a yeast sodium ribonucleatestandard (Nutritional Biochemicals Co., Cleveland,Ohio). For tandem chromatography, three 1.2-cm di-ameter columns were connected in series: first, ConA-Sepharose (20.4 ml); second, CM-Sephadex C50 (10.2ml); and third, DEAE-Sephadex A50 (12.4 ml). Allgels were obtained from Pharmacia. The ConA columnwas equilibrated with tris(hydroxymethyl)amino-methane-hydrochloride buffer (0.025 M, pH 7.2) con-taining 10-4 M each of MnCl2 and CaCl2. The DEAEand CM columns were equilibrated with the samebuffer minus added cations, and this served as thereservoir buffer for chromatography. The CA samplein 7.5 ml was applied to the ConA column, and frac-tions of 5 ml were collected from the effluent of theDEAE column. The effluent was monitored at 280 nm,and fractions were sampled for total carbohydrate andfor serological activity. Certain fractions were pooled,dialyzed versus deionized water, and lyophilized. Thecolumns were uncoupled, and the ConA column waseluted with a-MM as stated above. The DEAE column

GALACTOMANNAN ANTIGENEMIA 359

was eluted with a linear gradient of 0 to 0.5 M NaCl inbuffer, and the fractions were sampled for protein andcarbohydrate and then pooled, dialyzed, and lyophi-lized.The serologically active eluate from the ConA col-

umn was subjected to gel permeation chromatographyon Sephacryl S-200 superfine (Pharmacia). A column(2 by 95 cm) was packed with gel and equilibratedwith potassium phosphate buffer (0.025 M, pH 7.4)containing 0.07 M NaCl. The sample consisting of 29.9mg of ConA eluate was dissolved in 0.5 ml and appliedto the column. A flow rate of 20 ml/h was maintained,and fractions of 2.5 ml were collected, sampled forcarbohydrate and serological activity, and then pooledaccordingly, dialyzed, and lyophilized.

Ultrafiltration. A rapid estimate of molecular sizewas attempted by using membranes differing in theirnominal molecular weight limits. The antigens testedwere CA, the Sephacryl S200-included peak, and se-rum from an immunosuppressed, infected rabbit whichcontained AFSA. The membrane ultrafiltration de-vices were Minicons B15, exclusion limit 15,000; A75,75,000 limit; and S125, 125,000 limit (Amicon). Alsoemployed were 3-ml ultrafiltration cells requiring N2pressure (Millipore Corp., Bedford, Mass.) with mem-branes PSED, 25,000 limit, and PTJM, 100,000 limit.Antigens were dissolved in water or saline to give aconcentration of 0.05 mg/ml and then were concen-trated 1Ox. After diafiltration with an additional ali-quot of water or saline, the retentates were tested forserological activity in immunodiffusion versus anti-AFSA.Acid hydrolysis. Mild acid hydrolysis of CA in

0.01 N HCl (1 mg/0.5 ml) was carried out under N2 at100°C for 1, 2, and 3 h. In controls water was substi-tuted for acid. Samples then were dried under N2 at50°C, and traces of acid were removed in vacuo oversoda lime. Timed hydrolysis of CA in 2 N trifluoroac-etic acid (TFA) was used to determine conditions forthe optimal release of monosaccharides. Five milli-grams of CA and 1 ml of 2 N trifluoroacetic acid werecombined in a 3.7-ml vial, flushed with N2, fitted witha Teflon lined screw cap, and hydrolyzed for 1 to 4 h.Hydrolyzates were dried under N2, and traces of acidwere removed in vacuo. For amino sugar detection,samples were heated under N2 in 4N HCl for 6 h at1000C.Monosaccharide detection. The distribution of

monosaccharides was estimated by descending paperchromatography in which the upper phase of thesolvent system composed of n-butanol-pyridine-wa-ter-toluene (5:3:3:4, vol/vol) (11) was used. After de-velopment for 53 h, sugars were detected with alkalinesilver nitrate dip (24) or aniline phthalate spray (17)reagents. Galactostat and glucostat special reagenttest kits (Worthington Biochemicals Corp., Freehold,N.J.) were used according to the manufacturer's direc-tions. Amino sugar was detected by the Elson-Morganreaction (2) with respect to glucosamine-hydrochlo-ride. Gas-liquid chromatography of the trimethylsilylether derivatives of monosaccharides was carried outby the method of Sweeley et al. (23). Dry, neutralizedhydrolyzates were derivatized with 0.1 or 0.2 ml oftrimethylsilylimidazole in dry pyridine, (tri-sil Z,Pierce Chemical Co., Rockford, Ill.) and separated on

