INFECTION AND IMMUNITY, Jan. 1992, p. 143-149
Vol. 60, No. 1
0019-9567/92/010143-07$02.00/0 Copyright © 1992, American Society for Microbiology
A 34- to 38-Kilodalton Cryptococcus neoformans Glycoprotein Produced as an Exoantigen Bearing a Glycosylated Species-Specific Epitope HAMILTON,'* L. JEAVONS,1 P. HOBBY,2 AND R. J. HAY Dermatology Unit' and Renal Unit,2 Clinical Sciences Department, 18th Floor, Guy's Tower, Guy's Hospital, London Bridge, London SE] 9RT, United Kingdom A. J.
Received 15 August 1991/Accepted 17 October 1991
Three monoclonal antibodies (MAbs), all of the immunoglobulin Gl subclass, were raised against Cryptococcus neoformans by using the technique of cyclophosphamide ablation of B-cell responses against shared epitopes of the cross-reactive fungus Trichosporon beigelii. MAb 3C2 was reactive against the encapsulated and nonencapsulated isolates of C. neoformans var. neoformans by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot), and in addition to a 34- to 38-kDa deterniinant, it recognized a series of lower-molecular-weight species. 3C2 also reacted strongly with culture supernatant preparations of C. neoformans var. neoformans by ELISA. 3C2 showed no recognition of either T. beigelii or C. neoformans var. gaYii antigens. Enzymatic deglycosylation followed by reaction with 3C2 on Western blots revealed that sialic acid was an integral part of the determinant, together with N-acetylglucosaminylasparagine and a-mannose. Proteolytic digestion showed that the epitope was pepsin sensitive and that it also contained tryptophan and glycine and/or leucine as determinants of recognition by 3C2. The pl of the glycoprotein was 7.1. Affinity chromatography-purified antigen did not exhibit proteolytic activity on sodium dodecyl sulfate-polyacrylamide substrate gels. Indirect fluorescence antibody tests revealed that 3C2 labelling was confined to the cell membrane and cytoplasm of yeasts. The remaining MAbs, 7H4 and 5G5, recognized both capsulated and nonencapsulated strains of C. neoformans var. neoformans by both ELISA and Western Blot, identifying linear determinants with molecular masses of 36 and 30 kDa. They were unreactive against culture supernatant antigen (exoantigen) from either variant of C. neoformans.
The disease cryptococcosis is caused by either of the two varieties of the encapsulated yeast Cryptococcus neoformans, C. neoformans var. neoformans and C. neoformans var. gattii, and is characterized by dissemination from a pulmonary focus and a potentially fatal form of meningitis. Though the disease may occur in individuals who show no evidence of immunosuppression, it has recently attracted much interest because of its prevalence in patients infected with human immunodeficiency virus. For instance, a recent study in Zaire has shown, by screening for circulating cryptococcal antigen, that up to 11% of human immunodeficiency virus-positive individuals had been infected with the organism (20). At present, the diagnosis of the disease relies on either isolation of the organism or the detection of capsular polysaccharide antigen by using the latex agglutination test with either serum or cerebrospinal fluid (11, 16). Though the latex agglutination test has proved very useful for diagnosis, it is limited to some extent by the crossreactivity of the antigen with other fungal pathogens, such as Trichosporon beigelii (4, 6), and by the fact that some human immunodeficiency virus-positive individuals still show positive tests long after other signs of disease have disappeared. It is not clear whether this reflects failure to eliminate capsular polysaccharide or persistence of infection (5). Therefore, it would be useful to identify other speciesspecific exoantigens produced by C. neoformans, the occurrence and levels of which in serum or cerebrospinal fluid might more accurately reflect the number of viable yeast cells present. Recently, our laboratory has produced spe*
cies-specific monoclonal antibodies (MAbs) against a range of fungal pathogens by using the technique of cyclophosphamide ablation of shared B-cell responses (10, 12), and most recently, we have demonstrated the presence of a 110- to 120-kDa molecule in the culture filtrate of C. neoformans which bears a species-specific epitope (13). In this paper, we report the occurrence and preliminary biochemical characterization of a smaller glycoprotein (34 to 38 kDa) produced as an exoantigen which bears a species-specific epitope and as such has potential for use in serodiagnosis. MATERIALS AND METHODS
Antigen preparation. Lyophilized isolates of C. neoformans var. gattii (2), Cryptococcus laurentii (1), Cryptococcus albidus (1), Aspergillusfumigatus (2), Aspergillusflavus (2), Candida albicans (1) and Candida tropicalis (1) were obtained from the National Collection of Pathogenic Fungi (NCPF), Mycological Reference Laboratory, Colindale, London, United Kingdom (NCPF 3169 and NCPF 3170, NCPF 3141, NCPF 3140, NCPF 2010 and NCPF 2978, NCPF 2208 and NCPF 2617, NCPF 3343, and NCPF 3114, respectively). The isolates were reconstituted with 0.5 ml of sterile water, inoculated onto Sabouraud agar, and then expanded into larger (250 ml and 3 liters) volumes of Sabouraud liquid culture medium. Two additional strains of C. neoformans (B3501, encapsulated, and B4131, nonencapsulated) were obtained from June Kwon-Chung, Mycology Department, National Institutes of Health, Bethesda, Md., while four isolates of C. neoformans var. neoformans (NCPF 3171, serotype D; 3168, serotype A; 3409 and 3081, serotypes unknown), together with two isolates of T. beigelii
Corresponding author. 143
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(NCPF 4874 and 5246), were obtained from the Mycology Laboratory, St. John's Hospital for Diseases of the Skin, London, United Kingdom, and treated as described above. Mycelial isolates of Histoplasma capsulatum var. capsulatum (two isolates), Blastomyces dermatitidis (one), Paracoccidioides brasiliensis (three), and Sporothrix schenckii (two) (NCPF 4100 and NCPF 4088; NCPF 4076; NCPF 3285, NCPF 4115, and NCPF 4095; and NCPF 3181 and NCPF 3286, respectively) were also obtained from the National Collection of Pathogenic Fungi, transformed to the yeast phase at 37°C on slopes of brain heart infusion medium to which 0.2 mM L-cysteine was added, and subsequently subcultured in brain heart infusion broth at 37°C. All fungal preparations were collected by filtration (Whatman no. 2 filter paper), washed in phosphate-buffered saline (PBS; 0.01 M, pH 7.4), and divided into two subsamples, to one of which a cocktail of protease inhibitors was added (1, 2) for subsequent use as antigen for Western blotting (immunoblotting). Each sample was then homogenized on ice by using glass ball ballotini in a bead beater (Biospec Products, Bartlesville, Oklahoma) by using 20 1-min pulses separated by 5-min gaps, which had previously been shown by protein estimation and Western blotting to yield the largest amounts of undenatured protein. The soluble cell extracts were collected and centrifuged at 2,000 x g for 7 min, and the supernatants were retained to yield cytoplasmic antigens, which were divided into portions and stored at -70°C. For the various Cryptococcus strains, the culture filtrates were retained, dialyzed against polyethylene glycol 8000 to 1/100 of their original volumes, and then redialyzed overnight against PBS at 4°C to yield a crude exoantigen preparation. Protein concentration was estimated in each sample by using the Coomassie blue method (17). Immunization protocol. The immunization schedule incorporating cyclophosphamide was as previously described (13) with T. beigelii (NCPF 4874) and C. neoformans var. neoformans (B4131) as antigens, except that the test bleed and differential enzyme-linked immunosorbent assay (ELISA) took place on day 26. Fusion protocol. Cells of the myeloma line Sp2/0 were fused with spleen cells from the chosen mouse at a ratio of 1:10 by using polyethylene glycol 4000 according to a modification