INFECTION AND IMMUNITY, July 1979, p. 357-365 0019-9567/79/07-0357/09$2.00/0
Vol. 25, No. 1
Galactomannan Antigenemia in Invasive Aspergillosis E. 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 England Received for publication 18 April 1979
Galactomannan (GM) extracted from mycelia of Aspergillus fumigatus with cold dilute alkali reacted with antiserum specific for an antigen that circulated in invasive aspergillosis in rabbits and humans. The GM was purified by its affinity for concanavalin A and was separated from a nonantigenic glucan by gel permeation on Sephacryl S-200. The GM molecular weight of between 25,000 to 75,000 was smaller than the antigen present in infected rabbit serum which was retained by an ultrafiltration membrane that had a nominal molecular weight limit of 125,000. The ratio of galactose to mannose present in GM was 1:1.17. The serological activity of GM was stable to boiling but labile to 0.01 N HOl, implicating galactofuranose as an antigenic determinant. Analysis of purified GM by methylation-gas chromatography suggested a structure consisting of a 1 6linked mannan backbone with oligogalactoside side chains 3 units long, terminating in galactofuranose. The presence of mannose as a side chain component was also inferred. Another antigen of A. fumigatus, which did not bind to concanavalin A, was isolated after tandem chromatography on diethylaminoethyl- and carboxymethyl-Sephadex and was identified as a galactan. The galactan inhibited the immune precipitation of GM with specific antiserum. --
Invasive aspergillosis is encountered in some first demonstration of circulating antigen in a leukemia patients who are receiving intensive human patient with invasive aspergillosis was chemotherapy and broad spectrum antibiotics. made. Antisera produced by the conventional The trend towards heavy immunosuppression means of injecting rabbits with killed mycelium, and the success in treating previously lethal or with the fungus culture filtrate, were not opportunistic bacterial infections have led to an capable of detecting circulating A. fumigatus increase in the number of invasive aspergillosis antigen. Preliminary results (12) showed that an alkacases. One major cancer treatment center reported 93 cases of invasive aspergillosis in a 5.5- line borohydride extract (CA) of autoclaved whole mycelium contained a nondialyzable facyear period (16), as reviewed by Armstrong et al. (1). The demand for timely laboratory diag- tor capable of being absorbed and precipitated nosis of this infection has not been met because by the antibody directed against circulating anof the shortcomings of present methodology. An tigen. This antigenic activity was resistant to antemortem serodiagnosis is difficult because digestion by pronase, but was susceptible to the absence of precipitins may be linked to hy- periodate oxidation, suggesting the involvement pogamma-globulinemia (1). Only a few survivors of carbohydrate determinants. The CA extract have been reported, and treatment with the comigrated in immunoelectrophoresis with cirantifungal agent amphotericin B must be started culating antigen from immunosuppressed, inearly in the disease if it is to be successful (10). fected rabbits. With these observations as a In an earlier report from this laboratory anti- guide, a series of column chromatographic procedures was devised to further purify and chargen was detected in rabbits that were immunosuppressed with cortisone and cyclophospha- acterize this antigen. mide before infection with Aspergillus fumigaMATERIALS AND METHODS tus began (12). By using serum from an infected Antiserum production. Antiserum to circulating rabbit as the source of circulating A. fumigatus was produced in rabbits by the method deantigen, it was possible to produce, in a second antigen previously (12). Briefly, rabbits were immurabbit, an antiserum that detected the circulat- scribed by receiving 200 mg of cyclophosphanosuppressed ing antigen. Using this reference antiserum, the mide and 30 mg of cortisone, intravenously, 1 day
before intravenous injection of 5 x 10' A. fumigatus conidia. Within 6 days, mycelium was detected in the
t Present address: Department of Microbiology, Medical College of Ohio, CS 1008 Toledo, OH 43699. 357
358
INFECT. IMMUN.
REISS AND LEHMANN
liver, kidney, brain, and heart of each rabbit, as well as antigenemia. The serum containing fungal antigen was removed. Other rabbits were immunized with the serum in FCA to produce a reference antibody. Antigenemia was detected by counterimmunoelectrophoresis (CIE) (Fig. 1). The circulating antigen was designated AFSA (A. fumigatus serum antigen) and antisera, which reacted with it as anti-AFSA. Further batches of anti-AFSA were produced in rabbits, with precipitin lines used as sources of antigen (21). A precipitin line was formed between anti-AFSA and AFSA in the serum of an infected rabbit by using CIE (Fig. 1) (12). These lines were cut out of several gels and, after washing in cold saline eight times over 2 days, they were homogenized and injected intramuscularly in Freund complete adjuvant. After 3 weeks the rabbits were given booster injections intradermally in several sites on their backs. Culture and conditions of growth. A. fumigatus 2085 was obtained from the London School of Hygiene and Tropical Medicine, the United Kingdom. It was originally isolated in 1952 from a lung cavity of a patient with Friedlander's pneumonia. The culture is maintained in the culture collection of the Mycology Division, Center for Disease Control, as B2570. For the production of antigens, a 17-liter glass carboy was filled with 15 liters of Czapek-Dox medium (Difco Laboratories, Detroit, Mich.) supplemented with the following cations: MnCl2.4H20, FeSO4 *7H20, ZnS04. 7H20, 10-5 M; CuSO4 . 5H20, 10-6 M; CaCl2.2H20, 10-4 M. A 48-h starter culture, grown in two 1-liter flasks each containing 300 ml was incubated at 30'C and at 150 rpm on a gyratory shaker. The carboy was seeded with this growth, and incubation took place at 22 to 24°C for 54 h with forced aeration and magnetic stirring (Fig. 2). Then, 150 ml of 1% Merthiolate (sodium ethylmercurithiosalicylate, Aldrich Chemicals, Milwaukee, Wisc.) was added, and the culture was stirred for 18 h without aeration. The dense growth was poured over five layers of unbleached cotton muslin in a Buchner funnel, washed with 4 liters of 0.85% NaCl and 8 liters of deionized water. The washed fungus mat was peeled away from the filter, squeezed dry, and stored at -40°C. Antigen extraction. The washed mycelium was suspended in 2 liters of 0.02 M citric acid-NaOH buffer (pH 7.0) and autoclaved for 90 min at 121°C and 7.82 atmospheres of pressure. After cooling, the suspension was centrifuged (8,000 x g, 30 min) and the pellet was
Reference Artiserum
Antigenerric
.arrNple
FIG. 1. Detection of antigenemia
by CIE.
AIR
i~
ZL
VACUUM LINE
h
FIG. 2. Carboy device for growth of A. fumigatus. Filtered intake air flows through gas dispersion tube and circulates through magnetically stirred culture. Two traps are placed in outflow line, leading to vacuum source (design suggested by H. F. Hasenclever).
