Vol. 58, No. 3

INFECTION AND IMMUNITY, Mar. 1990, p. 625-631 0019-9567/90/030625-07$02.00/0 Copyright © 1990, American Society for Microbiology

A Monoclonal Antibody That Defines a Surface Antigen on Candida albicans Hyphae Cross-Reacts with Yeast Cell Protoplasts MARKUS W. OLLERTt AND RICHARD A. CALDERONE* Department of Microbiology, School of Medicine, Georgetown University, Washington, D.C. 20007 Received 11 August 1989/Accepted 1 December 1989

Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin Gl antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, P-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-a1-Nacetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, I-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.

ployed to characterize the high-molecular-mass species (180 and 260 kDa) of the germ tube-specific antigens (5). In the present study, BALB/c mice were immunized with a homogenate from whole hyphal cells for the production of MAbs. One of the hybridomas obtained produced a MAb which reacted exclusively with the surfaces of true hyphae and narrow, elongated pseudohyphae in an indirect immunofluorescence assay (IFA), whereas no reactivity was observed with yeast cells and pseudohyphal segments that originated directly from a parent blastoconidium. This germ tube-specific MAb was found to react with a lower-molecular-mass species of protein (55 to 60 kDa) and was selected for further characterization of the antigen which is recognized. (This work was presented in part previously [Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, F-26, p. 462].)

Infections with the opportunistic fungus Candida albicans are frequently found in immunocompromised patients (17, 23). C. albicans displays dimorphic growth, reproducing asexually either by budding, which results in the formation of new blastoconidia, or by germination, which is characterized by the formation of pseudohyphae or true hyphae (24). Although blastoconidia are commonly present in infected tissue (24), there is substantial evidence that the filamentous growth form plays a more critical role in the pathogenesis of candidiasis by mediating attachment to mammalian cells, promoting tissue invasion, and possibly enabling the organism to evade host defenses (2, 8, 9, 15, 16, 24, 33; R. A. Calderone and M. Ollert, in M. J. Kennedy, ed., Fungal Adhesion and Aggregation, in press). Therefore, numerous studies have been initiated in order to define and characterize antigens which are specific for the hyphal growth form of C. albicans. The presence of germ tube-specific antigens was first demonstrated with sera from infected patients (11, 12). In a series of systematic studies, yeast cell-absorbed, polyclonal antisera were used to characterize germ tube-specific antigens. These antigens were found to be sensitive to heat treatment, reducing agents, and proteolytic enzymes (32, 34). Antigens common to both blastoconidia and hyphae are resistant to these treatments (14, 34). Most of the mycelial phase-specific antigens identified by polyclonal antisera were found to have molecular masses of 155 kilodaltons (kDa) or higher (28, 35, 36), except for one wall-associated antigen which has a molecular mass of 19 kDa (28). More recently, monoclonal antibodies (MAbs) have been em-

MATERIALS AND METHODS Organisms and culture conditions. C. albicans wild-type (wt) strain 4918 was used in most experiments and has been described previously (3, 21). The other organisms used were C. albicans 4918-10, a less virulent strain derived from C. albicans 4918 (13), C. albicans A-9 (obtained from W. L. Whelan, National Institutes of Health, Bethesda, Md.), C. stellatoidea, C. tropicalis, and C. pseudotropicalis (all three obtained from K. J. Kwon-Chung, National Institutes of Health), and nine recent clinical isolates of C. albicans (subcultured once before use; obtained from the Georgetown University Hospital). The clinical isolates were classified by using the Yeast I-dent test (Abbott Laboratories, North Chicago, Ill.). Strains were maintained on modified Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). For most experiments, cultures were grown in the synthetic medium described by Lee et al. (19) either at 24°C (yeast

* Corresponding author. t Present address: Department of Biochemistry and Molecular Biology, School of Medicine, Georgetown University, Washington,