of an existing protocol (24), and the resulting hybridomas were plated onto 96-well microtiter plates. At 7 days postfusion, colonies were screened by ELISA with the immunizing antigens and the encapsulated C. neoformans isolate B3501 (13). Clones from wells differentiating between the three antigens were subcloned twice by limiting dilution before expansion into 25-ml flasks. Female BALB/c mice previously primed with pristane were given intraperitoneal injections of 104 hybridoma cells, and ascitic fluid was collected 14 to 21 days later. MAbs were purified from ascitic fluids using a protein A-Sepharose column (Pharmacia) (9). All three of the MAbs described in this study were effectively eluted by a 0.1 M citric acid buffer solution (pH 6), and MAb subclassing was then performed (13). Protein estimations of the eluted immunoglobulins were carried out as previously described (17). Purified MAbs (initial concentration, 100 ,ug ml-') in PBSTween were then assessed for reactivity by ELISA (100 ,ul per well) for all the cytoplasmic fungal antigens described above. All antigens were used at protein concentrations of 1 ,ug per well. In addition, MAbs were assessed for recognition of culture supernatant of C. neoformans by ELISA; the procedure was done as described previously (13) except that the antigen was diluted progressively by a ratio of 1:10,
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starting with an initial concentration of 1 ,ug per well, in order to determine the lowest detectable concentration of antigen. In all the ELISAs, either Histoplasma capsulatumspecific MAb C69 (12) or PBS-Tween was used as the negative control. Polyacrylamide gel electrophoresis (PAGE), electroblotting, and immunoenzyme development. Each of the C. neoformans var. neoformans, C. neoformans var. gattii, and T. beigelii antigens and one of each of the other antigens containing protease inhibitors (total of 100 ,ug of protein per gel) were electrophoresed on a sodium dodecyl sulfate (SDS)-7.5% (wt/vol) polyacrylamide gel at 200 V for 1 h. After semidry blotting, immunoenzyme development was performed with MAbs at a protein concentration of 25 ,ug per strip (12, 13). Affinity chromatography. One gram of CNBr-activated Sepharose 4B (Pharmacia) was swollen in 200 ml of ice-cold 1 mM HCl by washing on a sintered glass funnel for 15 min. Protein A-purified MAb 3C2 (15 mg) in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.5]) was added to the Sepharose, and the two were mixed by end-over-end rotation for 6 h at room temperature. After centrifugation (500 x g for 5 min), the supernatant was removed and active groups were blocked overnight at 4°C by the addition of 2 M ethanolamine (pH 8.0). The Sepharose was then poured into a 5-ml syringe blocked with glass wool and washed alternately with washing buffer at pH 4.0 (0.1 M glacial acetic acid, 0.5 M NaCl) and washing buffer at pH 8.0 (0.1 M Tris base, 0.5 M NaCl). Five micrograms of C. neoformans (capsular negative) cytoplasmic antigen was then added to the column in 20 ml of coupling buffer and recirculated overnight at 4°C in a closed system running at 0.5 ml/min. A280 was monitored as a baseline was established by flushing with coupling buffer. Bound antigen was then eluted by progressive washes with 0.2 M glycine HCI (pH 4.0, 3.0, and 2.2) in volumes of 25 ml each and immediately neutralized prior to analysis by ELISA, SDS-PAGE, and protein estimation. Substrate SDS-polyacrylamide gels. SDS-7.5% polyacrylamide gels were copolymerized with either 0.1% bacteriological gelatin (GIBCO) or 2% casein (BDH). Ten micrograms of affinity chromatography-purified 34- to 38-kDa antigen (unreduced and unboiled) was loaded into each of four wells together with a commercial preparation of pepsin (Sigma; 20 U total in 0.1 M citrate buffer, pH 2.0) and molecular weight markers (GIBCO). Gels were then electrophoresed quickly (25 mA per gel) at 4°C, placed in Triton X-100, and rocked at room temperature for 30 min to remove residual SDS. The gel was then further incubated in PBS for 60 min, the buffer was replaced, and the gel was placed overnight at 37°C prior to Coomassie brilliant blue staining and destaining. Isoelectric focusing. Samples of (10 ,ug) of cytoplasmic yeast (C. neoformans, capsular negative) were run in a 7.5% polyacrylamide gel containing 2% ampholytes (Pharmacia) in the pl range of 3 to 10 on a multiphor electrophoresis system (Pharmacia) with a thermostatic plate cooled to 10°C. The pH gradient of the gel was measured by using Electran colored protein markers (BDH). The gel was capillary blotted onto an immobilin membrane, and immunoenzyme development was performed by using 3C2. Enzymatic deglycosylation of the 34- to 38-kDa antigen.
Subsamples (10 ,ug) of C. neoformans (capsular-negative) cytoplasmic yeast antigen were incubated for 24 h with the following enzymes: a-mannosidase (Sigma) in 0.1 M sodium citrate buffer (pH 4.0) at 25°C, ,-mannosidase (Sigma) in 0.1
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M sodium citrate buffer (pH 4.0) at 25°C, neuraminidase (BDH) in 0.1 M potassium acetate buffer (pH 4.5) supplemented with 2 mM CaCl at 37°C, a-galactosidase (Sigma) in 0.1 M sodium citrate (pH 4.0) at room temperature, ,-galactosidase (Sigma) in 0.4 M sodium citrate (pH 4.0) at room temperature, a-glucosidase (Sigma) in 0.1 M sodium acetate supplemented with 0.05% EDTA (pH 6.0) at 37°C, and peptide N-glycanase F in 25 ,ul of digestion buffer consisting of 300 ,ul of 100 mM 1,10-phenanthroline, 500 ,ul of 7.5% NP-40, and 900 ,u1 of 550 mM sodium phosphate (pH 8.6) at 37°C. With peptide N-glycanase F, some of the antigen was reduced prior to digestion in 1% SDS and 1.6% 2-mercaptoethanol (boiled for 3 min). All enzymes were used in volumes equivalent to 10 U of activity. After digestion, all samples were boiled for 3 min in the presence of 1% SDS and 1.6% 2-mercaptoethanol, loaded onto SDS-7.5% polyacrylamide gels along with molecular weight markers, and electrophoresed at 200 V. Gels were then subsequently blotted onto Immobilin prior to immunoenzyme development with MAb 3C2. Protease digestion of the 34- to 38-kDa antigen. Subsamples (10 ,ug) of C. neoformans cytoplasmic antigen were treated with each of the following enzymes for 24 h (except where stated) under the following conditions: pepsin (Sigma) in 10 mM HCI at 37°C; protease V8 (Sigma) in 0.01 M PBS (pH 7.2) at 37°C; trypsin (Sigma), N-tosylamide-L-phenylamine chloromethyl ketone (TPCK) treated, in 0.67 M sodium phosphate buffer (pH 7.6) at room temperature; a-chymotrypsin (Sigma) in a solution of 80 ,u1 of 2 M CaCl and 3 ml of Tris-HCl buffer (pH 7.6) at room temperature; and papain (Sigma) in a solution of 1 ml of 3 M NaCl in 10 ml of a mixture of 20 mM EDTA and 50 mM L-cysteine (pH 6.2) at room temperature. All enzymes were used at concentrations equivalent to 50 U. Samples (10 ,ug) were also subjected to the following chemical treatments: hydroxylamine (Sigma), 50 mM in distilled water, at room temperature; 2-(2'-nitrophenylsulfonyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) (Pierce), 10 mg ml-' in 70% distilled acetic acid with 0.1% phenol for 48 h at room temperature and with a 10-fold excess of the reagent, followed by the addition of a 10-fold excess of 2-mercaptoethanol, a further incubation for 5 h at 37°C, and extraction of excess BNPS-skatole with ethyl acetate (14); pretreatment with BNPS-skatole followed by exposure of the antigen to a 12-fold excess of N-bromosuccinimide (Sigma), 10 mg ml-' in aqueous 50% distilled acetic acid for 2 h at 30°C; and cyanogen bromide (Sigma), 0.5 g in 10 ml of 0.1 M HCl for 48 h at room temperature. Samples were treated and analyzed by SDS-PAGE and Western blotting in the same way as those prepared for the deglycosylation study above. Immunofluorescent location of antigen. Immunofluorescent location of antigen was performed as previously described (13) with C. neoformans (B3501) as test organism and MAbs diluted 1:10 in PBS-Tween.