resuspended in 1 liter of the same buffer with the aid of a Waring blender. The mycelium was autoclaved again for 30 min and centrifuged, and the pellet, resistant to hot buffer extraction, was resuspended in 850 ml of freshly prepared and degassed alkaline borohydride (0.4 N NaOH, 0.1 M NaBH4). The suspension was placed in a 1-liter polypropylene bottle, flushed with nitrogen, then tightly stoppered and extracted with stirring for 21.5 h in an ice bath. After centrifugation (8,000 x g, 30 min) the turbid supernatant was recovered and brought to pH 6.5 with acetic acid. The resulting precipitate was removed by centrifugation (4,000 x g, 20 min), and the supernatant was concentrated in an ultrafiltration cell (TCF 10, Amicon Corp., Lexington, Mass.) under positive N2 pressure in the cold using a PM 10 membrance (Amicon) that had a nominal molecular weight limit for proteins of 10,000. The retentate, 125 ml, was dialyzed in the cold for 24 h versus four changes of 6 liters of deionized water. The material remaining in the dialysis sac was clarified by centrifugation (20,000 x g, 20 min), lyophilized, and stored at -40°C over a silica gel dessicant. The product 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
VOL. 25, 1979
unless otherwise indicated. Then the reaction mixture was tested in immunodiffusion versus antiserum AFSA. Lectins in buffer and no added CA were operated as controls. The following lectins were tested: concanavalin A (ConA, Miles Laboratories, Inc., Elkhart, Ind.) in tris(hydroxymethyl)aminomethane-hydrochloride (0.01 M, pH 7.2, containing 1 mM CaCl2, 1 mM MnCl2), Lens culinaris hemagglutinin B (MilesYeda), soybean agglutinin (Miles-Yeda), wheat germ agglutinin (Miles-Yeda), pokeweed mitogen (Sigma Chemical Co., St. Louis, Mo.), and phytohemagglutinin-P (Difco Laboratories, Detroit, Mich.). ConA-Sepharose chromatography. A column (1.2 by 25 cm) packed with ConA-Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) was equilibrated 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 of Tris buffer and applied to the column. The effluent was monitored at 280 nm with a flow analyzer, (Uvicord II, LKB Instruments, Bromma, Sweden), and 5ml fractions were collected. Material binding to ConA was eluted with 0.2 M a-methyl mannoside (a-MM). Fractions were screened for protein with the Folin phenol reagent (15) by using a bovine serum albumin fraction V standard. Carbohydrate in the effluent was determined with phenol-sulfuric acid with respect to glucose (9). The eluate was pooled, dialyzed versus deionized water, and lyophilized. Column chromatography. Small-scale binding in 3-ml plastic syringe columns was carried out to see if CA antigen would bind to either carboxymetbyl (CM)Sephadex C-25 or to diethylaminoethyl (DEAE)Sephadex A-50. The CM columns were operated at pH 3.9, pH 5.5 in acetic acid-sodium acetate (0.05 M); and pH 7.1 in potassium phosphate (0.05 M). The DEAE column was equilibrated with Tris-hydrochloride buffer (0.05 M, pH 7.1). Columns were charged with CA, and the first 20 ml of effluent was reserved. Then 20 ml of buffer containing 0.5 M NaCl was used for elution of bound solutes. The pooled effluents and eluates were assayed for serological activity versus anti-AFSA and for protein, carbohydrate, and ribonucleic acid (RNA). The orcinol method for determining RNA was used with a yeast sodium ribonucleate standard (Nutritional Biochemicals Co., Cleveland, Ohio). For tandem chromatography, three 1.2-cm diameter columns were connected in series: first, ConASepharose (20.4 ml); second, CM-Sephadex C50 (10.2 ml); and third, DEAE-Sephadex A50 (12.4 ml). All gels were obtained from Pharmacia. The ConA column was equilibrated with tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.025 M, pH 7.2) containing 10-4 M each of MnCl2 and CaCl2. The DEAE and CM columns were equilibrated with the same buffer minus added cations, and this served as the reservoir buffer for chromatography. The CA sample in 7.5 ml was applied to the ConA column, and fractions of 5 ml were collected from the effluent of the DEAE column. The effluent was monitored at 280 nm, and fractions were sampled for total carbohydrate and for serological activity. Certain fractions were pooled, dialyzed versus deionized water, and lyophilized. The columns were uncoupled, and the ConA column was eluted 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 in buffer, and the fractions were sampled for protein and carbohydrate and then pooled, dialyzed, and lyophilized. The serologically active eluate from the ConA column was subjected to gel permeation chromatography on Sephacryl S-200 superfine (Pharmacia). A column (2 by 95 cm) was packed with gel and equilibrated with potassium phosphate buffer (0.025 M, pH 7.4) containing 0.07 M NaCl. The sample consisting of 29.9 mg of ConA eluate was dissolved in 0.5 ml and applied to the column. A flow rate of 20 ml/h was maintained, and fractions of 2.5 ml were collected, sampled for carbohydrate and serological activity, and then pooled accordingly, dialyzed, and lyophilized. Ultrafiltration. A rapid estimate of molecular size was attempted by using membranes differing in their nominal molecular weight limits. The antigens tested were CA, the Sephacryl S200-included peak, and serum from an immunosuppressed, infected rabbit which contained AFSA. The membrane ultrafiltration devices were Minicons B15, exclusion limit 15,000; A75, 75,000 limit; and S125, 125,000 limit (Amicon). Also employed were 3-ml ultrafiltration cells requiring N2 pressure (Millipore Corp., Bedford, Mass.) with membranes PSED, 25,000 limit, and PTJM, 100,000 limit. Antigens were dissolved in water or saline to give a concentration of 0.05 mg/ml and then were concentrated 1Ox. After diafiltration with an additional aliquot of water or saline, the retentates were tested for serological activity in immunodiffusion versus antiAFSA. Acid hydrolysis. Mild acid hydrolysis of CA in 0.01 N HCl (1 mg/0.5 ml) was carried out under N2 at 100°C for 1, 2, and 3 h. In controls water was substituted for acid. Samples then were dried under N2 at 50°C, and traces of acid were removed in vacuo over soda lime. Timed hydrolysis of CA in 2 N trifluoroacetic acid (TFA) was used to determine conditions for the optimal release of monosaccharides. Five milligrams of CA and 1 ml of 2 N trifluoroacetic acid were combined in a 3.7-ml vial, flushed with N2, fitted with a Teflon lined screw cap, and hydrolyzed for 1 to 4 h. Hydrolyzates were dried under N2, and traces of acid were removed in vacuo. For amino sugar detection, samples were heated under N2 in 4N HCl for 6 h at
1000C. Monosaccharide detection. The distribution of monosaccharides was estimated by descending paper chromatography in which the upper phase of the solvent system composed of n-butanol-pyridine-water-toluene (5:3:3:4, vol/vol) (11) was used. After development for 53 h, sugars were detected with alkaline silver nitrate dip (24) or aniline phthalate spray (17) reagents. Galactostat and glucostat special reagent test kits (Worthington Biochemicals Corp., Freehold, N.J.) were used according to the manufacturer's directions. Amino sugar was detected by the Elson-Morgan reaction (2) with respect to glucosamine-hydrochloride. Gas-liquid chromatography of the trimethylsilyl ether derivatives of monosaccharides was carried out by the method of Sweeley et al. (23). Dry, neutralized hydrolyzates were derivatized with 0.1 or 0.2 ml of trimethylsilylimidazole in dry pyridine, (tri-sil Z, Pierce Chemical Co., Rockford, Ill.) and separated on
360
REISS AND LEHMANN
column (6.4 mm ID by 2 m) of SE52 (5% phenylmethylsilicone) on 100/120-mesh Supelcoport operated isothermally at 1500C in a Perkin Elmer 990 gas chromatograph. The N2 carrier gas flow rate was 71 ml/min, and typical retention times for standards were a-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 permethylated with dimethylsulfinyl-sodium and methyl iodide in the Hakomori procedure (13). The methylated polysaccharide 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 trifluoroacetic acid (12 h, 1000C, under N2) followed by volatilization of the acid and removal of traces in vacuo over soda lime. Alditol acetates of the methylated monosaccharides were prepared (13) and separated by gas chromatography on a column (6.4 mm ID by 3 m) of 3% ECNSS-M (ethylene succinate-phenylsilicone copolymer) coated on 100/120-mesh Gas Chrom Q operated isothermally at 190° C. The methylated monosaccharides were tentatively identified by calculating of retention times (T values) relative to an authentic standard of 2,3,4,6-tetra-O-methyl glucose (Supelco Inc., Bellefonte, Pa.) and comparing them with published T values (13). Serological tests. The criterion for determining antigenic activity was the ability of a column-derived fraction to form an immunoprecipitin arc with antiAFSA either in immunodiffusion or CIE. Conditions for immunodiffusion were in PBS (pH 7.2) polyethylene glycol 6,000 (2%, wt/vol) agar or, for CIE, in barbital buffer as previously described (12). In some cases fractions were shown to have antigenic activity by the inhibition of the reaction between anti-AFSA with CA. This was brought about by incubating antiAFSA with the antigenic fraction before using it in a
a
CIE test.