DC 20007. 625

626

OLLERT AND CALDERONE

cells) or at 37°C (hyphae). In some of the immunofluorescence studies the hyphal growth form was induced in RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) at 37°C. Yeast cells were also grown at 37°C in yeast nitrogen base (Difco) supplemented with 0.5% glucose. All cultures were grown in a Gyrotory shaker (New Brunswick Scientific Co., Inc., Edison, N.J.) at 150 rpm. Antigen preparation. C. albicans yeast cells were grown in Lee medium as described previously (19) and were allowed to germinate for 5 h at 37°C in the same medium. Cells were harvested by centrifugation, washed, and suspended in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.). Cells were homogenized with 0.45-mm glass beads in a Braun homogenizer (B. Braun Apparatebau, Melsungen, Federal Republic of Germany) under constant CO2 cooling. Subsequently, the cell homogenate was treated as described previously (2). This antigen preparation will be referred to as soluble hyphal cell extract. Dithiothreitol (DTT) extraction of hyphal surface components was performed as described by Chattaway et al. (7) with a final DTT concentration of 12 mM. Immunization. Six- to 10-week-old female BALB/c mice (Jackson Laboratories, Bar Harbor, Maine) were inoculated subcutaneously with 200 ,g of antigen (soluble hyphal cell extract from a 5-h culture in Lee medium at 37°C) emulsified in an equal amount of complete Freund adjuvant (GIBCO). Three weeks later each mouse was boosted by intraperitoneal injection of 100 ,ug of soluble antigen without adjuvant. Two weeks after the first booster injection anti-Candida titers were screened by enzyme-linked immunosorbent assay (ELISA) and Ouchterlony double diffusion. Selected mice received a final booster identical to the first one, and the spleens were removed for fusion 3 days following the last

injection. MAb production. For MAb production, a modification of the method described by Margulies et al. (22) was used. The mouse myeloma cell line NS-1 was grown in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum, 100 ,uM hypoxanthine, 16 ,M thymidine, 50 ,ug of gentamicin per ml, and 20 ,ug of 8-azaguanine (Sigma) per ml. Hybrid cells were selected in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 ,uM hypoxanthine, 16 ,uM thymidine, 0.4 ,uM aminopterin, 50 jig of gentamicin per ml, and 4.5% glucose. Subcloning of the hybridomas was performed in the same medium without aminopterin. Immune spleen cells from a selected mouse were fused with NS-1 myeloma cells at a 4:1 ratio with a 37% solution of polyethylene glycol (molecular weight, 1,300 to 1,600; Sigma). Seven to ten days later culture supematants from wells with positive growth were removed and screened for antibody production by ELISA. Cells from positive wells were subcloned twice by limiting dilution in 96-well plates (Costar, Cambridge, Mass.) with 5 x 105 normal mouse splenocytes per well as a feeder layer. After the second limiting dilution, hybrid cells were screened by IFA, which led to the selection of clone 1.183. For the production of MAb, pristane-primed BALB/c mice were injected intraperitoneally with 106 hybridomas per mouse. Five to fifteen days later the ascitic fluid was drained with an 18-gauge needle. The ascites was rescreened for antibody production by ELISA and IFA. ELISA. The ELISA was performed as described previously (4) with slight modifications. The 96-well plates (Costar) were precoated with 0.001% poly-L-lysine (Sigma) in PBS for 90 min at room temperature. After three washes with PBS the whole-cell homogenate was added (0.5 jig of

INFECT. IMMUN.