RESULTS The ELISA performed with the polyclonal serum samples from the four mice used for the immunization protocol identified one mouse with significantly higher titers to the C. neoformans (encapsulated) strain antigen than those to either the nonencapsulated C. neoformans or T. beigelii antigens used in the immunization protocol, and this animal was used for the fusion study. The other animals showed a limited differential response to the various antigens. Control mice, which underwent the same immunization protocol but
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3C2 MAbs FIG. 1. Reactivities of three MAbs against a range of antigens, as defined by absorbance values in ELISA. 0, C. neoformans nonencapsulated B4131; *, B3501; A, NCPF 3171; V, NCPF 3081; *, NCPF 3168; *, NCPF 3409; O, NCPF 3169; O, NCPF 3170; A, T. beigelii NCPF 4874 (NCPF 3169 and NCPF 3170 are C. neoformans var. gattii; the others are C. neoformans encapsulated isolates, except B4131). Isolates of all other fungi tested gave values below 0.1 (data not shown).
without the use of cyclophosphamide, showed no difference in serum reactivities in a similar ELISA (data not shown). Three hybridomas were chosen after a total of 1,500 were screened by ELISA. After successive subclonings, three MAbs, designated 3C2, 7H4, and 5G5, were obtained, and all three were shown to belong to the immunoglobulin Gl subclass. By ELISA, MAbs 3C2 and 7H4 recognized the nonencapsulated and capsular isolates of C. neoformans but showed no reactivity to T. beigelii, while MAb SG5 recognized all of these antigens (Fig. 1). None of the MAbs showed any reactivity to C. neoformans var. gattii or to any of the other fungal antigens tested. 3C2 was strongly reactive to culture supernatant antigen from C. neoformans var. neoformans isolates and was able to detect antigen at a protein concentration as low as 0.001 ng (Fig. 2). In contrast, 7H4 and 5G5 failed to recognize exoantigen. Western blot analysis demonstrated that 3C2 recognized a determinant at 34 to 38 kDa on reduced gels, two major bands at approximately 26 to 28 and 20 to 22 kDa, and four minor bands on blots of nonencapsulated. and encapsulated isolates of C. neoformans (Fig. 3) but showed no reaction to any other antigens except culture supernatant antigen from C. neoformans var. neoformans (data not shown). The pattern of recognition on nonreduced blots was exactly the same. MAbs 7H4 and 5G5 recognized two bands at 36 kDa and approximately 30 kDa and showed the same pattern of antigen recognition as that shown by ELISA. Isoelectric focusing of nonencapsulated C. neoformans cytoplasmic antigen followed by blotting and immunoenzyme development with 3C2 revealed a single strong band with a pl of 7.1, whereas over 80% of the remaining cytoplasmic antigens appeared to have pIs of 5.0 and less.
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A total of 35 ,ug of 3C2 antigen was eluted from affinity chromatography columns (two columns loaded with a total of 15 mg each of cytoplasmic antigen) with glycine-HCl buffer (pH 2.2). The fractions eluted with glycine-HCl at pH 4 and pH 3 were negative when reacted with MAb 3C2 by ELISA and contained undetectable amounts of protein. SDS-polyacrylamide gels stained with Coomassie brilliant blue revealed the predominance of the 34- to 38-kDa antigen in the eluent. However, when these samples were run on SDS-polyacrylamide substrate gels (with gelatin and casein),
26-28kD> 20-22kD> _
13
Antigen concentration (9)
FIG. 2. Reactivities of three MAbs to culture supernatant antigens by ELISA. encapsulated-isolate antigens gave a pattern similar to that shown here for B3501.
34-38kD>
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lo-11
0,
3C2; O, 5G5;
A,
7H4. Other C. neoformans
there was no evidence of proteolytic clearing compared with that of the positive controls. Figure 4 indicates the effect of deglycosylation on the recognition of the 34- to 38-kDa- glycoprotein and lowermolecular-weight species by 3C2 when visualized by Western blot. Neuraminidase completely destroys recognition as does peptide N-glycanase F. There is also a partial decrease in reactivity with treatment with a-mannosidase. ,B-Mannosidase, a-glycosidase, P-glycosidase, a-galactosidase, and 3-galactosidase had no obvious effect on either intensity or the molecular weight of species recognized by 3C2. Figure 5 shows the effect of proteolysis on epitope recognition by
A B C D E F G H I
c36kD ,c3OkD
' 'I
FIG. 3. Western blot reactivities of three MAbs. Lanes A, B, and C, reactivity of 3C2 with antigens B3501 and B4131 (C. neoformans) and NCPF 4874 (T. beigeliO), respectively; lanes D, E, and F, reactivity of 7H4 with same antigens; lanes G, H, and I, reactivity of 5G5 with same antigens.
34-38kD>
awi
__WI
FIG. 4. Effect of various deglycosylation enzymes on the reactivity of 3C2 as determined by Western blot. Lanes: A, untreated control; B, a-mannosidase; C, 1-mannosidase; D, neuraminidase; E, a-galactosidase; F, ,B-galactosidase; G, peptide N-glycanase F; H, a-glucosidase; I, ,B-glucosidase.
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of cell membrane and cytoplasm by all the MAbs with the four Cryptococcus isolates tested (Fig. 6), with no recognition of either the cell wall or capsule. DISCUSSION
34-38kD>Pw 20-22kD>¶
FIG. 5. Effect of various proteolytic enzymes on reactivity of 3C2 as determined by Western blot. Lanes: A, control; B, papain; C, pepsin; D, trypsin; E, hydroxylamine; F, V8 protease; G, a-chymotrypsin; H, CNBr; I, BNPS-skatole; J, BNPS-skatole followed by N-bromosuccinimide.
3C2. Of the enzymes used, papain, pepsin, and a-chymotrypsin prevented recognition by 3C2, whereas V8 protease and trypsin digestion did not. With the latter, low-molecularweight species were recognized, indicating digestion of the 34- to 38-kDa molecule and its smaller components but not of the epitope itself. Of the chemical treatments used, hydroxylamine and cyanogen bromide did not destroy the epitope, whereas BNPS-skatole did; it was not possible to deduce the effect of N-bromosuccinimide following treatment with BNPS-skatole. Immunofluorescence studies demonstrated strong staining
Until now, the MAbs raised against C. neoformans have been directed against capsular polysaccharide (7, 8, 19) since this material has been identified as the major exoantigen produced by this pathogen. Murphy et al. (15) defined three components within the culture filtrate from C. neoformans: high-molecular-weight glucuronoxylomannan, galactoxylomannan, which has been extensively characterized (18, 21) and against which MAbs have been raised (22), and a mannoprotein. None of the MAbs raised have been used to supplement the latex agglutination test, and while the test has proven to be generally effective, its detection of circulating antigen in patients suffering from AIDS, even after infection has apparently ceased, has made the isolation of other exoantigens, whose presence may more accurately reflect active infection in such situations, of some importance. In this regard, it is clear from our data that we have succeeded in raising a second MAb (3C2), in addition to the MAbs already detailed (13), that not only is capable of detecting an exoantigen of C. neoformans at extremely low concentrations by conventional ELISA but also is directed against some noncapsular component. It is clearly not related to either glucuronoxylomannan or galactoxylomannan, and any similarity to previously identified mannoprotein can be dismissed since mannoprotein has been shown to be a
FIG. 6. Immunofluorescence reactivity of 3C2 against cryostat sections of C. neoformans. Bar represents 150 ,um.
