Vol. 60, No. 8
INFECTION AND IMMUNITY, Aug. 1992, p. 3315-3324 0019-9567/92/083315-10$02.00/0 Copyright © 1992, American Society for Microbiology
Localization of the Tube Precipitin and Complement Fixation Antigens of Coccidioides immitis by Immunoelectron Microscopy with Murine Monoclonal Antibodies REBECCA A. COX,l* SUNG H. SUN,2 MATTHEW J. DOLAN,3 AND JEFFREY L. HARRISON2 Department of Research Immunology, San Antonio State Chest Hospital, San Antonio, Texas 782231 Audie L. Murphy Memorial Veterans Hospital, San Antonio, Te-xas 782842; and Wilford Hall Medical Center, Lackland Air Force Base, Texas 782363 Received 16 March 1992/Accepted 5 May 1992
The cellular localization of the tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides immitis was examined by immunoelectron microscopy with murine immunoglobulin Gl monoclonal antibodies directed against the TP and CF antigens, respectively. Immunoelectron microscopic analyses of saprobic- and parasitic-phase cells showed that the TP antigen is present at a high concentration within the inner cell wall layer and along the plasma membrane. The antigen was also detected, at a lesser concentration, within cytoplasmic vacuoles. In contrast to the predominant localization of the TP antigen in the cell walls, the CF antigen resides primarily within the cytoplasm, where it appears to be dispersed throughout the cytoplasm rather than associated with a specific cytoplasmic organelle. A sparse amount of the CF antigen within the inner cell walls was also demonstrable. The localization of the TP and CF antigens throughout the morphogenetic phases of C. immitis has important implications in antigen production and in analyses of host response in
coccidioidomycosis. Serologic detection of the tube precipitin (TP) and complement fixation (CF) antibodies to Coccidioides immitis provides an extremely valuable aid in establishing the diagnosis and clinical stage of coccidioidomycosis (17-19). Production of the TP antibody occurs early after primary infection, usually within 1 to 3 weeks of clinical onset, and diminishes to negligible levels by the 4th month of illness. The CF antibody is produced later in the disease, generally within 3 to 4 months, and persists throughout active infection. The magnitude of the CF antibody response, as measured by serial dilutions of the patient's serum or other biofluid, provides an index of disease involvement. Low titers generally reflect localized disease involving the lungs or a single extrapulmonary site, whereas high titers are consistent with extensive multifocal disease. Previous investigations have established that the TP antigen is a high-molecular-weight, heat-stable polysaccharide or polysaccharide-protein complex (4, 5, 8, 9, 15, 25) which can be extracted from cell walls by enzymatic treatment (7) or by incubating the walls in 1 N NaOH (10). The CF antigen has been characterized as a heat-labile protein or glycoprotein (8, 12, 23, 24), and although its cellular localization has not been established, evidence suggests that it is of cytoplasmic origin (7, 17, 23, 24). In order to more clearly define the cellular localization of the TP and CF antigens, we performed immunoelectron microscopic analyses of the morphogenetic phases of C. immitis by using a murine monoclonal antibody (MAb) which recognizes a heat-stable carbohydrate epitope on the TP antigen (13) and a murine MAb which is directed against a heat-labile peptide epitope on the CF antigen (12).