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360 REISS AND LEHMANN

a column (6.4 mm ID by 2 m) of SE52 (5% phenyl-methylsilicone) on 100/120-mesh Supelcoport oper-ated isothermally at 1500C in a Perkin Elmer 990 gaschromatograph. The N2 carrier gas flow rate was 71ml/min, and typical retention times for standards werea-mannose, 14 min; f?-mannose, 22.5 min; a-glucose,21.5 min; ,B-glucose, 37.5 min; y-galactose, 16.5 min;a-galactose, 19 min; and f-galactose, 23.5 min.Methylation analysis. GM (1 mg) was permeth-

ylated with dimethylsulfinyl-sodium and methyl io-dide in the Hakomori procedure (13). The methylatedpolysaccharide was treated with 88% formic acid (2 h,1000C), and then the acid was removed under N2.Next, hydrolysis was carried out with 0.3 N trifluo-roacetic acid (12 h, 1000C, under N2) followed byvolatilization of the acid and removal of traces invacuo over soda lime. Alditol acetates of the methyl-ated monosaccharides were prepared (13) and sepa-rated by gas chromatography on a column (6.4 mm IDby 3 m) of 3% ECNSS-M (ethylene succinate-phenyl-silicone copolymer) coated on 100/120-mesh GasChrom Q operated isothermally at 190° C. The meth-ylated monosaccharides were tentatively identified bycalculating of retention times (T values) relative to anauthentic standard of 2,3,4,6-tetra-O-methyl glucose(Supelco Inc., Bellefonte, Pa.) and comparing themwith published T values (13).

Serological tests. The criterion for determiningantigenic activity was the ability of a column-derivedfraction to form an immunoprecipitin arc with anti-AFSA either in immunodiffusion or CIE. Conditionsfor immunodiffusion were in PBS (pH 7.2) polyethyl-ene glycol 6,000 (2%, wt/vol) agar or, for CIE, inbarbital buffer as previously described (12). In some

cases fractions were shown to have antigenic activityby the inhibition of the reaction between anti-AFSAwith CA. This was brought about by incubating anti-AFSA with the antigenic fraction before using it in a

CIE test.

RESULTS

Yield of CA. The compressed filter cake ofA. fumigatus mycelium obtained from 15 litersof culture weighed 332 g. Sporulation was neg-

ligible in this submerged culture. From thisstarting material 447 mg of dialyzed and lyoph-ilized CA was derived containing 56.5% carbo-hydrate, 34.5% protein, and 1.47% moisture.Later, RNA was also found (see below).Lectin reactivity and ConA-Sepharose

chromatography. ConA was the only lectin, ofthose tested, that reacted with CA in immuno-diffusion. A separation based on affinity for in-solubilized ConA was then attempted. Of the100 mg of CA applied to the column, 25.5 mgwas recovered in the effluent fractions 5 to 7,and 4.6 mg was recovered in fractions 9 to 21(Fig. 3). The a-MM eluate, fractions 25 to 28,contained 20.5 mg (dry weight). All three pooledfractions were reactive in CIE versus anti-AFSA.Virtually all of the eluate was accounted for as

carbohydrate, with only 0.04% Lowry-positive

INFECT. IMMUN.