protein in PBS [pH 7.4] per well) and incubated for 18 h at 4°C. Control wells received only PBS. The plates were rinsed three times, and each well was filled with 3% bovine serum albumin in PBS (BSA-PBS) and incubated for 60 min. Each plate was washed three times with PBS containing 0.05% Tween 20 (PBST). Undiluted hybridoma culture supernatant or ascitic fluid and mouse serum diluted in BSAPBS were added to the wells (50 jil each). The plate was incubated for 2 h at 37°C and washed three times with PBST. An alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (Bio-Rad Laboratories, Richmond, Calif.) was used as the secondary antibody. This antibody was diluted 1:3,000 in BSA-PBST, and 100 ,ul was added to each well and incubated for 60 min. The plates were rinsed five times with PBST. The substrate mixture (100 ,ul of p-nitrophenylphosphate in five-times-concentrated diethanolamine buffer (BioRad) was added to each well and developed for 30 min at 37°C. The color reaction was stopped by the addition of an equal amount of 0.5 M NaOH. The color intensity was read at 405 nm with an automated plate reader (Flow Laboratories, Inc., McLean, Va.). Negative control wells received preimmune mouse serum, irrelevant ascites, or normal culture supernatant as the primary antibody, while positive control wells received a polyclonal antiserum raised against C. albicans wt 4918 (3). IFA. The IFA was modified from that described by Linehan et al. (20). Germination was induced either in Lee medium or in RPMI 1640 medium at 37°C for up to 16 h. Hyphae were washed twice in PBS, and the cell concentration was adjusted to approximately 5 x 105 cells per ml by reading the optical density at 550 nm. Twenty microliters of this suspension was added to each well of a Toxislide (Roboz Surgical Instrument Co., Washington, D.C.) and air dried for 60 min at 37°C. Cells were fixed for 5 min in cold methanol. For the primary antibody incubation, ascitic fluid from clone 1.183 was used in dilutions from 1:100 to 1:1,000 in PBS. Twenty microliters was added per well, and incubation was performed in a moist chamber for 30 min at 37°C. Slides were washed three times in PBS before 20 ,ul of fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Tago, Burlingame, Calif.) diluted 1:10 was added. Incubation was continued for another 30 min under the same conditions. The slides were washed again in PBS and were mounted in 50% glycerol in Veronal-buffered saline (pH 8.6). Cells were examined with a fluorescence microscope (Model MC-63; Carl Zeiss, New York, N.Y.), and pictures were taken with Kodak Ektachrome 400 slide film. The same procedure was used for immunofluorescence studies of yeast cells grown under various conditions and for blastoconidia and hyphae that were treated with different reagents. Controls included preimmune mouse sera, nonimmune murine ascites, normal mouse immunoglobulin G (IgG) buffer, and an anti-Candida MAb (CA-A) that has been described previously (2, 20). Preliminary antigen characterization. Hyphae of C. albicans wt 4918 were adjusted to approximately 5 x 106 cells per ml and treated with the reagents listed below. Following treatment, cells were processed for IFA as described above. Heat treatment was performed at 56°C in PBS for 30 min or at 100°C for 10 min. For enzymatic and chemical treatment, cells were incubated in 0.005 M Tris hydrochloride buffer for 90 min at 37°C with the following reagents: pronase (2.5 mg/ml, pH 7; Calbiochem-Behring, La Jolla, Calif.), trypsin (2.5 mg/ml, pH 8; Sigma), subtilisin (from Bacillus subtilis; 2.5 mg/ml, pH 8; Sigma), and P-mercaptoethanol (0.2 M, pH 7; Bio-Rad). Treatment of soluble hyphal cell extract was