*
MATERIALS AND METHODS Fungi. C. immitis Silveira (ATCC 28868) was cultured under previously described conditions for the production of saprobic and parasitic cells (6, 22). In brief, mycelia were grown in a 1% glucose-0.5% yeast extract broth at 25°C on a gyratory shaker for 7 days and collected by centrifugation. Arthroconidia were obtained from 6- to 8-week-old mycelial colonies grown at 25°C on 1% glucose-0.5% yeast extract agar by using a magnetic stir bar, as described by Sun and Huppert (21). Spherule-phase cells were produced in a modified liquid Converse medium (16) at 39°C under a 20% C02-80% air atmosphere. The spherule-phase cultures were incubated on a shaking platform and harvested 2 to 6 days thereafter to obtain young spherules, segmenting spherules, and first-generation endospores. Yeast-phase cultures of Histoplasma capsulatum G217B (1) and Blastomyces dermatitidis SCB-2 (ATCC 26199) were kindly provided by George S. Deepe (University of Cincinnati Medical Center, Cincinnati, Ohio). The yeasts were subcultured onto brain heart infusion agar slants (Difco Laboratories, Detroit, Mich.) and harvested after 7 days of incubation at 37°C under 5% CO2. MAbs. The production and characterization of a murine immunoglobulin Gl (IgGl) MAb which recognizes the TP antigen and a murine IgGl MAb which reacts with the CF antigen were reported in previous studies (12, 13). The anti-TP and anti-CF MAbs and, for an irrelevant control, a monoclonal IgGl produced by a murine myeloma cell line (ATCC TIB 8) were purified from ascites b3k solid-phase immunoadsorption on Affi-Gel Protein A MAPS II columns (Bio-Rad Laboratories, Richmond, Calif.). Immunoelectron microscopy. Fungal cells were embedded in 2% agarose and processed for immunoelectron microscopy as detailed previously (2, 6). In brief, specimens were fixed with 2% glutaraldehyde at 4°C overnight, washed three times in 0.1 M cacodylate (pH 7.3), and then postfixed with
Corresponding author. 3315
3316
COX ET AL.
INFECT. IMMUN.
2% osmium tetroxide for 1 h at 4°C. Samples were dehydrated through a graded series of ethanol, immersed in propylene oxide, and embedded in Spurr low-viscosity resin (20). Thin sections were prepared on an MT 6000 Ultramicrotome, and the sections were mounted on nickel grids and etched with 3% H202. Sections were incubated for 18 to 24 h at 4°C with 150 ,ul of the MAbs diluted in 1% ovalbumin. Following removal of nonreactive components, the sections were reacted for 1 h with a 1:200 dilution of rabbit antimouse IgG conjugated with colloidal gold particles (15-nm diameter; Sigma Chemical Co., St. Louis, Mo.) and then stained with uranyl acetate and lead citrate. The concentrations of the MAbs used were based upon preliminary immunoelectron microscopic analyses using C. immitis cells and, as a measure of specificity, yeast cells of H. capsulatum and B. dermatitidis. The anti-TP MAb showed optimal reactivity when used at a protein concentration of 0.1 pug/ml; the anti-CF MAb was optimally reactive when used at a concentration of 10.0 ,ug/ml. The irrelevant MAb control was used at a concentration of 7.5 ,ug of protein per ml.
Localization of the CF antigen. The localization of the CF antigen in saprobic- and parasitic-phase cells of C. immitis is shown in Fig. 4 and 5. Thin sections of young hyphal cells and newly released arthroconidia showed predominant deposition of label within the cytoplasm (Fig. 4A and B, respectively). The gold particles were observed to be dispersed throughout the cytoplasm and did not appear to be associated with a specific organelle. A low concentration of label was also observed within the inner cell wall layer. Analyses of presegmented spherules, segmenting spherules, and first-generation endospores revealed a pattern of labelling similar to that observed with the saprobic cells; namely, the immunogold was most concentrated within the cytoplasm and detected at a low concentration within the inner wall layer (Fig. 5A, B, and C, respectively). The anti-CF MAb did not react with yeast cells of H. capsulatum and B. dermatitidis yeast-phase cells (data not shown). This lack of reactivity is consistent with our previous report that the anti-CF MAb was nonreactive in enzymelinked immunosorbent and immunoblot assays of histoplasmin and blastomycin (12).