material. Effluent fractions 5 to 7 contained19.5% total carbohydrate, 28.3% protein, and35.7% RNA. This fraction was digested for 2 hwith enzite-agarose insolubilized ribonuclease.Under these conditions the digestion did not goto completion. Of the 22.3 mg of starting mate-rial, 17.4 mg was recovered after digestion, for aloss of 22% in dry weight.Ion-exchange and tandem chromatogra-

phy. Early experiments suggested that the an-tigen present in CA migrated cathodically duringimmunoelectrophoresis (12). Therefore, it wasconsidered worthwhile to test for binding to thecation exchanger CM-Sephadex. When 3 mg wasapplied to columns at pH 3.9, 5.6, 6.5, and 7.1,all serological activity was found in the effluentsand none was present in the 0.5 M NaCl eluates.When 3 mg of CA was applied to a DEAE-Sephadex column atpH 7.1, all antigenic activitywas present in the effluent and none was presentin the eluate. A purification was effected by ion-exchange chromatography since the CM eluatecontained 672 ,ug of protein and no carbohydrate.The DEAE eluate contained 260 ,ug of carbohy-drate, 72 ,ug of protein, and 451 1Lg of RNA.These results suggested that CM- and DEAE-Sephadex columns operated at pH 7.1 wouldallow antigen to pass through and would bindinert protein and RNA. The effluent profile,obtained from operating the ConA, CM, andDEAE columns in tandem, is shown in Fig. 4.Starting with 204 mg of CA, 8.03 mg was re-covered in effluent fractions 4 to 6, and 3.93 mgwas recovered in fractions 7 to 10. The columnswere uncoupled, and after elution with 0.5 MNaCl, 48.5 mg was recovered from the DEAEand 8.6 mg was recovered from the CM column.The yield from the ConA column eluted witha-MM was 40.1 mg. The combined recoveryfrom the tandem chromatography was 111.4 mg.Serological activity resided in three of the frac-tions. The ConA eluate formed an immunopre-cipitin arc in CIE with anti-AFSA. The tandemeffluent fractions 7 to 10 could inhibit the CIEreaction between CA and anti-AFSA, or be-tween infected rabbit serum and anti-AFSA, butwas incapable of producing an immune precipi-tate. A similar inhibitory behavior was found inthe eluate from the DEAE column.Gel permeation. The ConA eluate fraction

that formed a precipitin arc with anti-AFSA wasfurther characterized by gel permeation chro-matography on Sephacryl S-200 (Fig. 5). Thisgel had an exclusion limit of 80,000 for dextrans,according to the manufacturer. Of the 30 mg ofConA eluate applied to the column, 10.3 mg wasrecovered. This separated into a major excludedpeak, fractions 38 to 44, 6.7 mg; and a minor

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VOL. 25, 1979 GALACTOMANNAN ANTIGENEMIA 361

5-7 9-21 25-28

205001_

7000

6000-

Toa:

4000- -0.8I~~~~~~~~

3000- 0.6 E

F00

0~~~~~~~~~~~~~~~

2000- -0.4IU 0ae,/ \

ooo0- -l\0.2

5 l1 15 20 25 30 35FRACTION NO.

FIG. 3. ConA-Sepharose chromatography ofCA extract. A 0.2Mconcentration ofa-MM elated the materialin fr-actions 25 to 28. (-----) indicates strip chart recording fr-om ultraviolet flow analyzer shown. on rightvertical axis as optical density at 28 nm (O.D.ms.=).

4-6 7-11 included peak, fractions 50 to 64, 3.6 mg. All6000- serological activity resided in the minor included

peak.5000- Monosaccharide composition. Preliminary

ffi -% acid hydrolysis of the CA extract and paperA l chromatography showed that 2.5 h was the op-U;00- timal hydrolysis time and that the extract con-

!~~j4200004

z z \ ~~~~~~~~~~tainedglucose, mannose, galactose, and ribose.> > \ ~~~~~~~~TheConA equate contained glucose, mannose,

°0 3000-A and galactose, but the tandem chromatography8 ^ effluent fractions 7 to 10 had only galactose and8~~~glucose. Mild acid hydrolysis of 1 mg of CA in

X2000- Al l-0.2 ' 0.01 N HCI for 1, 2, and 3 h released galactoseI! | l | ~~~~~~~amounting to 22.5, 43.3, and 60 IL&, respectively.