VOL. 58, 1990

performed as described by Chaffin et al. (6) with both immobilized trypsin and pronase (Pierce Chemical Co., Rockford, Ill.) and with sodium periodate. Subsequently, the treated antigen was spotted onto a nitrocellulose membrane and processed for immunoblotting with MAb 1.183 as described below. Protoplast formation. C. albicans wt 4918 yeast cells were grown at 24°C in Lee medium and harvested in the exponential growth phase by centrifugation at 8,000 x g for 10 min. The incubation steps required for converting the cells to protoplasts were modified from the method described by Torres-Bauza and Riggsby (37). Briefly, cells were sequentially treated with 0.05 M EDTA (pH 7.5) for 10 min at 24°C, 0.35 M 3-mercaptoethanol (Bio-Rad) in 0.005 M EDTA (pH 9) for 30 min at 24°C, and Zymolase-20T (from Arthrobacter luteus; Seikagaku Kogyo Co., Tokyo, Japan; 2 mg/ml) in 0.6 M KCI-0.04 M K2HPO4 (pH 6) for 90 min at 37°C. The cells were spun after each step, and supernatants were saved for further analysis. Typically 90 to 93% of the cells were converted to protoplasts under the chosen conditions, as judged by light microscopy. Total-protoplast cell extracts were obtained by adding an equal amount of 10% sodium dodecyl sulfate (SDS) to the protoplast suspension in 0.6 M KCl-0.04 M K2HPO4 (pH 6). Incubation was continued for another 30 min at 24°C, and the supernatant was saved after centrifugation at 10,000 x g. SDS-PAGE and Western blotting (immunoblotting). SDSpolyacrylamide gel electrophoresis (PAGE) was performed under reducing conditions by the method of Laemmli (18) in a minigel system (Bio-Rad). Electrophoresis was carried out in 7.5 or 9% slab gels at 150 V for 40 min. Subsequently, the gels were either stained with Coomassie brilliant blue or were electrophoretically transferred. The following molecular mass standards (Bio-Rad) were used: myosin (200 kDa), P-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14 kDa). Electrophoretic transfers onto polyvinylidene difluoride membranes (Immobilon; Millipore Corp., Bedford, Mass.) were performed at 100 V for 90 min as described previously (38). Immediately following the transfer, the membranes were washed in Tris-buffered saline (TBS) for 15 min and subsequently blocked in 5% nonfat dry milk in TBS. The membranes were incubated with primary antibody (ascitic fluid diluted 1:1,000 or purified MAb 1.183 [0.003 ,ug/ml in 1% BSA-TBS]) at 4°C overnight. The remainder of the procedure was performed as described previously by Calderone et al. (2). Other methods. Total protein was estimated by the method of Peterson (26). The MAb isotype was determined by Ouchterlony double gel diffusion (25) in 1.2% agarose (BioRad) in PBS by using goat anti-mouse class-specific antisera (Tago). Radial immunodiffusion was performed as described by Fahey and McKelvey (10) with prepoured plates for the detection of mouse IgG (ICN, Costa Mesa, Calif.). MAb 1.183 was purified by protein A affinity chromatography (Pierce), taking advantage of a buffer system (MonoPure; Pierce) which allowed higher yields of mouse IgGl (1). Treatment of hyphal cell extracts with endo-a-N-acetylgalactosaminidase (O-glycanase; Genzyme, Boston, Mass.) and peptide:N-glycosidase F (N-glycanase; Genzyme) was performed as described previously (27). RESULTS MAb preparation and characterization. The fusion from which MAb 1.183 was derived yielded a total of 15 stable