RESULTS
Yield of CA. The compressed filter cake of A. fumigatus mycelium obtained from 15 liters of culture weighed 332 g. Sporulation was negligible in this submerged culture. From this starting material 447 mg of dialyzed and lyophilized CA was derived containing 56.5% carbohydrate, 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, of those tested, that reacted with CA in immunodiffusion. A separation based on affinity for insolubilized ConA was then attempted. Of the 100 mg of CA applied to the column, 25.5 mg was 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 pooled fractions 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 contained 19.5% total carbohydrate, 28.3% protein, and 35.7% RNA. This fraction was digested for 2 h with enzite-agarose insolubilized ribonuclease. Under these conditions the digestion did not go to completion. Of the 22.3 mg of starting material, 17.4 mg was recovered after digestion, for a loss of 22% in dry weight. Ion-exchange and tandem chromatography. Early experiments suggested that the antigen present in CA migrated cathodically during immunoelectrophoresis (12). Therefore, it was considered worthwhile to test for binding to the cation exchanger CM-Sephadex. When 3 mg was applied to columns at pH 3.9, 5.6, 6.5, and 7.1, all serological activity was found in the effluents and none was present in the 0.5 M NaCl eluates. When 3 mg of CA was applied to a DEAESephadex column at pH 7.1, all antigenic activity was present in the effluent and none was present in the eluate. A purification was effected by ionexchange chromatography since the CM eluate contained 672 ,ug of protein and no carbohydrate. The DEAE eluate contained 260 ,ug of carbohydrate, 72 ,ug of protein, and 451 1Lg of RNA. These results suggested that CM- and DEAESephadex columns operated at pH 7.1 would allow antigen to pass through and would bind inert protein and RNA. The effluent profile, obtained from operating the ConA, CM, and DEAE columns in tandem, is shown in Fig. 4. Starting with 204 mg of CA, 8.03 mg was recovered in effluent fractions 4 to 6, and 3.93 mg was recovered in fractions 7 to 10. The columns were uncoupled, and after elution with 0.5 M NaCl, 48.5 mg was recovered from the DEAE and 8.6 mg was recovered from the CM column. The yield from the ConA column eluted with a-MM was 40.1 mg. The combined recovery from the tandem chromatography was 111.4 mg. Serological activity resided in three of the fractions. The ConA eluate formed an immunoprecipitin arc in CIE with anti-AFSA. The tandem effluent fractions 7 to 10 could inhibit the CIE reaction between CA and anti-AFSA, or between infected rabbit serum and anti-AFSA, but was incapable of producing an immune precipitate. A similar inhibitory behavior was found in the eluate from the DEAE column. Gel permeation. The ConA eluate fraction that formed a precipitin arc with anti-AFSA was further characterized by gel permeation chromatography on Sephacryl S-200 (Fig. 5). This gel had an exclusion limit of 80,000 for dextrans, according to the manufacturer. Of the 30 mg of ConA eluate applied to the column, 10.3 mg was recovered. This separated into a major excluded peak, fractions 38 to 44, 6.7 mg; and a minor
GALACTOMANNAN ANTIGENEMIA
VOL. 25, 1979
361
25-28 5-7 9-21 205001_
7000
6000-
a: To
I~~~~~~~~
F0
4000-
-0.8
0 0~~~~~~~~~~~~~~~
0.6 E
3000-
\
ae,/
2000IU
-0.4
0
-l\ 0.2
ooo0-
5
l1
20
15
25
30
35
FRACTION NO.
4200004 FIG. 3. ConA-Sepharose chromatography of CA extract. A 0.2 M concentration of a-MM elated the material !~~j in fr-actions 25 to 28. (-----) indicates strip chart recording fr-om ultraviolet flow analyzer shown. on right vertical axis as optical density at 28 nm (O.D.ms.=).
4-6
7-11
6000A 100 l 000 ffi% 5000-l A
included peak, fractions 50 to 64, 3.6 mg. All
serological activity resided in the minor included
h erlgclatviyoMAwsMeue fe peak. Monosaccharide composition. Preliminary acid hydrolysis of the CA extract and paper chromatography showed that 2.5 h was the optimal hydrolysis time and that the extract conz \ z glucose, mannose, galactose, and ribose. ~~~~~~~~~~tained ConA equate contained glucose, mannose, > \ ~~~~~~~~The > °0 3000-A and galactose, but the tandem chromatography effluent fractions 7 to 10 had only galactose and ^ 8 glucose. Mild acid hydrolysis of 1 mg of CA in N HCI for 1, 2, and 3 h released galactose 0.01 Al l-0.2 ' X2000| ~~~~~~~amounting to 22.5, 43.3, and 60 IL&, respectively. l I !| h and abolished after 2 h of hydrolysis; whereas ° ~~~~~~1 \ ~~~~~~~~the is< controL boiled in water for 3 h, still retained 9,^___,__ ~~~~~serological activity. The molar ratios of mono5 510 20 1 255 saccharides determined as the trimethylsilyl FRACTION NO. S-2O-included ethers were, for the Sephacryl tandem chromatog- fraction, galactose-mannose glucose (1:1.16: FIG. 4. Effluent profile froma efConA-Sepharotes DEaE-Seph- 0.14); and for the tandem50 chromatography raphy of CA extract. to ose glucose (1: adex, and CM4-6phadex coludedfractions 7 0.23). The Sephacryl S200-excluded fraction and tandem (see text).I --0
U;00-
8~~~
362
INFECT. IMMUN.
REISS AND LEHMANN
tandem effluent fractions 4 to 6 contained only glucose. The absence of amino sugar in the Sephacryl S200-included and tandem fractions 7 to 10 was verified after hydrolysis in 4 N HCl for 6 h at 1000C. Only a trace, - 15004
C 1000
500L 30
N 50
40
60
l
in
c
0
70
80
FRACTION NUMBER
FIG. 5. Gelpermeation of ConA eluate on Sephacryl S-200. V. = void volume determined with blue dextran 2000. og 30-
L
man Q.
020
60
0)
0~Q. 0 I..
o 40-
2
.00)-
14
w
20-
gal
15
0
10
retention time, min. FIG. 6. Gas-liquid chromatography of Sephacryl S-200 included fraction as alditol acetate derivatives.
20
30
retention time, min. FIG. 7. Gas-liquid chromatography of Sephacryl S200-included fraction as permethylated alditol acetate derivatives. For tentative identifications see Table 1.
GALACTOMANNAN ANTIGENEMIA
VOL. 25, 1979
363
TABLE 1. Tentative identification of methylated monosaccharides derived from A. fumigatus 2085 GM Molar ratio Original linkage Methylated derivative T valueb 5 1 Man 2,3,4,6-tetra-O-methylmannose 0.98 1 3 Gal furc 1 2,3,5,6-tetra-O-methylgalactose 1.07 2 3 2 Man 1 3,4,6-tri-O-methylmannose 1.89 3 3,4,6 2.34 4 2 Gal 1 tri-O-methylgalactose or or 2,3,6 6 4 Gal 1 3.6 4 Man 1 2,3-di-O-methylmannose 4.32 5
Peak no.a
1
6
4.88
3,4-di-O-methylmannose
6 2 Man 1
2
1
6
See Fig. 7. b With respect to 2,3,4,6-tetra-O-methylglucose. e Man, Mannose; gal, galactose; fur, furanose.