--0000 100 Al h erlgclatviyoMAwsMeue fe

\ ° ~~~~~~1h and abolished after 2 h ofhydrolysis; whereasis< ~~~~~~~~thecontroL boiled in water for 3 h, still retained9,^___,__ ~~~~~serological activity. The molar ratios of mono-

510 1 520 255saccharides determined as the trimethylsilylFRACTION NO. ethers were, for the SephacrylS-2O-included

fraction, galactose-mannose glucose (1:1.16:FIG. 4. Effluent profile fromatandem chromatog-raphy of CA extract.ConA-Sepharotes DEaE-Seph- 0.14); and for the tandem chromatography ef-adex, and CM4-6phadexcoludedfractions 7 50 to ose glucose (1:tandem (see text). 0.23). The Sephacryl S200-excluded fraction andI

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362 REISS AND LEHMANN

tandem effluent fractions 4 to 6 contained onlyglucose. The absence of amino sugar in theSephacryl S200-included and tandem fractions7 to 10 was verified after hydrolysis in 4 N HClfor 6 h at 1000C. Only a trace, <1%, of aminosugar was detected in fractions 7 to 10. At thispoint it seemed justifiable to refer to the Seph-acryl S200-included fraction as a probable GMand to the tandem effluents 7 to 10 as a galactan.An additional amount ofGM was prepared by

separating 51.5 mg ofCA on Sephacryl S200 intoan excluded fraction (16.1 mg) and an includedfraction consisting of 14.8 mg of GM. The sepa-ration of glucan from the GM was judged com-plete, since galactose and mannose (1:1.17) werethe only sugars detected by gas chromatographyin the included fraction (Fig. 6).Methylation analysis. GM purified by re-

tention on ConA-Sepharose and inclusion on

3000

38-44 50-64

2500

t2000-Li

>- 1500-4

C 1000

500L N30 40 50 60 70 80

FRACTION NUMBER

FIG. 5. Gelpermeation ofConA eluate on Sephac-ryl S-200. V. = void volume determined with bluedextran 2000.

og 30-man

020 L galQ.

0

Sephacryl S200 was the starting material (Fig.7). Only galactose and mannose, not glucose,were present. Some structural features were es-timated as shown in Table 1. The observed ratioof galactopyranose to galactofuranose of 2:1 sug-gested oligogalactosides that were 3 units longin the original molecule. A large amount of aderivative corresponding to terminal nonreduc-ing mannose was found. Mannose branch pointsappeared to occur through the C6 position andeither at C2 or C4.

Ultrafiltration. The fraction ofmain serolog-ical interest, GM, was resolved by SephacrylS200, which has an exclusion limit for dextransof approximately 80,000. Another estimate ofmolecular size was obtained by ultrafiltration ofthe GM on membranes with different nominalmolecular weight limits. The antigenic activitywas retained by the PSED membrane, but notby the A75 membrane, suggesting a molecularweight range of between 25,000 and 75,000. Onthe other hand, the antigen in the serum ofinfected rabbits was retained by the S125 mem-brane, indicating a molecular weight exceeding125,000.Glucan bound to ConA. In addition to the

GM, a glucan fraction was observed to bind toConA and could be recovered in the a-MM

l 600)inc0Q.0~0I..

o 40-.0)-0w

20-

2

14

15

10 20 30retention time, min.

FIG. 7. Gas-liquid chromatography of SephacrylS200-included fraction as permethylated alditol ace-tate derivatives. For tentative identifications see Ta-ble 1.

retention time, min.

FIG. 6. Gas-liquid chromatography of SephacrylS-200 included fraction as alditol acetate derivatives.

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GALACTOMANNAN ANTIGENEMIA 363

TABLE 1. Tentative identification ofmethylated monosaccharides derived from A. fumigatus 2085 GM

Peak no.a T valueb Methylated derivative Original linkage Molar ratio

1 0.98 2,3,4,6-tetra-O-methylmannose Man 1 52 1.07 2,3,5,6-tetra-O-methylgalactose Gal furc 1 33 1.89 3,4,6-tri-O-methylmannose 2 Man 1 34 2.34 3,4,6

or tri-O-methylgalactose 2 Gal 12,3,6 or

4 Gal 1 65 4.32 2,3-di-O-methylmannose 4 Man 1 3.6

16

6 4.88 3,4-di-O-methylmannose 2 Man 1 216

a See Fig. 7.b With respect to 2,3,4,6-tetra-O-methylglucose.e Man, Mannose; gal, galactose; fur, furanose.