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627

anti-Candida antibody-producing hybridoma cell lines as determined by ELISA. One of the clones obtained (MAb 1.183) reacted exclusively with the hyphal growth form of C. albicans in IFA and was chosen for further investigation. The cloned cell line 1.183 was propagated in BALB/c mice for ascites production. The concentration of MAb 1.183 in the ascitic fluid averaged 3.0 mg/ml as determined by a radial immunodiffusion technique. MAb 1.183 belongs to the murine IgGl isotype subclass and has a kappa light chain. The MAb was purified from ascitic fluid by protein A affinity chromatography, which yielded IgGl recovery in the range of 85 to 90%. Antigen expression on intact cells. The surface expression of the epitope recognized by MAb 1.183 was examined by IFA. When a 16-h hyphal culture grown in Lee or RPMI 1640 medium at 37°C was assayed, immunofluorescence was typically found only on C. albicans hyphae and pseudohyphae. No fluorescent staining was detected on parent blastoconidia or on pseudohyphal segments directly originating from the parent yeast cell (Fig. 1). The percentage of hyphal cells expressing the antigen in C. albicans wt 4918 and A-9 was 85 to 90%. The distribution of the antigen on pseudohyphae was found to be uneven, with segments of strong immunofluorescence followed by segments showing less intense staining. Especially the pseudohyphal portions located distant from the first septum expressed high amounts of antigen (Fig. 1). When antigen expression was examined in a time course during hyphal outgrowth little staining was found in the first 3 h after germination. Thereafter a steady increase in antigen expression occurred over the next 1 to 3 h. After 6 h the staining signals were comparable to those obtained with 16-h cultures (data not shown). To further elucidate the surface expression of the antigen, we examined yeast cells grown under various conditions, as well as additional isolates of C. albicans and other Candida spp. (Table 1). Eight of nine recent clinical C. albicans isolates tested were positive to various degrees. Of the other Candida spp. examined, only C. stellatoidea expressed the antigen. No antigen expression was found on yeast cells of strain wt 4918 when grown at 30 or 37°C in yeast nitrogen base to exponential and stationary growth phases. Surface reactivity of treated cells and preliminary antigen characterization. Hyphae of wt 4918 were treated under different chemical and physical conditions and subsequently screened in IFA. The epitope recognized by MAb 1.183 was susceptible to treatment with heat at 56 and 100°C, proteolytic enzymes (pronase, trypsin, and subtilisin), and ,Bmercaptoethanol, resulting in a complete loss of fluorescent staining. This suggests the possibility of a heat-labile protein epitope which is recognized by MAb 1.183. The results of the cellular assay were confirmed by dot blot analysis of soluble hyphal cell extract treated with proteolytic enzymes and sodium periodate. A loss of antigen recognition with MAb 1.183 was obtained by treatment with trypsin and pronase, whereas treatment with periodate had only a slight reducing effect on antigen recognition (data not shown). Although the antigen was not expressed on the surface of yeast cells, its presence within the cell wall, on the plasma membrane, or in the cytoplasm of blastoconidia was examined. For this reason, yeast cells were sequentially treated until protoplasts were formed. After each step treated cells were subjected to IFA. No reactivity was obtained after treatment with EDTA and P-mercaptoethanol, the two steps which removed outer cell wall layers (data not shown). After protoplast formation with Zymolase for 90 min, 40 to 60% of the cells showed a positive signal (Fig. 2). Between 7 and

628

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FIG. 1. Bright-field (A and C) and immunofluorescence (B and D) optics of intact mycelial cells (16-h culture in RPMI 1640 medium) of C. albicans wt 4918, incubated with MAb 1.183 (1:100 dilution) and subsequently stained with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin. Small arrows indicate parent blastoconidia and pseudohyphal segments originating from the parent cell, both of which were not stained with the MAb. The antibody, in contrast, bound to hyphal segments distant from the first septum (larger arrowheads in panels C and D). Magnifications: A and B, x1,120; C and D, x2,100.

10% of the yeast cells did not convert to protoplasts under these conditions. These cells did not react with MAb 1.183

(Fig. 2).

Western blot analysis. Soluble cell extracts obtained by mechanical disruption of hyphae from C. albicans wt 4918 and A-9 were separated by SDS-PAGE and analyzed by immunoblotting with MAb 1.183. In both strains a major protein with a molecular mass of 55 kDa was detected (Fig. 3, lanes 1 and 2). An additional though less prominent band with a mass of 60 kDa was blotted in both strains. In strain A-9 there was also some reactive material in the region of 115 kDa which could have been caused by protein aggregation. DTT-extracted material from whole hyphae of strain wt 4918 was also examined by immunoblotting (Fig. 3, lane 3). MAb 1.183 recognized the same proteins in this preparation, though the staining signal was less intense than that obtained with the homogenized cell material of the same strain (Fig. 3, lane 2). Both preparations from strain wt 4918, the wholecell homogenate and the DTT extract, contained highmolecular-mass antigens (200 kDa) that cross-reacted with MAb 1.183 (Fig. 3, lanes 2 and 3). When preimmune mouse serum was used for primary antibody incubation, no positive signal was obtained (Fig. 3, lane 4). Treatment of the whole-cell hyphal extract with O-glycanase reduced the molecular mass of the 60-kDa protein by approximately 2.5 kDa but did not affect recognition by MAb 1.183 (Fig. 3,