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nonstructural element of the cell wall by immunoelectron microscopy (23), whereas 3C2 is reactive by immunofluorescence to the cytoplasm but not the cell wall or capsule of C. neoformans. Any relationship between the 34- to 38-kDa glycoprotein recognized by 3C2 and the 110- to 120-kDa molecule identified by our previously defined MAb 7C9 (13) is unclear, although there appears to be some overlap in molecular weight recognition since 7C9 is also reactive to a series of lower-molecular-weight species, including one at 36 to 38 kDa. Our unpublished data suggest that 7C9 recognizes a nonglycosylated epitope on a glycoprotein, though our studies of this are at a preliminary stage. Some aspects of the composition of the epitope which is recognized by 3C2 can be deduced from the effects of various deglycosylation and proteolytic enzymes. Neuraminidase specifically removes terminal sialic acids, the result being the abolition of recognition by 3C2, indicating the primacy of this carbohydrate in the composition of the epitope. The effect of peptide N-glycanase F demonstrates the presence of N-acetylglucosaminyl asparagine residue(s) to which the sialic acid(s) will be linked, either directly or via an oligosaccharide chain, though it may be that the importance of the acetylglucosaminyl residue is only as a linkage group to which the major determinant, sialic acid, is joined. There is also evidence for the existence of a-mannose in the epitope, though its presence does not appear to be an absolute determinant for the recognition by 3C2. a-Mannose probably exists as side chains extending from the N-acetylglucosaminyl group; only in this sort of format would they be accessible to the exoglycosidic action of ot-mannosidase, which leads to the reduction in recognition by 3C2. It is conceivable that abolition of recognition by 3C2 after deglycosylation is a result of conformational change; however, conformationally defining carbohydrates are likely to be internal and so would not be accessible to the various enzymes employed in this study. When the specificities of the enzymatic and chemical cleavage of the peptide backbone are interpreted, it becomes immediately clear that tryptophan is an integral part of the epitope, since BNPS-skatole and a-chymotrypsin destroy the reactivity of 3C2. The latter agent also cleaves at tyrosine and phenylalanine as well as tryptophan, so the existence of one or both of these residues cannot be ruled out. Similarly, whereas the broadly active enzyme papain cuts at any one of lysine, arginine, leucine, and glycine and clearly destroys the epitope, the fact that trypsin, which cuts at arginine and lysine, does not prevent recognition by 3C2 suggests that leucine or glycine or both are important residues within the epitope. Unfortunately, little can be deduced from the loss of 3C2 reactivity by using N-bromosuccinimide since, in addition to cleaving at tyrosine and histidine, it is also active at tryptophan. Finally, the absence of any effect by CNBr and V8 protease indicates the absence of methionine, aspartic acid, and glutamic acid in the epitope, though the fact that the molecular weight of the species recognized by 3C2 declines after treatment suggests the presence of such residues elsewhere in the molecule. While 7H4 and 5G5 recognized only two bands by Western blot at 36 and 30 kDa, 3C2 was reactive to a large number of epitopes with molecular masses below 34 kDa. This may suggest that 3C2 recognizes an epitope shared by a range of low-molecular-weight antigens, but we think it more likely that 3C2 is reacting with breakdown products of the 34- to 38-kDa molecule, despite the fact that protease inhibitors were added to the antigen used in Western blotting. Preliminary attempts to directly detect circulating antigen
INFECT. IMMUN.
in patients' sera and cerebrospinal fluid by both ELISA and Western blot have proved only partially successful, and so we are presently attempting to isolate the antigen to which 3C2 binds in large quantities via a combination of ionexchange (using the pl data disclosed) and affinity chromatography, with a view to raising an immunoglobulin M MAb against it. When combined with 3C2, the latter would form the basis for a specific-antigen-trap ELISA similar to that developed for other pathogens (3). Such a test would have to be both highly sensitive and reproducible, though it would immediately have the advantage of enabling infections by the two varieties of C. neoformans to be distinguished since 3C2 is only reactive to C. neoformans var. neoformans. It is also hoped that highly purified antigen can be subjected to peptide sequencing in an attempt to define a function for the molecule since it appears not to perform the most obvious role of fungal exoantigens, that of proteolytic enzyme. The partial information on the amino acid composition of the determinant recognized by 3C2, when combined with peptide sequence data, will allow us to deduce the location of this species-specific epitope and so determine its full amino acid sequence. ACKNOWLEDGMENTS We thank Colin Campbell of the medical Mycology Reference Laboratory, PHLS Laboratory, Colindale, London, and K. J. Kwon-Chung, National Institutes of Health, Bethesda, Md., for supplying us with various fungal isolates. We also thank Mary Moore of the Mycology Department, St. John's Hospital, St. Thomas Hospital, London, for also supplying isolates. This research was funded by a grant from the Wellcome Trust. REFERENCES 1. Bergmeyer, H. U. 1984. Methods in enzymatic analysis. V. Enzymes 3: peptidases, proteinases and their inhibitors, 3rd ed., p. 140-156. Academic Press, New York. 2. Birk, Y. 1976. Protease inhibitors. Methods Enzymol. 45:701703. 3. Burkot, T. R., F. Zavala, R. W. Gwadz, F. H. Collins, R. S. Nussensweig, and D. R. Roberts. 1984. Identification of malaria
infected mosquitoes by a two-site enzyme-linked immunosorbent assay. Am. J. Trop. Med. Hyg. 33:227-231. 4. Campbell, C. K., A. L. Payne, A. J. Teall, A. Brownell, and D. W. R. Mackenzie. 1985. Cryptococcal latex antigen test positive in a patient with Trichosporon beigelii infection. Lancet
ii:43-44.
5. Dismukes, W. E. 1988. Cryptococcal meningitis in patients with AIDS. J. Infect. Dis. 157:624-627. 6. Dolan, C. T. 1972. Specificity of the latex-cryptococcal antigen test. Am. J. Clin. Pathol. 58:358-364. 7. Dromer, F., J. Salamero, A. Contrepois, C. Carbon, and P. Yeni. 1987. Production, characterization, and antibody specificity of a mouse monoclonal antibody reactive with Cryptococcus neoformans capsular polysaccharide. Infect. Immun. 55:742-748. 8. Eckert, T. F., and T. R. Kozel. 1987. Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide. Infect. Immun. 55:18951899. 9. Ey, P. L., S. J. Prowse, and C. R. Jenkin. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Biochemistry 15:429-436. 10. Figueroa, J., A. J. Hamilton, M. A. Bartholomew, T. Harada, L. E. Fenelon, and R. J. Hay. 1990. Preparation of speciesspecific murine monoclonal antibodies against the yeast phase of Paracoccidioides brasiliensis. J. Clin. Microbiol. 28:17661769. 11. Goodman, J. S., L. Kaufman, and R. Keonig. 1971. Diagnosis of cryptococcal meningitis. Value of immunologic detection of cryptococcal antigen. N. Engl. J. Med. 285:434-436. 12. Hamilton, A. J., M. A. Bartholomew, J. Figueroa, L. E. Fenelon,
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and R. J. Hay. 1990. A murine monoclonal antibody exhibiting high species specificity for Histoplasma capsulatum var. capsulatum. J. Gen. Microbiol. 136:331-335. 13. Hamilton, A. J., M. A. Bartholomew, J. Figueroa, L. E. Fenelon, and R. J. Hay. 1991. Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen. J. Clin. Microbiol. 29:980-
immunoassay detection of IgM to galactoxylomannan of Crypneoformans. Diagn. Immunol. 2:109-15. Spiropulu, C., R. A. Eppard, E. Otteson, and T. R. Kozel. 1989. Antigenic variation within serotypes of Cryptococcus neoformans detected by monoclonal antibodies specific for the capsular polysaccharide. Infect. Immun. 57:3240-3242. Swinne, D., and C. de Vroey. 1987. Epidemiologie de la cryptococcuse. Rev. Mycol. 4:77083. Turner, S. H., R. Cherniak, and E. Reiss. 1984. Fractionation and characterization of galactoxylomannan from Cryptococcus neoformans. Carbohydr. Res. 125:343-349. Van de Moer, A., S. L. Salhi, R. Cherniak, B. Pan, M. L. Garrigues, and J. M. Bastide. 1990. An anti-Cryptococcus neoformans monoclonal antibody directed against galactoxylomannan. Res. Immunol. 141:33-42. Vartivarian, S. E., G. H. Reyes, E. S. Jacobson, P. G. James, R. Cherniak, and V. R. Muman. 1989. Localization of mannoprotein in Cryptococcus neoformans. J. Bacteriol. 171:6850-6852. Zola, H., and D. Brooks. 1982. Techniques for the production and characterization of monoclonal antibodies, p. 1-57. In J. Hurrell (ed.), Monoclonal hybridoma antibodies: techniques and applications. CRC Press, Boca Raton, Fla.