a
eluate. It was subsequently resolved in the excluded portion of the Sephacryl S200 column. This material had no detectable serological activity. It was precipitated upon acidification to pH 2. When a glucan sample, 460 ILg, was incubated overnight at 370C with a(1 -- 3)glucanase (20), 142 ,jg of glucose was released, giving a 30.9% digestion. Premortem diagnosis of human invasive aspergillosis. Three persons with invasive aspergillosis, all of whom were leukemics, whose disease was diagnosed by histopathology, have been shown to have circulating antigen detectable by CIE. Another three human patients suspected of having invasive aspergillosis, but not confirmed by histopathology, were positive for antigenemia. At present, a total of 6 persons having aspergillosis out of 40 to 60 persons suspected of having this disease gave CIE evidence of circulating antigen. DISCUSSION The objective of the present work was to characterize the antigenic portion of the mycelial extract of A. fumigatus that could react with an antiserum produced in rabbits immunized with antigenemic serum from an immunosuppressed, infected rabbit. It was hoped, by this means to identify the antigen that circulated in experimental rabbit aspergillosis and in invasive human aspergillosis. The evidence presented indicates that this antigen is a GM polysaccharide (galactose-mannose, 1:1.17) with a molecular weight of between 25,000 and 75,000. Proof is based on the ability of the ConA eluate-Sephacryl S200-included fraction to react in CIE with anti-AFSA and to absorb the antibody that would otherwise react with serum from an immunosuppressed, infected rabbit. It was also
possible to show that another component of the CA extract was capable of absorbing anti-AFSA, but was insufficient to act as a precipitinogen in the CIE test. This moiety did not bind ConA and was present in the pH 7.1 effluent of DEAE and CM columns operated in tandem. After acid hydrolysis, it was identified as a galactan with a trace of glucose by three methods: paper chromatography, galactostat, and gas-liquid chromatography. The fortuitous separation of the galactan from a glucan present in the tandem chromatography effluent was probably due to a molecular sieving effect. The main interest was focussed on the ConA eluate because of its strong serological activity. The eluate contained a nonantigenic glucan as the major component. This impurity was easily removed on Sephacryl S200 in the void volume and was presumed to be a(1 -- 3)glucan because of its ConA-binding properties, susceptibility to a(1 -+ 3)glucanase, and acid precipitability (18). Other members of the genus Aspergillus, especially A. niger, produce mycodextran, a glucan composed of alternating a(1 -- 3) and a(1 -* 4) bonds (8). The A. fumigatus culture studied herein was not capable of producing mycodextran, even when grown under the nitrogen-deficient conditions reported by Gold et al. (8) to be favorable to large yields of this glucan (T. F. Bobbitt, personal communication). This is probably the case for A. fumigatus cultures in general. The antigenic activity of CA resided in a minor component of the ConA eluate that was included on Sephacryl S200. This GM was presumed to contain terminal galactofuranosyl antigenic determinants because of its lability to 0.01 N HCl, with concomitant release of galactose and the loss of serological activity. The differences in apparent molecular weight of the serum antigen AFSA (>125,000) and GM ex-
364
REISS AND LEHMANN
tracted from mycelia (25,000 < GM < 75,000) suggest that the GM in AFSA exists in some form of complex. An interpretation for this disparity is that GM are commonly complexed with protein by either dilute alkali stable N-glycosylamine-asparagine bonds or alkali labile O-glycosyl-serine or -threonine bonds (14). The necessity for using alkali as an extraction milieu to dislodge the GM from its location in the cell wall of A. niger was described by Bardalaye and Nordin (4) and was in accord with our experience with A. fumigatus. The low level of protein in the GM suggests that it was eliminated from an O-glycosylhydroxyamino acid ester linkage to the peptide moiety (22). This hypothetical loss of peptide count accounts for the inability of CA to act as an antigen for producing anti-AFSA in rabbits, whereas the circulating material was antigenic (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. granulosum (1:2.4) as reported by others (4, 5) and differs from the GMI (1:12.3) of T. mentagrophytes var. granulosum (6). A further similarity was noted between A. fumigatus GM and Trichophyton GMII in that neither bound to DEAE-based ion exchanger, though the latter could bind in borate buffer (5). The structure of A. niger GM, studied by Bardalaye and Nordin (4), consisted of a (1 -- 2)-linked mannan, with (1 -+ 6) branch points to the galactose oligosaccharides (3 to 4 units long). The terminal galactose occurred in the furanoside configuration. The most notable difference observed in the methylation analysis of A. fumigatus GM was a substantial amount of terminal nonreducing mannose. The galactose side chains in A. fumigatus appear to be no more than 3 units long. The binding of A. fumigatus GM to ConA is evidence that galactose is covalently linked to mannose: moreover, the terminal mannose units that were detected would be available for binding to ConA, since this lectin's specificity is directed to the axial C4 position (20). Another difference between the two GMs was that the GM of A. niger had a single type of branch point, but the GM of A. fumigatus had two types. The structural picture of A. fumigatus GM that seems most compatible with these data is a 1-6-linked mannan backbone heavily substituted with oligogalactoside side chains. The terminal mannose units that were indicated by methylation analysis may occur as single side substituents or as oligomannosides suspended from the main chain. Partial acetolysis may facilitate isolation of oligomannoside side chains
INFECT. IMMUN.
for further sequence and hapten inhibition analysis. At present it is not known ifA. fumigatus GM and the galactan, that was also isolated, form a macromolecular complex in the cell wall. Preliminary studies showed that the antigenic portion of GM appeared to be resistant to a number of glycosidases (exo-a-mannanse, a-mannosidase, and ,f-galactosidase) in that the ability to react with anti-AFSA was unchanged by exposure to these enzymes. Perhaps this resistance is one of the reasons that the AFSA survives in the host's circulation. From the standpoint of the immunodiagnosis of invasive aspergillosis, it has not been possible, thus far, to raise high antibody titers, a requirement for sensitive assays, such as the sandwich enzyme-linked immunosorbent assay or hemagglutination tests. The low rate of antigen detection in humans known to have invasive aspergillosis may be largely due to the insensitivity of the CIE test as used to detect AFSA (12). Even in experimental infections, antigenemia is not always detectable in mice that are heavily infected with A. fumigatus 2085. Certain strains do not produce detectable levels of AFSA in vivo. (P. F. Lehmann and E. Reiss, Bull. Soc. Fr. Mycol. Med., in press). The isolation and characterization of a purified GM which reacts with anti-AFSA will facilitate the synthesis of GM conjugates that can be used to stimulate production of high titers of antibodies for detecting AFSA with assays more sensitive than CIE. Further structural studies will also be useful in identifying the sugar sequences and glycosidic bond arrangements that differentiate the antigens of this fungus. ACKNOWLEDGMENTS This 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 Mycology Division, Center for Disease Control. LITERATURE CITED 1. Armstrong, D., H. Chmel, C. Singer, M. Tapper, and P. P. Rosen. 1975. Nonbacterial infections associated with neoplastic disease. Eur. J. Cancer 11(Suppl.):7994. 2. Ashwell, G. 1966. New colorimetric methods of sugar analysis. Methods Enzymol. 8:85-95. 3. Azuma, I., H. Kimura, F. Hirao, E. Tsubura, Y. Yamamura, and A. Misaki. 1971. Biochemical and immunological studies on Aspergillus. III. Chemical and immunological properties of glycopeptide obtained from Aspergillus fumigatus. Jpn. J. Microbiol. 15:237-246. 4. Bardalaye, P. C., and J. H. Nordin. 1977. Chemical structure of the galactomannan from the cell wall of Aspergillus niger. J. Biol. Chem. 252:2584-2591. 5. Bishop, C. T., M. B. Perry, and F. Blank. 1966. The
VOL. 25, 1979
6.
7.
8.
9.
10. 11.
water-soluble polysaccharides of dermatophytes. V. Galactomannans II from Trichophyton granulosum, Trichophyton interdigitale, Microsporum quinckeanum, Trichophyton rubrum, and Trichophyton schonkinii. Can. J. Chem. 44:2291-2297. Bishop, C. T., M. B. Perry, F. Blank, and F. P. Cooper. 1965. The water-soluble polysaccharides of dermatophytes. IV. Galactomannans I from Trichophytongranulosum, Trichophyton interdigitale, Microsporum quinckeanum, Trichophyton rubrum and Trichophyton schonkinii. Can. J. Chem. 43:30-39. Ellsworth, J. H., E. Reiss, R. L Bradley, H. Chmel, and D. Armstrong. 1977. Comparative serological and cutaneous reactivity of candidal cytoplasmic proteins and mannan separated by affinity for concanavalin A. J. Clin. Microbiol. 5:91-99. Gold, M. H., S. Larson, L. H. Segal, and C. R. Stocking. 1974. Intracellular localization of nigeran in the wall of Aspergillus aculeatus by autoradiography with the electron microscope. J. Bacteriol. 118:1176-1178. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates. Methods Carbohydr. Chem. 1:380-394. Krick, J. A., and J. S. Remington. 1976. Opportunistic invasive fungal infections in patients with leukaemia and lymphoma. Clin. Haematol. 5:249-310. Lechevalier, M. P. 1968. Identification of aerobic actinomycetes of clinical importance. J. Lab. Clin. Med. 71:
934-944. 12. Lehmann, P. F., and E. Reiss. 1978. Invasive aspergillosis: antiserum for circulating antigen produced after immunization with serum from infected rabbits. Infect. Immun. 20:570-572. 13. Lindberg, B. 1972. Methylation analysis of polysaccharides. Methods Enzymol. 28:178-195.