eluate. It was subsequently resolved in the ex-cluded portion of the Sephacryl S200 column.This material had no detectable serological ac-tivity. It was precipitated upon acidification topH 2. When a glucan sample, 460 ILg, was incu-bated overnight at 370C with a(1 -- 3)glucanase(20), 142 ,jg of glucose was released, giving a30.9% digestion.Premortem diagnosis of human invasive

aspergillosis. Three persons with invasive as-pergillosis, all of whom were leukemics, whosedisease was diagnosed by histopathology, havebeen shown to have circulating antigen detect-able by CIE.Another three human patients suspected of

having invasive aspergillosis, but not confirmedby histopathology, were positive for antigene-mia. At present, a total of 6 persons havingaspergillosis out of 40 to 60 persons suspected ofhaving this disease gave CIE evidence of circu-lating antigen.

DISCUSSIONThe objective of the present work was to

characterize the antigenic portion of the myce-lial extract ofA. fumigatus that could react withan antiserum produced in rabbits immunizedwith antigenemic serum from an immunosup-pressed, infected rabbit. It was hoped, by thismeans to identify the antigen that circulated inexperimental rabbit aspergillosis and in invasivehuman aspergillosis. The evidence presented in-dicates that this antigen is a GM polysaccharide(galactose-mannose, 1:1.17) with a molecularweight of between 25,000 and 75,000. Proof isbased on the ability of the ConA eluate-Sephac-ryl S200-included fraction to react in CIE withanti-AFSA and to absorb the antibody thatwould otherwise react with serum from an im-munosuppressed, infected rabbit. It was also

possible to show that another component of theCA extract was capable of absorbing anti-AFSA,but was insufficient to act as a precipitinogen inthe CIE test. This moiety did not bind ConAand was present in the pH 7.1 effluent ofDEAEand CM columns operated in tandem. After acidhydrolysis, it was identified as a galactan with atrace of glucose by three methods: paper chro-matography, galactostat, and gas-liquid chro-matography. The fortuitous separation of thegalactan from a glucan present in the tandemchromatography effluent was probably due to amolecular sieving effect.The main interest was focussed on the ConA

eluate because of its strong serological activity.The eluate contained a nonantigenic glucan asthe major component. This impurity was easilyremoved on Sephacryl S200 in the void volumeand was presumed to be a(1 -- 3)glucan becauseof its ConA-binding properties, susceptibility toa(1 -+ 3)glucanase, and acid precipitability (18).Other members of the genus Aspergillus, espe-cially A. niger, produce mycodextran, a glucancomposed of alternating a(1 -- 3) and a(1 -* 4)bonds (8). The A. fumigatus culture studiedherein was not capable of producing mycodex-tran, even when grown under the nitrogen-defi-cient conditions reported by Gold et al. (8) to befavorable to large yields of this glucan (T. F.Bobbitt, personal communication). This is prob-ably the case for A. fumigatus cultures in gen-eral. The antigenic activity of CA resided in aminor component of the ConA eluate that wasincluded on Sephacryl S200. This GM was pre-sumed to contain terminal galactofuranosyl an-tigenic determinants because of its lability to0.01 N HCl, with concomitant release of galac-tose and the loss of serological activity. Thedifferences in apparent molecular weight of theserum antigen AFSA (>125,000) and GM ex-

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364 REISS AND LEHMANN

tracted from mycelia (25,000 < GM < 75,000)suggest that the GM in AFSA exists in someform of complex. An interpretation for this dis-parity is thatGM are commonly complexed withprotein by either dilute alkali stable N-glyco-sylamine-asparagine bonds or alkali labile O-gly-cosyl-serine or -threonine bonds (14). The ne-cessity for using alkali as an extraction milieu todislodge the GM from its location in the cell wallof A. niger was described by Bardalaye andNordin (4) and was in accord with our experiencewith A. fumigatus. The low level of protein inthe GM suggests that it was eliminated from anO-glycosylhydroxyamino acid ester linkage tothe peptide moiety (22). This hypothetical lossof peptide count accounts for the inability ofCAto act as an antigen for producing anti-AFSA inrabbits, whereas the circulating material wasantigenic (12).