984.
14. Hunziker, P. E., G. J. Hughes, and K. J. Wilson. 1980. Peptide fragmentation for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole (3-bromo-3-methyl-2-((2-nitrophenyl)thio)-3H-indole). Biochem. J. 187:515-519. 15. Murphy, J. W., R. L. Mosley, R. Cherniak, G. H. Reyes, T. R. Kozel, and E. Reiss. 1988. Serological, electrophoretic, and biological properties of Cryptococcus neoformans antigens. Infect. Immun. 56:424-431. 16. Prevost, E., and R. NeweUl. 1978. Commercial cryptococcal latex kit: clinical evaluation in a medical center hospital. J. Clin. Microbiol. 8:529-533. 17. Read, S. M., and D. H. Northcote. 1981. Minimization of variation in the responses to different proteins of the Coomassie blue dye-binding assay for proteins. Ann. Biochem. 116:53- 64. 18. Reiss, E., R. Cherniak, R. Eby, and C. Kaufman. 1984. Enzyme
tococcus
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20. 21. 22.
23. 24.
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A 34- to 38-Kilodalton Cryptococcus neoformans Glycoprotein Produced as an Exoantigen Bearing a Glycosylated Species-Specific Epitope HAMILTON,'* L. JEAVONS,1 P. HOBBY,2 AND R. J. HAY Dermatology Unit' and Renal Unit,2 Clinical Sciences Department, 18th Floor, Guy's Tower, Guy's Hospital, London Bridge, London SE] 9RT, United Kingdom A. J.
Received 15 August 1991/Accepted 17 October 1991
Three monoclonal antibodies (MAbs), all of the immunoglobulin Gl subclass, were raised against Cryptococcus neoformans by using the technique of cyclophosphamide ablation of B-cell responses against shared epitopes of the cross-reactive fungus Trichosporon beigelii. MAb 3C2 was reactive against the encapsulated and nonencapsulated isolates of C. neoformans var. neoformans by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot), and in addition to a 34- to 38-kDa deterniinant, it recognized a series of lower-molecular-weight species. 3C2 also reacted strongly with culture supernatant preparations of C. neoformans var. neoformans by ELISA. 3C2 showed no recognition of either T. beigelii or C. neoformans var. gaYii antigens. Enzymatic deglycosylation followed by reaction with 3C2 on Western blots revealed that sialic acid was an integral part of the determinant, together with N-acetylglucosaminylasparagine and a-mannose. Proteolytic digestion showed that the epitope was pepsin sensitive and that it also contained tryptophan and glycine and/or leucine as determinants of recognition by 3C2. The pl of the glycoprotein was 7.1. Affinity chromatography-purified antigen did not exhibit proteolytic activity on sodium dodecyl sulfate-polyacrylamide substrate gels. Indirect fluorescence antibody tests revealed that 3C2 labelling was confined to the cell membrane and cytoplasm of yeasts. The remaining MAbs, 7H4 and 5G5, recognized both capsulated and nonencapsulated strains of C. neoformans var. neoformans by both ELISA and Western Blot, identifying linear determinants with molecular masses of 36 and 30 kDa. They were unreactive against culture supernatant antigen (exoantigen) from either variant of C. neoformans.
The disease cryptococcosis is caused by either of the two varieties of the encapsulated yeast Cryptococcus neoformans, C. neoformans var. neoformans and C. neoformans var. gattii, and is characterized by dissemination from a pulmonary focus and a potentially fatal form of meningitis. Though the disease may occur in individuals who show no evidence of immunosuppression, it has recently attracted much interest because of its prevalence in patients infected with human immunodeficiency virus. For instance, a recent study in Zaire has shown, by screening for circulating cryptococcal antigen, that up to 11% of human immunodeficiency virus-positive individuals had been infected with the organism (20). At present, the diagnosis of the disease relies on either isolation of the organism or the detection of capsular polysaccharide antigen by using the latex agglutination test with either serum or cerebrospinal fluid (11, 16). Though the latex agglutination test has proved very useful for diagnosis, it is limited to some extent by the crossreactivity of the antigen with other fungal pathogens, such as Trichosporon beigelii (4, 6), and by the fact that some human immunodeficiency virus-positive individuals still show positive tests long after other signs of disease have disappeared. It is not clear whether this reflects failure to eliminate capsular polysaccharide or persistence of infection (5). Therefore, it would be useful to identify other speciesspecific exoantigens produced by C. neoformans, the occurrence and levels of which in serum or cerebrospinal fluid might more accurately reflect the number of viable yeast cells present. Recently, our laboratory has produced spe*
cies-specific monoclonal antibodies (MAbs) against a range of fungal pathogens by using the technique of cyclophosphamide ablation of shared B-cell responses (10, 12), and most recently, we have demonstrated the presence of a 110- to 120-kDa molecule in the culture filtrate of C. neoformans which bears a species-specific epitope (13). In this paper, we report the occurrence and preliminary biochemical characterization of a smaller glycoprotein (34 to 38 kDa) produced as an exoantigen which bears a species-specific epitope and as such has potential for use in serodiagnosis. MATERIALS AND METHODS
Antigen preparation. Lyophilized isolates of C. neoformans var. gattii (2), Cryptococcus laurentii (1), Cryptococcus albidus (1), Aspergillusfumigatus (2), Aspergillusflavus (2), Candida albicans (1) and Candida tropicalis (1) were obtained from the National Collection of Pathogenic Fungi (NCPF), Mycological Reference Laboratory, Colindale, London, United Kingdom (NCPF 3169 and NCPF 3170, NCPF 3141, NCPF 3140, NCPF 2010 and NCPF 2978, NCPF 2208 and NCPF 2617, NCPF 3343, and NCPF 3114, respectively). The isolates were reconstituted with 0.5 ml of sterile water, inoculated onto Sabouraud agar, and then expanded into larger (250 ml and 3 liters) volumes of Sabouraud liquid culture medium. Two additional strains of C. neoformans (B3501, encapsulated, and B4131, nonencapsulated) were obtained from June Kwon-Chung, Mycology Department, National Institutes of Health, Bethesda, Md., while four isolates of C. neoformans var. neoformans (NCPF 3171, serotype D; 3168, serotype A; 3409 and 3081, serotypes unknown), together with two isolates of T. beigelii
Corresponding author. 143