GALACTOMANNAN ANTIGENEMIA
365
14. Uoyd, K. 0. 1972. Molecular organization of a covalent peptidophosphopolysaccharide complex from the yeast form 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 Folin phenol 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 a spraying 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 to mycodextranase, p. 735-742. In International Fermentation Symposium, 4th, Kyoto. 19. Reiss, E. 1977. Serial enzymatic hydrolysis of cell walls of two serotypes of yeast-form Histoplasma capsulatum with 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-agglutinating and sugar-specific proteins. Science 177:949-959. 21. Shivers, C. A., and J. M. James. 1967. Specific antibodies 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 Enzymol. 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.
Vol. 25, No. 1
Galactomannan Antigenemia in Invasive Aspergillosis E. 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 England Received for publication 18 April 1979
Galactomannan (GM) extracted from mycelia of Aspergillus fumigatus with cold dilute alkali reacted with antiserum specific for an antigen that circulated in invasive aspergillosis in rabbits and humans. The GM was purified by its affinity for concanavalin A and was separated from a nonantigenic glucan by gel permeation on Sephacryl S-200. The GM molecular weight of between 25,000 to 75,000 was smaller than the antigen present in infected rabbit serum which was retained by an ultrafiltration membrane that had a nominal molecular weight limit of 125,000. The ratio of galactose to mannose present in GM was 1:1.17. The serological activity of GM was stable to boiling but labile to 0.01 N HOl, implicating galactofuranose as an antigenic determinant. Analysis of purified GM by methylation-gas chromatography suggested a structure consisting of a 1 6linked mannan backbone with oligogalactoside side chains 3 units long, terminating in galactofuranose. The presence of mannose as a side chain component was also inferred. Another antigen of A. fumigatus, which did not bind to concanavalin A, was isolated after tandem chromatography on diethylaminoethyl- and carboxymethyl-Sephadex and was identified as a galactan. The galactan inhibited the immune precipitation of GM with specific antiserum. --
Invasive aspergillosis is encountered in some first demonstration of circulating antigen in a leukemia patients who are receiving intensive human patient with invasive aspergillosis was chemotherapy and broad spectrum antibiotics. made. Antisera produced by the conventional The trend towards heavy immunosuppression means of injecting rabbits with killed mycelium, and the success in treating previously lethal or with the fungus culture filtrate, were not opportunistic bacterial infections have led to an capable of detecting circulating A. fumigatus increase in the number of invasive aspergillosis antigen. Preliminary results (12) showed that an alkacases. One major cancer treatment center reported 93 cases of invasive aspergillosis in a 5.5- line borohydride extract (CA) of autoclaved whole mycelium contained a nondialyzable facyear period (16), as reviewed by Armstrong et al. (1). The demand for timely laboratory diag- tor capable of being absorbed and precipitated nosis of this infection has not been met because by the antibody directed against circulating anof the shortcomings of present methodology. An tigen. This antigenic activity was resistant to antemortem serodiagnosis is difficult because digestion by pronase, but was susceptible to the absence of precipitins may be linked to hy- periodate oxidation, suggesting the involvement pogamma-globulinemia (1). Only a few survivors of carbohydrate determinants. The CA extract have been reported, and treatment with the comigrated in immunoelectrophoresis with cirantifungal agent amphotericin B must be started culating antigen from immunosuppressed, inearly in the disease if it is to be successful (10). fected rabbits. With these observations as a In an earlier report from this laboratory anti- guide, a series of column chromatographic procedures was devised to further purify and chargen was detected in rabbits that were immunosuppressed with cortisone and cyclophospha- acterize this antigen. mide before infection with Aspergillus fumigaMATERIALS AND METHODS tus began (12). By using serum from an infected Antiserum production. Antiserum to circulating rabbit as the source of circulating A. fumigatus was produced in rabbits by the method deantigen, it was possible to produce, in a second antigen previously (12). Briefly, rabbits were immurabbit, an antiserum that detected the circulat- scribed by receiving 200 mg of cyclophosphanosuppressed ing antigen. Using this reference antiserum, the mide and 30 mg of cortisone, intravenously, 1 day
before intravenous injection of 5 x 10' A. fumigatus conidia. Within 6 days, mycelium was detected in the
t Present address: Department of Microbiology, Medical College of Ohio, CS 1008 Toledo, OH 43699. 357
358
INFECT. IMMUN.
REISS AND LEHMANN
liver, kidney, brain, and heart of each rabbit, as well as antigenemia. The serum containing fungal antigen was removed. Other rabbits were immunized with the serum in FCA to produce a reference antibody. Antigenemia was detected by counterimmunoelectrophoresis (CIE) (Fig. 1). The circulating antigen was designated AFSA (A. fumigatus serum antigen) and antisera, which reacted with it as anti-AFSA. Further batches of anti-AFSA were produced in rabbits, with precipitin lines used as sources of antigen (21). A precipitin line was formed between anti-AFSA and AFSA in the serum of an infected rabbit by using CIE (Fig. 1) (12). These lines were cut out of several gels and, after washing in cold saline eight times over 2 days, they were homogenized and injected intramuscularly in Freund complete adjuvant. After 3 weeks the rabbits were given booster injections intradermally in several sites on their backs. Culture and conditions of growth. A. fumigatus 2085 was obtained from the London School of Hygiene and Tropical Medicine, the United Kingdom. It was originally isolated in 1952 from a lung cavity of a patient with Friedlander's pneumonia. The culture is maintained in the culture collection of the Mycology Division, Center for Disease Control, as B2570. For the production of antigens, a 17-liter glass carboy was filled with 15 liters of Czapek-Dox medium (Difco Laboratories, Detroit, Mich.) supplemented with the following cations: MnCl2.4H20, FeSO4 *7H20, ZnS04. 7H20, 10-5 M; CuSO4 . 5H20, 10-6 M; CaCl2.2H20, 10-4 M. A 48-h starter culture, grown in two 1-liter flasks each containing 300 ml was incubated at 30'C and at 150 rpm on a gyratory shaker. The carboy was seeded with this growth, and incubation took place at 22 to 24°C for 54 h with forced aeration and magnetic stirring (Fig. 2). Then, 150 ml of 1% Merthiolate (sodium ethylmercurithiosalicylate, Aldrich Chemicals, Milwaukee, Wisc.) was added, and the culture was stirred for 18 h without aeration. The dense growth was poured over five layers of unbleached cotton muslin in a Buchner funnel, washed with 4 liters of 0.85% NaCl and 8 liters of deionized water. The washed fungus mat was peeled away from the filter, squeezed dry, and stored at -40°C. Antigen extraction. The washed mycelium was suspended in 2 liters of 0.02 M citric acid-NaOH buffer (pH 7.0) and autoclaved for 90 min at 121°C and 7.82 atmospheres of pressure. After cooling, the suspension was centrifuged (8,000 x g, 30 min) and the pellet was
Reference Artiserum
Antigenerric
.arrNple
FIG. 1. Detection of antigenemia
by CIE.
AIR
i~
ZL
VACUUM LINE
h
FIG. 2. Carboy device for growth of A. fumigatus. Filtered intake air flows through gas dispersion tube and circulates through magnetically stirred culture. Two traps are placed in outflow line, leading to vacuum source (design suggested by H. F. Hasenclever).