RESULTS Localization of the TP antigen. The reactivities of the saprobic forms of C. immitis with the anti-TP MAb are shown in Fig. 1. Hyphal cells harvested from 7-day-old broth cultures showed deposition of the immunolabel throughout the electron-translucent inner cell wall layer and along the plasma membrane (Fig. 1A). There was no detectable label in the thin, electron-opaque outer wall layer or within the cytoplasm. Newly released arthroconidia showed a similar pattern of reactivity, with localization of the immunogold within the inner cell wall layer and along the plasmalemma (Fig. 1B). A few gold particles were also observed within cytoplasmic vacuoles. The results obtained when cells of the parasitic spheruleendospore phases were probed with the anti-TP MAb are presented in Fig. 2. Young, presegmented spherules showed deposition of immunogold within the inner cell wall and along the plasma membrane (Fig. 2A). A sparse deposition of label was also detected within the cytoplasm. Thin sections prepared from segmenting spherules showed gold label within the inner wall layer and dispersed throughout the contiguous segmentation apparatus which is formed by an ingrowth of the inner cell wall (Fig. 2B). Immunogold was also deposited within cytoplasmic vacuoles. Following maturation of the compartmentalized cells and rupture of the mature spherules, the newly developed endospores showed a uniform deposition of label throughout their cell walls and, to a lesser extent, in cytoplasmic organelles (Fig. 2C). It is noteworthy that the walls of the first-generation endospores appeared as a single, electron-transparent layer, indicating that the electron-dense outer wall layer develops later during endospore maturation. The epitope recognized by the anti-TP MAb was previously reported to be specific to C. immitis, as assessed by the lack of reactivity of MAb in immunoblot and enzymelinked immunosorbent assays of histoplasmin and blastomycin (13). When the anti-TP MAb was used to probe yeast cells of H. capsulatum and B. dennatitidis, only an occasional gold particle was detected (Fig. 3A and B, respectively). The sparse deposition of label was judged to be nonspecific, since it was also observed in sections reacted with the irrelevant MAb or with the colloidal gold-conjugated rabbit anti-mouse IgG alone.
DISCUSSION Immunoelectron microscopic studies with murine MAbs directed against the TP and CF antigens of C. immitis established differences in the cellular localizations of these two antigens. The TP antigen is shown to be localized predominantly to the inner cell wall layer of saprobic- and parasitic-phase cells and present at a low concentration within cytoplasmic vacuoles. By contrast, the CF antigen is a predominantly cytoplasmic component and is demonstrable at a low concentration within the cell walls. The immunolocalization of the TP antigen has been evaluated in a previous study by Cole et al. (4) using human anti-TP antibodies. The antibodies were isolated from patients' sera by immunoadsorption on 3-O-methylmannose, a carbohydrate which has been shown to react with anti-TP antibodies (4, 5). In immunoelectron microscopic analyses of presegmented spherules, the investigators reported that the TP antigen was present within the inner cell wall, being detected at the highest concentration along the plasmalemma (4). They also observed immunolabel within cytoplasmic vesicles. This pattern of reactivity is similar to that which we observed with the murine anti-TP MAb. Since the human anti-TP had been purified by using 3-O-methylmannose as a ligand, we conducted experiments to determine whether the MAb might also recognize this methylated carbohydrate. Preincubation of the anti-TP MAb with 3-O-methylmannose, but not with sterically related carbohydrates, inhibited its reactivity with the TP antigen (11). These results, taken together, provide evidence that both the MAb and the human anti-TP recognize an epitope containing 3-0-methylmannose. In an earlier study, we reported that the anti-TP MAb was weakly reactive with a second coccidioidin component which has been designated antigen 2 (Ag2) (13). This reactivity was demonstrable by the binding of radiolabelled
anti-TP MAb with the cathodal peak of the Ag2 polymer in two-dimensional immunoelectrophoresis. We do not know whether this binding is attributed to the presence of the epitope on Ag2 or, alternatively, to the association of the TP antigen with Ag2. Until this issue is resolved, the possibility that the MAb detected both the TP antigen and Ag2 cannot be excluded. In contrast to the reactivity of the anti-TP MAb prepon-
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FIG. 1. Immunoelectron micrographs of saprobic-phase mycelium (A) and arthroconidium (B) of C. immitis reacted with a murine anti-TP MAb (0.1 p.g/ml) and then with colloidal-gold-labelled rabbit anti-mouse IgG. Bar = 1.0 ,um. 3317
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INFECTION AND IMMUNITY, Aug. 1992, p. 3315-3324 0019-9567/92/083315-10$02.00/0 Copyright © 1992, American Society for Microbiology