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(NCPF 4874 and 5246), were obtained from the Mycology Laboratory, St. John's Hospital for Diseases of the Skin, London, United Kingdom, and treated as described above. Mycelial isolates of Histoplasma capsulatum var. capsulatum (two isolates), Blastomyces dermatitidis (one), Paracoccidioides brasiliensis (three), and Sporothrix schenckii (two) (NCPF 4100 and NCPF 4088; NCPF 4076; NCPF 3285, NCPF 4115, and NCPF 4095; and NCPF 3181 and NCPF 3286, respectively) were also obtained from the National Collection of Pathogenic Fungi, transformed to the yeast phase at 37°C on slopes of brain heart infusion medium to which 0.2 mM L-cysteine was added, and subsequently subcultured in brain heart infusion broth at 37°C. All fungal preparations were collected by filtration (Whatman no. 2 filter paper), washed in phosphate-buffered saline (PBS; 0.01 M, pH 7.4), and divided into two subsamples, to one of which a cocktail of protease inhibitors was added (1, 2) for subsequent use as antigen for Western blotting (immunoblotting). Each sample was then homogenized on ice by using glass ball ballotini in a bead beater (Biospec Products, Bartlesville, Oklahoma) by using 20 1-min pulses separated by 5-min gaps, which had previously been shown by protein estimation and Western blotting to yield the largest amounts of undenatured protein. The soluble cell extracts were collected and centrifuged at 2,000 x g for 7 min, and the supernatants were retained to yield cytoplasmic antigens, which were divided into portions and stored at -70°C. For the various Cryptococcus strains, the culture filtrates were retained, dialyzed against polyethylene glycol 8000 to 1/100 of their original volumes, and then redialyzed overnight against PBS at 4°C to yield a crude exoantigen preparation. Protein concentration was estimated in each sample by using the Coomassie blue method (17). Immunization protocol. The immunization schedule incorporating cyclophosphamide was as previously described (13) with T. beigelii (NCPF 4874) and C. neoformans var. neoformans (B4131) as antigens, except that the test bleed and differential enzyme-linked immunosorbent assay (ELISA) took place on day 26. Fusion protocol. Cells of the myeloma line Sp2/0 were fused with spleen cells from the chosen mouse at a ratio of 1:10 by using polyethylene glycol 4000 according to a modification of an existing protocol (24), and the resulting hybridomas were plated onto 96-well microtiter plates. At 7 days postfusion, colonies were screened by ELISA with the immunizing antigens and the encapsulated C. neoformans isolate B3501 (13). Clones from wells differentiating between the three antigens were subcloned twice by limiting dilution before expansion into 25-ml flasks. Female BALB/c mice previously primed with pristane were given intraperitoneal injections of 104 hybridoma cells, and ascitic fluid was collected 14 to 21 days later. MAbs were purified from ascitic fluids using a protein A-Sepharose column (Pharmacia) (9). All three of the MAbs described in this study were effectively eluted by a 0.1 M citric acid buffer solution (pH 6), and MAb subclassing was then performed (13). Protein estimations of the eluted immunoglobulins were carried out as previously described (17). Purified MAbs (initial concentration, 100 ,ug ml-') in PBSTween were then assessed for reactivity by ELISA (100 ,ul per well) for all the cytoplasmic fungal antigens described above. All antigens were used at protein concentrations of 1 ,ug per well. In addition, MAbs were assessed for recognition of culture supernatant of C. neoformans by ELISA; the procedure was done as described previously (13) except that the antigen was diluted progressively by a ratio of 1:10,
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starting with an initial concentration of 1 ,ug per well, in order to determine the lowest detectable concentration of antigen. In all the ELISAs, either Histoplasma capsulatumspecific MAb C69 (12) or PBS-Tween was used as the negative control. Polyacrylamide gel electrophoresis (PAGE), electroblotting, and immunoenzyme development. Each of the C. neoformans var. neoformans, C. neoformans var. gattii, and T. beigelii antigens and one of each of the other antigens containing protease inhibitors (total of 100 ,ug of protein per gel) were electrophoresed on a sodium dodecyl sulfate (SDS)-7.5% (wt/vol) polyacrylamide gel at 200 V for 1 h. After semidry blotting, immunoenzyme development was performed with MAbs at a protein concentration of 25 ,ug per strip (12, 13). Affinity chromatography. One gram of CNBr-activated Sepharose 4B (Pharmacia) was swollen in 200 ml of ice-cold 1 mM HCl by washing on a sintered glass funnel for 15 min. Protein A-purified MAb 3C2 (15 mg) in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.5]) was added to the Sepharose, and the two were mixed by end-over-end rotation for 6 h at room temperature. After centrifugation (500 x g for 5 min), the supernatant was removed and active groups were blocked overnight at 4°C by the addition of 2 M ethanolamine (pH 8.0). The Sepharose was then poured into a 5-ml syringe blocked with glass wool and washed alternately with washing buffer at pH 4.0 (0.1 M glacial acetic acid, 0.5 M NaCl) and washing buffer at pH 8.0 (0.1 M Tris base, 0.5 M NaCl). Five micrograms of C. neoformans (capsular negative) cytoplasmic antigen was then added to the column in 20 ml of coupling buffer and recirculated overnight at 4°C in a closed system running at 0.5 ml/min. A280 was monitored as a baseline was established by flushing with coupling buffer. Bound antigen was then eluted by progressive washes with 0.2 M glycine HCI (pH 4.0, 3.0, and 2.2) in volumes of 25 ml each and immediately neutralized prior to analysis by ELISA, SDS-PAGE, and protein estimation. Substrate SDS-polyacrylamide gels. SDS-7.5% polyacrylamide gels were copolymerized with either 0.1% bacteriological gelatin (GIBCO) or 2% casein (BDH). Ten micrograms of affinity chromatography-purified 34- to 38-kDa antigen (unreduced and unboiled) was loaded into each of four wells together with a commercial preparation of pepsin (Sigma; 20 U total in 0.1 M citrate buffer, pH 2.0) and molecular weight markers (GIBCO). Gels were then electrophoresed quickly (25 mA per gel) at 4°C, placed in Triton X-100, and rocked at room temperature for 30 min to remove residual SDS. The gel was then further incubated in PBS for 60 min, the buffer was replaced, and the gel was placed overnight at 37°C prior to Coomassie brilliant blue staining and destaining. Isoelectric focusing. Samples of (10 ,ug) of cytoplasmic yeast (C. neoformans, capsular negative) were run in a 7.5% polyacrylamide gel containing 2% ampholytes (Pharmacia) in the pl range of 3 to 10 on a multiphor electrophoresis system (Pharmacia) with a thermostatic plate cooled to 10°C. The pH gradient of the gel was measured by using Electran colored protein markers (BDH). The gel was capillary blotted onto an immobilin membrane, and immunoenzyme development was performed by using 3C2. Enzymatic deglycosylation of the 34- to 38-kDa antigen.