lanes 7 and 8). N-Glycanase had no effect on the antigen (data not shown). The cell wall material which was released during the steps of protoplast formation by exponentially growing wt 4918 yeast cells and the total-cell extract from lysed protoplasts were also examined by immunoblotting. However, none of the yeast cell wall preparations showed any reactivity when blotted with MAb 1.183 (Fig. 3, lane 5). In the SDS-extracted material from yeast cell protoplasts, MAb 1.183 recognized a protein of ca. 20 kDa (Fig. 3, lane 6). In control experiments, concentrated samples of Zymolase (up to 20 mg/ml) showed no reaction in immunoblots with MAb 1.183 (data not shown). To rule out the possibility that the low molecular weight of the antigen detected in yeast cell protoplasts was due to degradation by various enzymes in the Zymolase preparation, a control experiment with Zymolase-treated hyphal cell extract was performed. In an immunoblot, MAb 1.183 recognized the same antigens as in the untreated hyphal cell extract and no low-molecular-weight antigen was detectable (data not shown). DISCUSSION An important question in all studies involving germ tubespecific antigens on C. albicans has been whether these components reflect de novo synthesis of proteins, morpho-

C. ALBICANS ANTIGENS

VOL. 58, 1990 TABLE 1. IFA of MAb 1.183 with different Candida species Degree of reactivity of hyphaea

Organism

C. albicans Wt 4918b .......................... ++++ wt

4918C

A_gb

.......................... ND

..............

........................................

4918-10b ............................................ C. albicans recent clinical isolateSb 982 .......................... 975 .......................... 837 .......................... 989 .......................... 615 .......................... 688 .......................... 847 .......................... 897 .......................... 612 ..........................

++++ +

++

+++ ++++ +++ ++++ -

++++

++ +++

++

Other Candida Spp.b C. stellatoidea ......................... ++++ C. tropicalis ......................... C. pseudotropicalis ......................... a No reactivity was observed with yeast cells of any of the organisms tested. The reactivity of hyphae is indicated as follows: -, no reactivity; +, >0 to 25% of hyphae reactive; + +, >25 to 50% of hyphae reactive; +++, >50 to 75% of hyphae reactive; + + + +, >75 to 100% of hyphae reactive. ND, Not determined. b Yeast cell culture in Lee medium at 24°C (exponential and stationary growth stages); hyphal cell cultures at 37°C for 16 h in Lee or RPMI 1640 medium. ' Yeast cell culture in yeast nitrogen base at 30 or 37°C (exponential and stationary growth stages).

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logical and topological rearrangement of yeast cell components on the surface of mycelia, or, as a third possibility, major quantitative differences in the composition of surface components on both growth forms (5, 28, 32, 34, 35). The high-molecular-weight species of the germ tube-specific antigens have been shown to be specific for the mycelial cell wall (5, 34), although other investigators have pointed out that there may be quantitative differences in the same antigen on blastoconidia and mycelia (32). Our findings support the results obtained with germ tube-specific highmolecular-weight mannoproteins: the antigen recognized by MAb 1.183 was present on the surface of intact hyphae and in DTT extracts and whole-cell homogenates of hyphal cells as early as 4 to 6 h after germination was initiated but was not found on or within the outermost layers of the yeast cell wall, as determined by IFA and immunoblotting. Surprisingly, however, in the IFA, MAb 1.183 reacted specifically with protoplasts formed from exponentially growing yeast cells, thus suggesting the presence of a cross-reacting protein associated with the innermost layer of the cell wall, with the plasma membrane, or with the cytoplasm of yeast cells. In a Western blot with SDS-extracted material from yeast cell protoplasts, MAb 1.183 recognized a low-molecularmass protein of ca. 20 kDa, although the signal obtained was weaker than the one obtained with hyphal cell extracts. It still remains to be determined whether the specific reactivity of yeast cell protoplasts with MAb 1.183 in IFA and Western blotting is the result of cross-reactivity with an unrelated protein or whether it represents a precursor protein which is expressed by mycelial cells following induction of germination. The latter would support the hypothesis that the same gene products are expressed in blastoconidia and hyphae but are located differently within the cells (24). In this context the recent finding by Li et al. (R. K. Li, P. M. Glee, and J. E. Cutler, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989,

N

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FIG. 2. Bright-field (A) and immunofluorescence (B) optics of yeast cell protoplasts formed from exponentially growing cells of C. albicans wt 4918. Cells were incubated with MAb 1.183 (1:100 dilution) and stained with a fluorescein isothiocyanate-conjugated secondary antibody. Note that incomplete protoplasts with most of the cell wall still present (arrows) did not stain with MAb 1.183. Magnification: x 1,600.