In its high galactose to mannose ratio (1:1.17)the GM of A. fumigatus resembled that of A.niger (1:1.05) and the galactomannan II (GMII)of Trichophyton mentagrophytes var. granu-losum (1:2.4) as reported by others (4, 5) anddiffers from the GMI (1:12.3) of T. mentagro-phytes var. granulosum (6). A further similaritywas noted between A. fumigatus GM and Tri-chophyton GMII in that neither bound toDEAE-based ion exchanger, though the lattercould bind in borate buffer (5). The structure ofA. niger GM, studied by Bardalaye and Nordin(4), consisted of a (1 -- 2)-linked mannan, with(1 -+ 6) branch points to the galactose oligosac-charides (3 to 4 units long). The terminal galac-tose occurred in the furanoside configuration.The most notable difference observed in themethylation analysis of A. fumigatus GM was asubstantial amount of terminal nonreducingmannose. The galactose side chains in A. fumi-gatus appear to be no more than 3 units long.The binding of A. fumigatus GM to ConA isevidence that galactose is covalently linked tomannose: moreover, the terminal mannose unitsthat were detected would be available for bind-ing to ConA, since this lectin's specificity isdirected to the axial C4 position (20). Anotherdifference between the two GMs was that theGM of A. niger had a single type of branchpoint, but the GM of A. fumigatus had twotypes. The structural picture of A. fumigatusGM that seems most compatible with these datais a 1-6-linked mannan backbone heavily substi-tuted with oligogalactoside side chains. The ter-minal mannose units that were indicated bymethylation analysis may occur as single sidesubstituents or as oligomannosides suspendedfrom the main chain. Partial acetolysis may fa-cilitate isolation of oligomannoside side chains

INFECT. IMMUN.

for further sequence and hapten inhibition anal-ysis.At present it is not known ifA. fumigatus GM

and the galactan, that was also isolated, form amacromolecular complex in the cell wall. Prelim-inary studies showed that the antigenic portionof GM appeared to be resistant to a number ofglycosidases (exo-a-mannanse, a-mannosidase,and ,f-galactosidase) in that the ability to reactwith anti-AFSA was unchanged by exposure tothese enzymes. Perhaps this resistance is one ofthe reasons that the AFSA survives in the host'scirculation.From the standpoint of the immunodiagnosis

of invasive aspergillosis, it has not been possible,thus far, to raise high antibody titers, a require-ment for sensitive assays, such as the sandwichenzyme-linked immunosorbent assay or hemag-glutination tests. The low rate of antigen detec-tion in humans known to have invasive aspergil-losis may be largely due to the insensitivity ofthe CIE test as used to detect AFSA (12). Evenin experimental infections, antigenemia is notalways detectable in mice that are heavily in-fected with A. fumigatus 2085. Certain strainsdo not produce detectable levels of AFSA invivo. (P. F. Lehmann and E. Reiss, Bull. Soc. Fr.Mycol. Med., in press). The isolation and char-acterization of a purified GM which reacts withanti-AFSA will facilitate the synthesis of GMconjugates that can be used to stimulate pro-duction of high titers of antibodies for detectingAFSA with assays more sensitive than CIE.Further structural studies will also be useful inidentifying the sugar sequences and glycosidicbond arrangements that differentiate the anti-gens of this fungus.

ACKNOWLEDGMENTSThis paper is dedicated to the memory of our esteemed

colleague, H. F. Hasenclever.P.F.L. was the recipient of a Wellcome Trust Research

Fellowship and was a Visiting Scientist in the MycologyDivision, Center for Disease Control.

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2. Ashwell, G. 1966. New colorimetric methods of sugaranalysis. Methods Enzymol. 8:85-95.