resuspended in 1 liter of the same buffer with the aid of a Waring blender. The mycelium was autoclaved again for 30 min and centrifuged, and the pellet, resistant to hot buffer extraction, was resuspended in 850 ml of freshly prepared and degassed alkaline borohydride (0.4 N NaOH, 0.1 M NaBH4). The suspension was placed in a 1-liter polypropylene bottle, flushed with nitrogen, then tightly stoppered and extracted with stirring for 21.5 h in an ice bath. After centrifugation (8,000 x g, 30 min) the turbid supernatant was recovered and brought to pH 6.5 with acetic acid. The resulting precipitate was removed by centrifugation (4,000 x g, 20 min), and the supernatant was concentrated in an ultrafiltration cell (TCF 10, Amicon Corp., Lexington, Mass.) under positive N2 pressure in the cold using a PM 10 membrance (Amicon) that had a nominal molecular weight limit for proteins of 10,000. The retentate, 125 ml, was dialyzed in the cold for 24 h versus four changes of 6 liters of deionized water. The material remaining in the dialysis sac was clarified by centrifugation (20,000 x g, 20 min), lyophilized, and stored at -40°C over a silica gel dessicant. The product 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
VOL. 25, 1979
unless otherwise indicated. Then the reaction mixture was tested in immunodiffusion versus antiserum AFSA. Lectins in buffer and no added CA were operated as controls. The following lectins were tested: concanavalin A (ConA, Miles Laboratories, Inc., Elkhart, Ind.) in tris(hydroxymethyl)aminomethane-hydrochloride (0.01 M, pH 7.2, containing 1 mM CaCl2, 1 mM MnCl2), Lens culinaris hemagglutinin B (MilesYeda), soybean agglutinin (Miles-Yeda), wheat germ agglutinin (Miles-Yeda), pokeweed mitogen (Sigma Chemical Co., St. Louis, Mo.), and phytohemagglutinin-P (Difco Laboratories, Detroit, Mich.). ConA-Sepharose chromatography. A column (1.2 by 25 cm) packed with ConA-Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) was equilibrated 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 of Tris buffer and applied to the column. The effluent was monitored at 280 nm with a flow analyzer, (Uvicord II, LKB Instruments, Bromma, Sweden), and 5ml fractions were collected. Material binding to ConA was eluted with 0.2 M a-methyl mannoside (a-MM). Fractions were screened for protein with the Folin phenol reagent (15) by using a bovine serum albumin fraction V standard. Carbohydrate in the effluent was determined with phenol-sulfuric acid with respect to glucose (9). The eluate was pooled, dialyzed versus deionized water, and lyophilized. Column chromatography. Small-scale binding in 3-ml plastic syringe columns was carried out to see if CA antigen would bind to either carboxymetbyl (CM)Sephadex C-25 or to diethylaminoethyl (DEAE)Sephadex A-50. The CM columns were operated at pH 3.9, pH 5.5 in acetic acid-sodium acetate (0.05 M); and pH 7.1 in potassium phosphate (0.05 M). The DEAE column was equilibrated with Tris-hydrochloride buffer (0.05 M, pH 7.1). Columns were charged with CA, and the first 20 ml of effluent was reserved. Then 20 ml of buffer containing 0.5 M NaCl was used for elution of bound solutes. The pooled effluents and eluates were assayed for serological activity versus anti-AFSA and for protein, carbohydrate, and ribonucleic acid (RNA). The orcinol method for determining RNA was used with a yeast sodium ribonucleate standard (Nutritional Biochemicals Co., Cleveland, Ohio). For tandem chromatography, three 1.2-cm diameter columns were connected in series: first, ConASepharose (20.4 ml); second, CM-Sephadex C50 (10.2 ml); and third, DEAE-Sephadex A50 (12.4 ml). All gels were obtained from Pharmacia. The ConA column was equilibrated with tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.025 M, pH 7.2) containing 10-4 M each of MnCl2 and CaCl2. The DEAE and CM columns were equilibrated with the same buffer minus added cations, and this served as the reservoir buffer for chromatography. The CA sample in 7.5 ml was applied to the ConA column, and fractions of 5 ml were collected from the effluent of the DEAE column. The effluent was monitored at 280 nm, and fractions were sampled for total carbohydrate and for serological activity. Certain fractions were pooled, dialyzed versus deionized water, and lyophilized. The columns were uncoupled, and the ConA column was eluted 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 in buffer, and the fractions were sampled for protein and carbohydrate and then pooled, dialyzed, and lyophilized. The serologically active eluate from the ConA column was subjected to gel permeation chromatography on Sephacryl S-200 superfine (Pharmacia). A column (2 by 95 cm) was packed with gel and equilibrated with potassium phosphate buffer (0.025 M, pH 7.4) containing 0.07 M NaCl. The sample consisting of 29.9 mg of ConA eluate was dissolved in 0.5 ml and applied to the column. A flow rate of 20 ml/h was maintained, and fractions of 2.5 ml were collected, sampled for carbohydrate and serological activity, and then pooled accordingly, dialyzed, and lyophilized. Ultrafiltration. A rapid estimate of molecular size was attempted by using membranes differing in their nominal molecular weight limits. The antigens tested were CA, the Sephacryl S200-included peak, and serum from an immunosuppressed, infected rabbit which contained AFSA. The membrane ultrafiltration devices were Minicons B15, exclusion limit 15,000; A75, 75,000 limit; and S125, 125,000 limit (Amicon). Also employed were 3-ml ultrafiltration cells requiring N2 pressure (Millipore Corp., Bedford, Mass.) with membranes PSED, 25,000 limit, and PTJM, 100,000 limit. Antigens were dissolved in water or saline to give a concentration of 0.05 mg/ml and then were concentrated 1Ox. After diafiltration with an additional aliquot of water or saline, the retentates were tested for serological activity in immunodiffusion versus antiAFSA. Acid hydrolysis. Mild acid hydrolysis of CA in 0.01 N HCl (1 mg/0.5 ml) was carried out under N2 at 100°C for 1, 2, and 3 h. In controls water was substituted for acid. Samples then were dried under N2 at 50°C, and traces of acid were removed in vacuo over soda lime. Timed hydrolysis of CA in 2 N trifluoroacetic acid (TFA) was used to determine conditions for the optimal release of monosaccharides. Five milligrams of CA and 1 ml of 2 N trifluoroacetic acid were combined in a 3.7-ml vial, flushed with N2, fitted with a Teflon lined screw cap, and hydrolyzed for 1 to 4 h. Hydrolyzates were dried under N2, and traces of acid were removed in vacuo. For amino sugar detection, samples were heated under N2 in 4N HCl for 6 h at
1000C. Monosaccharide detection. The distribution of monosaccharides was estimated by descending paper chromatography in which the upper phase of the solvent system composed of n-butanol-pyridine-water-toluene (5:3:3:4, vol/vol) (11) was used. After development for 53 h, sugars were detected with alkaline silver nitrate dip (24) or aniline phthalate spray (17) reagents. Galactostat and glucostat special reagent test kits (Worthington Biochemicals Corp., Freehold, N.J.) were used according to the manufacturer's directions. Amino sugar was detected by the Elson-Morgan reaction (2) with respect to glucosamine-hydrochloride. Gas-liquid chromatography of the trimethylsilyl ether derivatives of monosaccharides was carried out by the method of Sweeley et al. (23). Dry, neutralized hydrolyzates were derivatized with 0.1 or 0.2 ml of trimethylsilylimidazole in dry pyridine, (tri-sil Z, Pierce Chemical Co., Rockford, Ill.) and separated on
360
REISS AND LEHMANN
column (6.4 mm ID by 2 m) of SE52 (5% phenylmethylsilicone) on 100/120-mesh Supelcoport operated isothermally at 1500C in a Perkin Elmer 990 gas chromatograph. The N2 carrier gas flow rate was 71 ml/min, and typical retention times for standards were a-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 permethylated with dimethylsulfinyl-sodium and methyl iodide in the Hakomori procedure (13). The methylated polysaccharide 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 trifluoroacetic acid (12 h, 1000C, under N2) followed by volatilization of the acid and removal of traces in vacuo over soda lime. Alditol acetates of the methylated monosaccharides were prepared (13) and separated by gas chromatography on a column (6.4 mm ID by 3 m) of 3% ECNSS-M (ethylene succinate-phenylsilicone copolymer) coated on 100/120-mesh Gas Chrom Q operated isothermally at 190° C. The methylated monosaccharides were tentatively identified by calculating of retention times (T values) relative to an authentic standard of 2,3,4,6-tetra-O-methyl glucose (Supelco Inc., Bellefonte, Pa.) and comparing them with published T values (13). Serological tests. The criterion for determining antigenic activity was the ability of a column-derived fraction to form an immunoprecipitin arc with antiAFSA either in immunodiffusion or CIE. Conditions for immunodiffusion were in PBS (pH 7.2) polyethylene glycol 6,000 (2%, wt/vol) agar or, for CIE, in barbital buffer as previously described (12). In some cases fractions were shown to have antigenic activity by the inhibition of the reaction between anti-AFSA with CA. This was brought about by incubating antiAFSA with the antigenic fraction before using it in a
a
CIE test.