Localization of the Tube Precipitin and Complement Fixation Antigens of Coccidioides immitis by Immunoelectron Microscopy with Murine Monoclonal Antibodies REBECCA A. COX,l* SUNG H. SUN,2 MATTHEW J. DOLAN,3 AND JEFFREY L. HARRISON2 Department of Research Immunology, San Antonio State Chest Hospital, San Antonio, Texas 782231 Audie L. Murphy Memorial Veterans Hospital, San Antonio, Te-xas 782842; and Wilford Hall Medical Center, Lackland Air Force Base, Texas 782363 Received 16 March 1992/Accepted 5 May 1992
The cellular localization of the tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides immitis was examined by immunoelectron microscopy with murine immunoglobulin Gl monoclonal antibodies directed against the TP and CF antigens, respectively. Immunoelectron microscopic analyses of saprobic- and parasitic-phase cells showed that the TP antigen is present at a high concentration within the inner cell wall layer and along the plasma membrane. The antigen was also detected, at a lesser concentration, within cytoplasmic vacuoles. In contrast to the predominant localization of the TP antigen in the cell walls, the CF antigen resides primarily within the cytoplasm, where it appears to be dispersed throughout the cytoplasm rather than associated with a specific cytoplasmic organelle. A sparse amount of the CF antigen within the inner cell walls was also demonstrable. The localization of the TP and CF antigens throughout the morphogenetic phases of C. immitis has important implications in antigen production and in analyses of host response in
coccidioidomycosis. Serologic detection of the tube precipitin (TP) and complement fixation (CF) antibodies to Coccidioides immitis provides an extremely valuable aid in establishing the diagnosis and clinical stage of coccidioidomycosis (17-19). Production of the TP antibody occurs early after primary infection, usually within 1 to 3 weeks of clinical onset, and diminishes to negligible levels by the 4th month of illness. The CF antibody is produced later in the disease, generally within 3 to 4 months, and persists throughout active infection. The magnitude of the CF antibody response, as measured by serial dilutions of the patient's serum or other biofluid, provides an index of disease involvement. Low titers generally reflect localized disease involving the lungs or a single extrapulmonary site, whereas high titers are consistent with extensive multifocal disease. Previous investigations have established that the TP antigen is a high-molecular-weight, heat-stable polysaccharide or polysaccharide-protein complex (4, 5, 8, 9, 15, 25) which can be extracted from cell walls by enzymatic treatment (7) or by incubating the walls in 1 N NaOH (10). The CF antigen has been characterized as a heat-labile protein or glycoprotein (8, 12, 23, 24), and although its cellular localization has not been established, evidence suggests that it is of cytoplasmic origin (7, 17, 23, 24). In order to more clearly define the cellular localization of the TP and CF antigens, we performed immunoelectron microscopic analyses of the morphogenetic phases of C. immitis by using a murine monoclonal antibody (MAb) which recognizes a heat-stable carbohydrate epitope on the TP antigen (13) and a murine MAb which is directed against a heat-labile peptide epitope on the CF antigen (12).
*
MATERIALS AND METHODS Fungi. C. immitis Silveira (ATCC 28868) was cultured under previously described conditions for the production of saprobic and parasitic cells (6, 22). In brief, mycelia were grown in a 1% glucose-0.5% yeast extract broth at 25°C on a gyratory shaker for 7 days and collected by centrifugation. Arthroconidia were obtained from 6- to 8-week-old mycelial colonies grown at 25°C on 1% glucose-0.5% yeast extract agar by using a magnetic stir bar, as described by Sun and Huppert (21). Spherule-phase cells were produced in a modified liquid Converse medium (16) at 39°C under a 20% C02-80% air atmosphere. The spherule-phase cultures were incubated on a shaking platform and harvested 2 to 6 days thereafter to obtain young spherules, segmenting spherules, and first-generation endospores. Yeast-phase cultures of Histoplasma capsulatum G217B (1) and Blastomyces dermatitidis SCB-2 (ATCC 26199) were kindly provided by George S. Deepe (University of Cincinnati Medical Center, Cincinnati, Ohio). The yeasts were subcultured onto brain heart infusion agar slants (Difco Laboratories, Detroit, Mich.) and harvested after 7 days of incubation at 37°C under 5% CO2. MAbs. The production and characterization of a murine immunoglobulin Gl (IgGl) MAb which recognizes the TP antigen and a murine IgGl MAb which reacts with the CF antigen were reported in previous studies (12, 13). The anti-TP and anti-CF MAbs and, for an irrelevant control, a monoclonal IgGl produced by a murine myeloma cell line (ATCC TIB 8) were purified from ascites b3k solid-phase immunoadsorption on Affi-Gel Protein A MAPS II columns (Bio-Rad Laboratories, Richmond, Calif.). Immunoelectron microscopy. Fungal cells were embedded in 2% agarose and processed for immunoelectron microscopy as detailed previously (2, 6). In brief, specimens were fixed with 2% glutaraldehyde at 4°C overnight, washed three times in 0.1 M cacodylate (pH 7.3), and then postfixed with
Corresponding author. 3315
3316
COX ET AL.