Subsamples (10 ,ug) of C. neoformans (capsular-negative) cytoplasmic yeast antigen were incubated for 24 h with the following enzymes: a-mannosidase (Sigma) in 0.1 M sodium citrate buffer (pH 4.0) at 25°C, ,-mannosidase (Sigma) in 0.1
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M sodium citrate buffer (pH 4.0) at 25°C, neuraminidase (BDH) in 0.1 M potassium acetate buffer (pH 4.5) supplemented with 2 mM CaCl at 37°C, a-galactosidase (Sigma) in 0.1 M sodium citrate (pH 4.0) at room temperature, ,-galactosidase (Sigma) in 0.4 M sodium citrate (pH 4.0) at room temperature, a-glucosidase (Sigma) in 0.1 M sodium acetate supplemented with 0.05% EDTA (pH 6.0) at 37°C, and peptide N-glycanase F in 25 ,ul of digestion buffer consisting of 300 ,ul of 100 mM 1,10-phenanthroline, 500 ,ul of 7.5% NP-40, and 900 ,u1 of 550 mM sodium phosphate (pH 8.6) at 37°C. With peptide N-glycanase F, some of the antigen was reduced prior to digestion in 1% SDS and 1.6% 2-mercaptoethanol (boiled for 3 min). All enzymes were used in volumes equivalent to 10 U of activity. After digestion, all samples were boiled for 3 min in the presence of 1% SDS and 1.6% 2-mercaptoethanol, loaded onto SDS-7.5% polyacrylamide gels along with molecular weight markers, and electrophoresed at 200 V. Gels were then subsequently blotted onto Immobilin prior to immunoenzyme development with MAb 3C2. Protease digestion of the 34- to 38-kDa antigen. Subsamples (10 ,ug) of C. neoformans cytoplasmic antigen were treated with each of the following enzymes for 24 h (except where stated) under the following conditions: pepsin (Sigma) in 10 mM HCI at 37°C; protease V8 (Sigma) in 0.01 M PBS (pH 7.2) at 37°C; trypsin (Sigma), N-tosylamide-L-phenylamine chloromethyl ketone (TPCK) treated, in 0.67 M sodium phosphate buffer (pH 7.6) at room temperature; a-chymotrypsin (Sigma) in a solution of 80 ,u1 of 2 M CaCl and 3 ml of Tris-HCl buffer (pH 7.6) at room temperature; and papain (Sigma) in a solution of 1 ml of 3 M NaCl in 10 ml of a mixture of 20 mM EDTA and 50 mM L-cysteine (pH 6.2) at room temperature. All enzymes were used at concentrations equivalent to 50 U. Samples (10 ,ug) were also subjected to the following chemical treatments: hydroxylamine (Sigma), 50 mM in distilled water, at room temperature; 2-(2'-nitrophenylsulfonyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) (Pierce), 10 mg ml-' in 70% distilled acetic acid with 0.1% phenol for 48 h at room temperature and with a 10-fold excess of the reagent, followed by the addition of a 10-fold excess of 2-mercaptoethanol, a further incubation for 5 h at 37°C, and extraction of excess BNPS-skatole with ethyl acetate (14); pretreatment with BNPS-skatole followed by exposure of the antigen to a 12-fold excess of N-bromosuccinimide (Sigma), 10 mg ml-' in aqueous 50% distilled acetic acid for 2 h at 30°C; and cyanogen bromide (Sigma), 0.5 g in 10 ml of 0.1 M HCl for 48 h at room temperature. Samples were treated and analyzed by SDS-PAGE and Western blotting in the same way as those prepared for the deglycosylation study above. Immunofluorescent location of antigen. Immunofluorescent location of antigen was performed as previously described (13) with C. neoformans (B3501) as test organism and MAbs diluted 1:10 in PBS-Tween.
RESULTS The ELISA performed with the polyclonal serum samples from the four mice used for the immunization protocol identified one mouse with significantly higher titers to the C. neoformans (encapsulated) strain antigen than those to either the nonencapsulated C. neoformans or T. beigelii antigens used in the immunization protocol, and this animal was used for the fusion study. The other animals showed a limited differential response to the various antigens. Control mice, which underwent the same immunization protocol but
CRYPTOCOCCUS NEOFORMANS EXOANTIGEN
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1-2 0 U
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0
0 0 a
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3C2 MAbs FIG. 1. Reactivities of three MAbs against a range of antigens, as defined by absorbance values in ELISA. 0, C. neoformans nonencapsulated B4131; *, B3501; A, NCPF 3171; V, NCPF 3081; *, NCPF 3168; *, NCPF 3409; O, NCPF 3169; O, NCPF 3170; A, T. beigelii NCPF 4874 (NCPF 3169 and NCPF 3170 are C. neoformans var. gattii; the others are C. neoformans encapsulated isolates, except B4131). Isolates of all other fungi tested gave values below 0.1 (data not shown).
without the use of cyclophosphamide, showed no difference in serum reactivities in a similar ELISA (data not shown). Three hybridomas were chosen after a total of 1,500 were screened by ELISA. After successive subclonings, three MAbs, designated 3C2, 7H4, and 5G5, were obtained, and all three were shown to belong to the immunoglobulin Gl subclass. By ELISA, MAbs 3C2 and 7H4 recognized the nonencapsulated and capsular isolates of C. neoformans but showed no reactivity to T. beigelii, while MAb SG5 recognized all of these antigens (Fig. 1). None of the MAbs showed any reactivity to C. neoformans var. gattii or to any of the other fungal antigens tested. 3C2 was strongly reactive to culture supernatant antigen from C. neoformans var. neoformans isolates and was able to detect antigen at a protein concentration as low as 0.001 ng (Fig. 2). In contrast, 7H4 and 5G5 failed to recognize exoantigen. Western blot analysis demonstrated that 3C2 recognized a determinant at 34 to 38 kDa on reduced gels, two major bands at approximately 26 to 28 and 20 to 22 kDa, and four minor bands on blots of nonencapsulated. and encapsulated isolates of C. neoformans (Fig. 3) but showed no reaction to any other antigens except culture supernatant antigen from C. neoformans var. neoformans (data not shown). The pattern of recognition on nonreduced blots was exactly the same. MAbs 7H4 and 5G5 recognized two bands at 36 kDa and approximately 30 kDa and showed the same pattern of antigen recognition as that shown by ELISA. Isoelectric focusing of nonencapsulated C. neoformans cytoplasmic antigen followed by blotting and immunoenzyme development with 3C2 revealed a single strong band with a pl of 7.1, whereas over 80% of the remaining cytoplasmic antigens appeared to have pIs of 5.0 and less.
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1-21
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wc
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10
A total of 35 ,ug of 3C2 antigen was eluted from affinity chromatography columns (two columns loaded with a total of 15 mg each of cytoplasmic antigen) with glycine-HCl buffer (pH 2.2). The fractions eluted with glycine-HCl at pH 4 and pH 3 were negative when reacted with MAb 3C2 by ELISA and contained undetectable amounts of protein. SDS-polyacrylamide gels stained with Coomassie brilliant blue revealed the predominance of the 34- to 38-kDa antigen in the eluent. However, when these samples were run on SDS-polyacrylamide substrate gels (with gelatin and casein),
26-28kD> 20-22kD> _
13
Antigen concentration (9)
FIG. 2. Reactivities of three MAbs to culture supernatant antigens by ELISA. encapsulated-isolate antigens gave a pattern similar to that shown here for B3501.
34-38kD>
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lo-11
0,
3C2; O, 5G5;
A,
7H4. Other C. neoformans
there was no evidence of proteolytic clearing compared with that of the positive controls. Figure 4 indicates the effect of deglycosylation on the recognition of the 34- to 38-kDa- glycoprotein and lowermolecular-weight species by 3C2 when visualized by Western blot. Neuraminidase completely destroys recognition as does peptide N-glycanase F. There is also a partial decrease in reactivity with treatment with a-mannosidase. ,B-Mannosidase, a-glycosidase, P-glycosidase, a-galactosidase, and 3-galactosidase had no obvious effect on either intensity or the molecular weight of species recognized by 3C2. Figure 5 shows the effect of proteolysis on epitope recognition by
A B C D E F G H I
c36kD ,c3OkD
' 'I
FIG. 3. Western blot reactivities of three MAbs. Lanes A, B, and C, reactivity of 3C2 with antigens B3501 and B4131 (C. neoformans) and NCPF 4874 (T. beigeliO), respectively; lanes D, E, and F, reactivity of 7H4 with same antigens; lanes G, H, and I, reactivity of 5G5 with same antigens.
34-38kD>
awi
__WI
FIG. 4. Effect of various deglycosylation enzymes on the reactivity of 3C2 as determined by Western blot. Lanes: A, untreated control; B, a-mannosidase; C, 1-mannosidase; D, neuraminidase; E, a-galactosidase; F, ,B-galactosidase; G, peptide N-glycanase F; H, a-glucosidase; I, ,B-glucosidase.