RESULTS
Yield of CA. The compressed filter cake of A. fumigatus mycelium obtained from 15 liters of culture weighed 332 g. Sporulation was negligible in this submerged culture. From this starting material 447 mg of dialyzed and lyophilized CA was derived containing 56.5% carbohydrate, 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, of those tested, that reacted with CA in immunodiffusion. A separation based on affinity for insolubilized ConA was then attempted. Of the 100 mg of CA applied to the column, 25.5 mg was 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 pooled fractions 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 contained 19.5% total carbohydrate, 28.3% protein, and 35.7% RNA. This fraction was digested for 2 h with enzite-agarose insolubilized ribonuclease. Under these conditions the digestion did not go to completion. Of the 22.3 mg of starting material, 17.4 mg was recovered after digestion, for a loss of 22% in dry weight. Ion-exchange and tandem chromatography. Early experiments suggested that the antigen present in CA migrated cathodically during immunoelectrophoresis (12). Therefore, it was considered worthwhile to test for binding to the cation exchanger CM-Sephadex. When 3 mg was applied to columns at pH 3.9, 5.6, 6.5, and 7.1, all serological activity was found in the effluents and none was present in the 0.5 M NaCl eluates. When 3 mg of CA was applied to a DEAESephadex column at pH 7.1, all antigenic activity was present in the effluent and none was present in the eluate. A purification was effected by ionexchange chromatography since the CM eluate contained 672 ,ug of protein and no carbohydrate. The DEAE eluate contained 260 ,ug of carbohydrate, 72 ,ug of protein, and 451 1Lg of RNA. These results suggested that CM- and DEAESephadex columns operated at pH 7.1 would allow antigen to pass through and would bind inert protein and RNA. The effluent profile, obtained from operating the ConA, CM, and DEAE columns in tandem, is shown in Fig. 4. Starting with 204 mg of CA, 8.03 mg was recovered in effluent fractions 4 to 6, and 3.93 mg was recovered in fractions 7 to 10. The columns were uncoupled, and after elution with 0.5 M NaCl, 48.5 mg was recovered from the DEAE and 8.6 mg was recovered from the CM column. The yield from the ConA column eluted with a-MM was 40.1 mg. The combined recovery from the tandem chromatography was 111.4 mg. Serological activity resided in three of the fractions. The ConA eluate formed an immunoprecipitin arc in CIE with anti-AFSA. The tandem effluent fractions 7 to 10 could inhibit the CIE reaction between CA and anti-AFSA, or between infected rabbit serum and anti-AFSA, but was incapable of producing an immune precipitate. A similar inhibitory behavior was found in the eluate from the DEAE column. Gel permeation. The ConA eluate fraction that formed a precipitin arc with anti-AFSA was further characterized by gel permeation chromatography on Sephacryl S-200 (Fig. 5). This gel had an exclusion limit of 80,000 for dextrans, according to the manufacturer. Of the 30 mg of ConA eluate applied to the column, 10.3 mg was recovered. This separated into a major excluded peak, fractions 38 to 44, 6.7 mg; and a minor
GALACTOMANNAN ANTIGENEMIA
VOL. 25, 1979
361
25-28 5-7 9-21 205001_
7000
6000-
a: To
I~~~~~~~~
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4000-
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3000-
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0
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15
25
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FRACTION NO.
4200004 FIG. 3. ConA-Sepharose chromatography of CA extract. A 0.2 M concentration of a-MM elated the material !~~j in fr-actions 25 to 28. (-----) indicates strip chart recording fr-om ultraviolet flow analyzer shown. on right vertical axis as optical density at 28 nm (O.D.ms.=).
4-6
7-11
6000A 100 l 000 ffi% 5000-l A
included peak, fractions 50 to 64, 3.6 mg. All
serological activity resided in the minor included
h erlgclatviyoMAwsMeue fe peak. Monosaccharide composition. Preliminary acid hydrolysis of the CA extract and paper chromatography showed that 2.5 h was the optimal hydrolysis time and that the extract conz \ z glucose, mannose, galactose, and ribose. ~~~~~~~~~~tained ConA equate contained glucose, mannose, > \ ~~~~~~~~The > °0 3000-A and galactose, but the tandem chromatography effluent fractions 7 to 10 had only galactose and ^ 8 glucose. Mild acid hydrolysis of 1 mg of CA in N HCI for 1, 2, and 3 h released galactose 0.01 Al l-0.2 ' X2000| ~~~~~~~amounting to 22.5, 43.3, and 60 IL&, respectively. l I !| h and abolished after 2 h of hydrolysis; whereas ° ~~~~~~1 \ ~~~~~~~~the is< controL boiled in water for 3 h, still retained 9,^___,__ ~~~~~serological activity. The molar ratios of mono5 510 20 1 255 saccharides determined as the trimethylsilyl FRACTION NO. S-2O-included ethers were, for the Sephacryl tandem chromatog- fraction, galactose-mannose glucose (1:1.16: FIG. 4. Effluent profile froma efConA-Sepharotes DEaE-Seph- 0.14); and for the tandem50 chromatography raphy of CA extract. to ose glucose (1: adex, and CM4-6phadex coludedfractions 7 0.23). The Sephacryl S200-excluded fraction and tandem (see text).I --0
U;00-
8~~~
362
INFECT. IMMUN.
REISS AND LEHMANN
tandem effluent fractions 4 to 6 contained only glucose. The absence of amino sugar in the Sephacryl S200-included and tandem fractions 7 to 10 was verified after hydrolysis in 4 N HCl for 6 h at 1000C. Only a trace, - 15004
C 1000
500L 30
N 50
40
60
l
in
c
0
70
80
FRACTION NUMBER
FIG. 5. Gelpermeation of ConA eluate on Sephacryl S-200. V. = void volume determined with blue dextran 2000. og 30-
L
man Q.
020
60
0)
0~Q. 0 I..
o 40-
2
.00)-
14
w
20-
gal
15
0
10
retention time, min. FIG. 6. Gas-liquid chromatography of Sephacryl S-200 included fraction as alditol acetate derivatives.
20
30
retention time, min. FIG. 7. Gas-liquid chromatography of Sephacryl S200-included fraction as permethylated alditol acetate derivatives. For tentative identifications see Table 1.
GALACTOMANNAN ANTIGENEMIA
VOL. 25, 1979
363
TABLE 1. Tentative identification of methylated monosaccharides derived from A. fumigatus 2085 GM Molar ratio Original linkage Methylated derivative T valueb 5 1 Man 2,3,4,6-tetra-O-methylmannose 0.98 1 3 Gal furc 1 2,3,5,6-tetra-O-methylgalactose 1.07 2 3 2 Man 1 3,4,6-tri-O-methylmannose 1.89 3 3,4,6 2.34 4 2 Gal 1 tri-O-methylgalactose or or 2,3,6 6 4 Gal 1 3.6 4 Man 1 2,3-di-O-methylmannose 4.32 5
Peak no.a
1
6
4.88
3,4-di-O-methylmannose
6 2 Man 1
2
1
6
See Fig. 7. b With respect to 2,3,4,6-tetra-O-methylglucose. e Man, Mannose; gal, galactose; fur, furanose.