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another group, which found a hyphal surface-specific antigen of 68 kDa (D. A. Cortlandt, W. L. Chaffin, and K. J. Morrow, Jr., Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, F-27, p. 462) clearly demonstrate that important rearrangements other than in the high-molecular-weight mannoprotein complex are occurring on the surface of C. albicans during germination. The relationship of these proteins in the pathogenesis of candidiasis has yet to be examined.

B M M

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FIG. 3. (A) Westerm blot analysis with MAb 1.183 of extracts from either yeast cells (Y) or mycelial cells (M) of C. albicans wt 4918 and A-9. The following antigens were examined: whole-cell hyphal extracts of C. albicans A-9 (lane 1) and C. albicans wt 4918 (lane 2), DTT hyphal cell extract of C. albicans wt 4918 (lane 3), Zymolase-released material from blastoconidia of C. albicans wt 4918 (lane 5), and SDS-extracted material from yeast cell protoplasts of C. albicans wt 4918 (lane 6). Lane 4 represents a control with preimmune mouse serum for primary antibody incubation and whole-cell hyphal extract from strain A-9 as an antigen. (B) Western blot analysis with MAb 1.183 of O-glycanase-treated whole-cell hyphal extract from C. albicans wt 4918. Shown are the control incubation mixture (lane 7) and treated material (lane 8).

F-40, p. 464) might be of interest. These investigators showed that a yeast cell cytoplasmic antigen is expressed on the surface of blastoconidia and hyphae 6 to 8 h after germination is induced. Another group found that a membrane-bound C. albicans proteinase with a molecular mass of 86 kDa has many properties in common with a secreted proteinase, and the former might therefore represent a precursor form (29). The two major proteins recognized by MAb 1.183 have molecular masses of about 55 and 60 kDa, as determined by SDS-PAGE under reducing conditions. The 60-kDa protein made a slight shift (approximately 2.5 kDa) to a lower molecular mass after treatment with O-glycanase, an enzyme which cleaves serine- or threonine-linked oligosaccharides from glycoproteins, but its recognition by MAb 1.183 was unaffected. The presence of 0-linked oligosaccharide units in C. albicans mannoproteins is in agreement with previous findings (30, 31). Treatment with N-glycanase, which removes asparagine-linked high-mannose oligosaccharides, had no effect on the recognized proteins. This, however, does not rule out the presence of N-linked oligosaccharide units, as the cleavage site of the enzyme could have been masked, as has been shown for other mannoproteins (36). The susceptibility of the epitope recognized by MAb 1.183 to heat treatment, proteolytic enzymes, and ,-mercaptoethanol, as determined by IFA in a cellular system, and to proteolytic enzymes but not to sodium periodate in the fluid phase, as determined by dot blot analysis, suggests that MAb 1.183 binds to a peptide moiety. This is in agreement with previous findings on other germ tube-specific antigens (34). However, except for one lowmolecular-weight germ tube-specific antigen (28) that was not expressed on the surface of mycelia, all of the reported epitopes specific for the hyphal phase of C. albicans are found in the high-molecular-mass mannoproteins (5, 28, 35, 36). The present finding of germ tube-specific surface antigens with lower molecular weights and the recent report by

ACKNOWLEDGMENTS We thank A. Saxena and E. Wadsworth of our laboratory for providing some of the antigen preparations and for valuable discussion of the data. This study was supported in part by Public Health Service grant Al 25738 from the National Institutes of Health (to R.A.C.). M.W.O. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Bigbee, W. L., M. Vanderlaan, S. S. N. Fong, and R. H. Jensen. 1983. Monoclonal antibodies specific for the M- and N-forms of

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