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of cell membrane and cytoplasm by all the MAbs with the four Cryptococcus isolates tested (Fig. 6), with no recognition of either the cell wall or capsule. DISCUSSION
34-38kD>Pw 20-22kD>¶
FIG. 5. Effect of various proteolytic enzymes on reactivity of 3C2 as determined by Western blot. Lanes: A, control; B, papain; C, pepsin; D, trypsin; E, hydroxylamine; F, V8 protease; G, a-chymotrypsin; H, CNBr; I, BNPS-skatole; J, BNPS-skatole followed by N-bromosuccinimide.
3C2. Of the enzymes used, papain, pepsin, and a-chymotrypsin prevented recognition by 3C2, whereas V8 protease and trypsin digestion did not. With the latter, low-molecularweight species were recognized, indicating digestion of the 34- to 38-kDa molecule and its smaller components but not of the epitope itself. Of the chemical treatments used, hydroxylamine and cyanogen bromide did not destroy the epitope, whereas BNPS-skatole did; it was not possible to deduce the effect of N-bromosuccinimide following treatment with BNPS-skatole. Immunofluorescence studies demonstrated strong staining
Until now, the MAbs raised against C. neoformans have been directed against capsular polysaccharide (7, 8, 19) since this material has been identified as the major exoantigen produced by this pathogen. Murphy et al. (15) defined three components within the culture filtrate from C. neoformans: high-molecular-weight glucuronoxylomannan, galactoxylomannan, which has been extensively characterized (18, 21) and against which MAbs have been raised (22), and a mannoprotein. None of the MAbs raised have been used to supplement the latex agglutination test, and while the test has proven to be generally effective, its detection of circulating antigen in patients suffering from AIDS, even after infection has apparently ceased, has made the isolation of other exoantigens, whose presence may more accurately reflect active infection in such situations, of some importance. In this regard, it is clear from our data that we have succeeded in raising a second MAb (3C2), in addition to the MAbs already detailed (13), that not only is capable of detecting an exoantigen of C. neoformans at extremely low concentrations by conventional ELISA but also is directed against some noncapsular component. It is clearly not related to either glucuronoxylomannan or galactoxylomannan, and any similarity to previously identified mannoprotein can be dismissed since mannoprotein has been shown to be a
FIG. 6. Immunofluorescence reactivity of 3C2 against cryostat sections of C. neoformans. Bar represents 150 ,um.
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nonstructural element of the cell wall by immunoelectron microscopy (23), whereas 3C2 is reactive by immunofluorescence to the cytoplasm but not the cell wall or capsule of C. neoformans. Any relationship between the 34- to 38-kDa glycoprotein recognized by 3C2 and the 110- to 120-kDa molecule identified by our previously defined MAb 7C9 (13) is unclear, although there appears to be some overlap in molecular weight recognition since 7C9 is also reactive to a series of lower-molecular-weight species, including one at 36 to 38 kDa. Our unpublished data suggest that 7C9 recognizes a nonglycosylated epitope on a glycoprotein, though our studies of this are at a preliminary stage. Some aspects of the composition of the epitope which is recognized by 3C2 can be deduced from the effects of various deglycosylation and proteolytic enzymes. Neuraminidase specifically removes terminal sialic acids, the result being the abolition of recognition by 3C2, indicating the primacy of this carbohydrate in the composition of the epitope. The effect of peptide N-glycanase F demonstrates the presence of N-acetylglucosaminyl asparagine residue(s) to which the sialic acid(s) will be linked, either directly or via an oligosaccharide chain, though it may be that the importance of the acetylglucosaminyl residue is only as a linkage group to which the major determinant, sialic acid, is joined. There is also evidence for the existence of a-mannose in the epitope, though its presence does not appear to be an absolute determinant for the recognition by 3C2. a-Mannose probably exists as side chains extending from the N-acetylglucosaminyl group; only in this sort of format would they be accessible to the exoglycosidic action of ot-mannosidase, which leads to the reduction in recognition by 3C2. It is conceivable that abolition of recognition by 3C2 after deglycosylation is a result of conformational change; however, conformationally defining carbohydrates are likely to be internal and so would not be accessible to the various enzymes employed in this study. When the specificities of the enzymatic and chemical cleavage of the peptide backbone are interpreted, it becomes immediately clear that tryptophan is an integral part of the epitope, since BNPS-skatole and a-chymotrypsin destroy the reactivity of 3C2. The latter agent also cleaves at tyrosine and phenylalanine as well as tryptophan, so the existence of one or both of these residues cannot be ruled out. Similarly, whereas the broadly active enzyme papain cuts at any one of lysine, arginine, leucine, and glycine and clearly destroys the epitope, the fact that trypsin, which cuts at arginine and lysine, does not prevent recognition by 3C2 suggests that leucine or glycine or both are important residues within the epitope. Unfortunately, little can be deduced from the loss of 3C2 reactivity by using N-bromosuccinimide since, in addition to cleaving at tyrosine and histidine, it is also active at tryptophan. Finally, the absence of any effect by CNBr and V8 protease indicates the absence of methionine, aspartic acid, and glutamic acid in the epitope, though the fact that the molecular weight of the species recognized by 3C2 declines after treatment suggests the presence of such residues elsewhere in the molecule. While 7H4 and 5G5 recognized only two bands by Western blot at 36 and 30 kDa, 3C2 was reactive to a large number of epitopes with molecular masses below 34 kDa. This may suggest that 3C2 recognizes an epitope shared by a range of low-molecular-weight antigens, but we think it more likely that 3C2 is reacting with breakdown products of the 34- to 38-kDa molecule, despite the fact that protease inhibitors were added to the antigen used in Western blotting. Preliminary attempts to directly detect circulating antigen
INFECT. IMMUN.
in patients' sera and cerebrospinal fluid by both ELISA and Western blot have proved only partially successful, and so we are presently attempting to isolate the antigen to which 3C2 binds in large quantities via a combination of ionexchange (using the pl data disclosed) and affinity chromatography, with a view to raising an immunoglobulin M MAb against it. When combined with 3C2, the latter would form the basis for a specific-antigen-trap ELISA similar to that developed for other pathogens (3). Such a test would have to be both highly sensitive and reproducible, though it would immediately have the advantage of enabling infections by the two varieties of C. neoformans to be distinguished since 3C2 is only reactive to C. neoformans var. neoformans. It is also hoped that highly purified antigen can be subjected to peptide sequencing in an attempt to define a function for the molecule since it appears not to perform the most obvious role of fungal exoantigens, that of proteolytic enzyme. The partial information on the amino acid composition of the determinant recognized by 3C2, when combined with peptide sequence data, will allow us to deduce the location of this species-specific epitope and so determine its full amino acid sequence. ACKNOWLEDGMENTS We thank Colin Campbell of the medical Mycology Reference Laboratory, PHLS Laboratory, Colindale, London, and K. J. Kwon-Chung, National Institutes of Health, Bethesda, Md., for supplying us with various fungal isolates. We also thank Mary Moore of the Mycology Department, St. John's Hospital, St. Thomas Hospital, London, for also supplying isolates. This research was funded by a grant from the Wellcome Trust. REFERENCES 1. Bergmeyer, H. U. 1984. Methods in enzymatic analysis. V. Enzymes 3: peptidases, proteinases and their inhibitors, 3rd ed., p. 140-156. Academic Press, New York. 2. Birk, Y. 1976. Protease inhibitors. Methods Enzymol. 45:701703. 3. Burkot, T. R., F. Zavala, R. W. Gwadz, F. H. Collins, R. S. Nussensweig, and D. R. Roberts. 1984. Identification of malaria
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and R. J. Hay. 1990. A murine monoclonal antibody exhibiting high species specificity for Histoplasma capsulatum var. capsulatum. J. Gen. Microbiol. 136:331-335. 13. Hamilton, A. J., M. A. Bartholomew, J. Figueroa, L. E. Fenelon, and R. J. Hay. 1991. Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen. J. Clin. Microbiol. 29:980-
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