3. Azuma, I., H. Kimura, F. Hirao, E. Tsubura, Y. Ya-mamura, and A. Misaki. 1971. Biochemical and im-munological studies on Aspergillus. III. Chemical andimmunological properties of glycopeptide obtained fromAspergillus fumigatus. Jpn. J. Microbiol. 15:237-246.

4. Bardalaye, P. C., and J. H. Nordin. 1977. Chemicalstructure of the galactomannan from the cell wall ofAspergillus niger. J. Biol. Chem. 252:2584-2591.

5. Bishop, C. T., M. B. Perry, and F. Blank. 1966. The

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water-soluble polysaccharides of dermatophytes. V.Galactomannans II from Trichophyton granulosum,Trichophyton interdigitale, Microsporum quinck-eanum, Trichophyton rubrum, and Trichophytonschonkinii. Can. J. Chem. 44:2291-2297.

6. Bishop, C. T., M. B. Perry, F. Blank, and F. P. Cooper.1965. The water-soluble polysaccharides of dermato-phytes. IV. Galactomannans I from Trichophytongran-ulosum, Trichophyton interdigitale, Microsporumquinckeanum, Trichophyton rubrum and Trichophytonschonkinii. Can. J. Chem. 43:30-39.

7. Ellsworth, J. H., E. Reiss, R. L Bradley, H. Chmel,and D. Armstrong. 1977. Comparative serological andcutaneous reactivity of candidal cytoplasmic proteinsand mannan separated by affinity for concanavalin A.J. Clin. Microbiol. 5:91-99.

8. Gold, M. H., S. Larson, L. H. Segal, and C. R. Stocking.1974. Intracellular localization of nigeran in the wall ofAspergillus aculeatus by autoradiography with theelectron microscope. J. Bacteriol. 118:1176-1178.

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10. Krick, J. A., and J. S. Remington. 1976. Opportunisticinvasive fungal infections in patients with leukaemiaand lymphoma. Clin. Haematol. 5:249-310.

11. Lechevalier, M. P. 1968. Identification of aerobic acti-nomycetes of clinical importance. J. Lab. Clin. Med. 71:934-944.

12. Lehmann, P. F., and E. Reiss. 1978. Invasive aspergil-losis: antiserum for circulating antigen produced afterimmunization with serum from infected rabbits. Infect.Immun. 20:570-572.

13. Lindberg, B. 1972. Methylation analysis of polysaccha-rides. Methods Enzymol. 28:178-195.

14. Uoyd, K. 0. 1972. Molecular organization of a covalentpeptidophosphopolysaccharide complex from the yeastform of Cladosporium werneckii. Biochemistry 11:3884-3890.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

16. Meyer, R. D., L. S. Young, D. Armstrong, and B. Yu.1973. Aspergillosis complicating neoplastic disease. Am.J. Med. 64:6-15.

17. Partridge, S. M. 1949. Aniline hydrogen phthalate as aspraying reagent for chromatography of sugars. Nature(London) 164:443.

18. Reese, E. T., A. Maguire, and F. W. Parrish. 1972.Alpha-1, 3-glucanases of fungi and their relationship tomycodextranase, p. 735-742. In International Fermen-tation Symposium, 4th, Kyoto.

19. Reiss, E. 1977. Serial enzymatic hydrolysis of cell walls oftwo serotypes of yeast-form Histoplasma capsulatumwith a(1 -- 3)-glucanase, B(1 -* 3)-glucanase, Pronase,and chitinase. Infect. Immun. 16:181-188.

20. Sharon, N., and H. Lis. 1972. Lectins: cell-agglutinatingand sugar-specific proteins. Science 177:949-959.

21. Shivers, C. A., and J. M. James. 1967. Specific antibod-ies produced against antigens of agar-gel precipitates.Immunology 13:547-554.

22. Spiro, R. G. 1973. Glycoproteins. Adv. Protein Chem. 27:349-467.

23. Sweeley, C. C., W. W. Wells, and R. Bentley. 1966.Gas chromatography of carbohydrates. Methods En-zymol. 8:95-108.

24. Trevelyan, W. E., D. P. Procter, and J. S. Harrison.1950. Detection of sugars on paper chromatograms.Nature (London) 166:444-445.

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