a
eluate. It was subsequently resolved in the excluded portion of the Sephacryl S200 column. This material had no detectable serological activity. It was precipitated upon acidification to pH 2. When a glucan sample, 460 ILg, was incubated overnight at 370C with a(1 -- 3)glucanase (20), 142 ,jg of glucose was released, giving a 30.9% digestion. Premortem diagnosis of human invasive aspergillosis. Three persons with invasive aspergillosis, all of whom were leukemics, whose disease was diagnosed by histopathology, have been shown to have circulating antigen detectable by CIE. Another three human patients suspected of having invasive aspergillosis, but not confirmed by histopathology, were positive for antigenemia. At present, a total of 6 persons having aspergillosis out of 40 to 60 persons suspected of having this disease gave CIE evidence of circulating antigen. DISCUSSION The objective of the present work was to characterize the antigenic portion of the mycelial extract of A. fumigatus that could react with an antiserum produced in rabbits immunized with antigenemic serum from an immunosuppressed, infected rabbit. It was hoped, by this means to identify the antigen that circulated in experimental rabbit aspergillosis and in invasive human aspergillosis. The evidence presented indicates that this antigen is a GM polysaccharide (galactose-mannose, 1:1.17) with a molecular weight of between 25,000 and 75,000. Proof is based on the ability of the ConA eluate-Sephacryl S200-included fraction to react in CIE with anti-AFSA and to absorb the antibody that would otherwise react with serum from an immunosuppressed, infected rabbit. It was also
possible to show that another component of the CA extract was capable of absorbing anti-AFSA, but was insufficient to act as a precipitinogen in the CIE test. This moiety did not bind ConA and was present in the pH 7.1 effluent of DEAE and CM columns operated in tandem. After acid hydrolysis, it was identified as a galactan with a trace of glucose by three methods: paper chromatography, galactostat, and gas-liquid chromatography. The fortuitous separation of the galactan from a glucan present in the tandem chromatography effluent was probably due to a molecular sieving effect. The main interest was focussed on the ConA eluate because of its strong serological activity. The eluate contained a nonantigenic glucan as the major component. This impurity was easily removed on Sephacryl S200 in the void volume and was presumed to be a(1 -- 3)glucan because of its ConA-binding properties, susceptibility to a(1 -+ 3)glucanase, and acid precipitability (18). Other members of the genus Aspergillus, especially A. niger, produce mycodextran, a glucan composed of alternating a(1 -- 3) and a(1 -* 4) bonds (8). The A. fumigatus culture studied herein was not capable of producing mycodextran, even when grown under the nitrogen-deficient conditions reported by Gold et al. (8) to be favorable to large yields of this glucan (T. F. Bobbitt, personal communication). This is probably the case for A. fumigatus cultures in general. The antigenic activity of CA resided in a minor component of the ConA eluate that was included on Sephacryl S200. This GM was presumed to contain terminal galactofuranosyl antigenic determinants because of its lability to 0.01 N HCl, with concomitant release of galactose and the loss of serological activity. The differences in apparent molecular weight of the serum antigen AFSA (>125,000) and GM ex-
364
REISS AND LEHMANN
tracted from mycelia (25,000 < GM < 75,000) suggest that the GM in AFSA exists in some form of complex. An interpretation for this disparity is that GM are commonly complexed with protein by either dilute alkali stable N-glycosylamine-asparagine bonds or alkali labile O-glycosyl-serine or -threonine bonds (14). The necessity for using alkali as an extraction milieu to dislodge the GM from its location in the cell wall of A. niger was described by Bardalaye and Nordin (4) and was in accord with our experience with A. fumigatus. The low level of protein in the GM suggests that it was eliminated from an O-glycosylhydroxyamino acid ester linkage to the peptide moiety (22). This hypothetical loss of peptide count accounts for the inability of CA to act as an antigen for producing anti-AFSA in rabbits, whereas the circulating material was antigenic (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. granulosum (1:2.4) as reported by others (4, 5) and differs from the GMI (1:12.3) of T. mentagrophytes var. granulosum (6). A further similarity was noted between A. fumigatus GM and Trichophyton GMII in that neither bound to DEAE-based ion exchanger, though the latter could bind in borate buffer (5). The structure of A. niger GM, studied by Bardalaye and Nordin (4), consisted of a (1 -- 2)-linked mannan, with (1 -+ 6) branch points to the galactose oligosaccharides (3 to 4 units long). The terminal galactose occurred in the furanoside configuration. The most notable difference observed in the methylation analysis of A. fumigatus GM was a substantial amount of terminal nonreducing mannose. The galactose side chains in A. fumigatus appear to be no more than 3 units long. The binding of A. fumigatus GM to ConA is evidence that galactose is covalently linked to mannose: moreover, the terminal mannose units that were detected would be available for binding to ConA, since this lectin's specificity is directed to the axial C4 position (20). Another difference between the two GMs was that the GM of A. niger had a single type of branch point, but the GM of A. fumigatus had two types. The structural picture of A. fumigatus GM that seems most compatible with these data is a 1-6-linked mannan backbone heavily substituted with oligogalactoside side chains. The terminal mannose units that were indicated by methylation analysis may occur as single side substituents or as oligomannosides suspended from the main chain. Partial acetolysis may facilitate isolation of oligomannoside side chains
INFECT. IMMUN.
for further sequence and hapten inhibition analysis. At present it is not known ifA. fumigatus GM and the galactan, that was also isolated, form a macromolecular complex in the cell wall. Preliminary studies showed that the antigenic portion of GM appeared to be resistant to a number of glycosidases (exo-a-mannanse, a-mannosidase, and ,f-galactosidase) in that the ability to react with anti-AFSA was unchanged by exposure to these enzymes. Perhaps this resistance is one of the reasons that the AFSA survives in the host's circulation. From the standpoint of the immunodiagnosis of invasive aspergillosis, it has not been possible, thus far, to raise high antibody titers, a requirement for sensitive assays, such as the sandwich enzyme-linked immunosorbent assay or hemagglutination tests. The low rate of antigen detection in humans known to have invasive aspergillosis may be largely due to the insensitivity of the CIE test as used to detect AFSA (12). Even in experimental infections, antigenemia is not always detectable in mice that are heavily infected with A. fumigatus 2085. Certain strains do not produce detectable levels of AFSA in vivo. (P. F. Lehmann and E. Reiss, Bull. Soc. Fr. Mycol. Med., in press). The isolation and characterization of a purified GM which reacts with anti-AFSA will facilitate the synthesis of GM conjugates that can be used to stimulate production of high titers of antibodies for detecting AFSA with assays more sensitive than CIE. Further structural studies will also be useful in identifying the sugar sequences and glycosidic bond arrangements that differentiate the antigens of this fungus. ACKNOWLEDGMENTS This 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 Mycology Division, Center for Disease Control. LITERATURE CITED 1. Armstrong, D., H. Chmel, C. Singer, M. Tapper, and P. P. Rosen. 1975. Nonbacterial infections associated with neoplastic disease. Eur. J. Cancer 11(Suppl.):7994. 2. Ashwell, G. 1966. New colorimetric methods of sugar analysis. Methods Enzymol. 8:85-95. 3. Azuma, I., H. Kimura, F. Hirao, E. Tsubura, Y. Yamamura, and A. Misaki. 1971. Biochemical and immunological studies on Aspergillus. III. Chemical and immunological properties of glycopeptide obtained from Aspergillus fumigatus. Jpn. J. Microbiol. 15:237-246. 4. Bardalaye, P. C., and J. H. Nordin. 1977. Chemical structure of the galactomannan from the cell wall of Aspergillus niger. J. Biol. Chem. 252:2584-2591. 5. Bishop, C. T., M. B. Perry, and F. Blank. 1966. The
VOL. 25, 1979
6.
7.
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water-soluble polysaccharides of dermatophytes. V. Galactomannans II from Trichophyton granulosum, Trichophyton interdigitale, Microsporum quinckeanum, Trichophyton rubrum, and Trichophyton schonkinii. Can. J. Chem. 44:2291-2297. Bishop, C. T., M. B. Perry, F. Blank, and F. P. Cooper. 1965. The water-soluble polysaccharides of dermatophytes. IV. Galactomannans I from Trichophytongranulosum, Trichophyton interdigitale, Microsporum quinckeanum, Trichophyton rubrum and Trichophyton schonkinii. Can. J. Chem. 43:30-39. Ellsworth, J. H., E. Reiss, R. L Bradley, H. Chmel, and D. Armstrong. 1977. Comparative serological and cutaneous reactivity of candidal cytoplasmic proteins and mannan separated by affinity for concanavalin A. J. Clin. Microbiol. 5:91-99. Gold, M. H., S. Larson, L. H. Segal, and C. R. Stocking. 1974. Intracellular localization of nigeran in the wall of Aspergillus aculeatus by autoradiography with the electron microscope. J. Bacteriol. 118:1176-1178. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates. Methods Carbohydr. Chem. 1:380-394. Krick, J. A., and J. S. Remington. 1976. Opportunistic invasive fungal infections in patients with leukaemia and lymphoma. Clin. Haematol. 5:249-310. Lechevalier, M. P. 1968. Identification of aerobic actinomycetes of clinical importance. J. Lab. Clin. Med. 71:
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14. Uoyd, K. 0. 1972. Molecular organization of a covalent peptidophosphopolysaccharide complex from the yeast form 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 Folin phenol 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 a spraying 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 to mycodextranase, p. 735-742. In International Fermentation Symposium, 4th, Kyoto. 19. Reiss, E. 1977. Serial enzymatic hydrolysis of cell walls of two serotypes of yeast-form Histoplasma capsulatum with 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-agglutinating and sugar-specific proteins. Science 177:949-959. 21. Shivers, C. A., and J. M. James. 1967. Specific antibodies 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 Enzymol. 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.