INFECT. IMMUN.
2% osmium tetroxide for 1 h at 4°C. Samples were dehydrated through a graded series of ethanol, immersed in propylene oxide, and embedded in Spurr low-viscosity resin (20). Thin sections were prepared on an MT 6000 Ultramicrotome, and the sections were mounted on nickel grids and etched with 3% H202. Sections were incubated for 18 to 24 h at 4°C with 150 ,ul of the MAbs diluted in 1% ovalbumin. Following removal of nonreactive components, the sections were reacted for 1 h with a 1:200 dilution of rabbit antimouse IgG conjugated with colloidal gold particles (15-nm diameter; Sigma Chemical Co., St. Louis, Mo.) and then stained with uranyl acetate and lead citrate. The concentrations of the MAbs used were based upon preliminary immunoelectron microscopic analyses using C. immitis cells and, as a measure of specificity, yeast cells of H. capsulatum and B. dermatitidis. The anti-TP MAb showed optimal reactivity when used at a protein concentration of 0.1 pug/ml; the anti-CF MAb was optimally reactive when used at a concentration of 10.0 ,ug/ml. The irrelevant MAb control was used at a concentration of 7.5 ,ug of protein per ml.
Localization of the CF antigen. The localization of the CF antigen in saprobic- and parasitic-phase cells of C. immitis is shown in Fig. 4 and 5. Thin sections of young hyphal cells and newly released arthroconidia showed predominant deposition of label within the cytoplasm (Fig. 4A and B, respectively). The gold particles were observed to be dispersed throughout the cytoplasm and did not appear to be associated with a specific organelle. A low concentration of label was also observed within the inner cell wall layer. Analyses of presegmented spherules, segmenting spherules, and first-generation endospores revealed a pattern of labelling similar to that observed with the saprobic cells; namely, the immunogold was most concentrated within the cytoplasm and detected at a low concentration within the inner wall layer (Fig. 5A, B, and C, respectively). The anti-CF MAb did not react with yeast cells of H. capsulatum and B. dermatitidis yeast-phase cells (data not shown). This lack of reactivity is consistent with our previous report that the anti-CF MAb was nonreactive in enzymelinked immunosorbent and immunoblot assays of histoplasmin and blastomycin (12).
RESULTS Localization of the TP antigen. The reactivities of the saprobic forms of C. immitis with the anti-TP MAb are shown in Fig. 1. Hyphal cells harvested from 7-day-old broth cultures showed deposition of the immunolabel throughout the electron-translucent inner cell wall layer and along the plasma membrane (Fig. 1A). There was no detectable label in the thin, electron-opaque outer wall layer or within the cytoplasm. Newly released arthroconidia showed a similar pattern of reactivity, with localization of the immunogold within the inner cell wall layer and along the plasmalemma (Fig. 1B). A few gold particles were also observed within cytoplasmic vacuoles. The results obtained when cells of the parasitic spheruleendospore phases were probed with the anti-TP MAb are presented in Fig. 2. Young, presegmented spherules showed deposition of immunogold within the inner cell wall and along the plasma membrane (Fig. 2A). A sparse deposition of label was also detected within the cytoplasm. Thin sections prepared from segmenting spherules showed gold label within the inner wall layer and dispersed throughout the contiguous segmentation apparatus which is formed by an ingrowth of the inner cell wall (Fig. 2B). Immunogold was also deposited within cytoplasmic vacuoles. Following maturation of the compartmentalized cells and rupture of the mature spherules, the newly developed endospores showed a uniform deposition of label throughout their cell walls and, to a lesser extent, in cytoplasmic organelles (Fig. 2C). It is noteworthy that the walls of the first-generation endospores appeared as a single, electron-transparent layer, indicating that the electron-dense outer wall layer develops later during endospore maturation. The epitope recognized by the anti-TP MAb was previously reported to be specific to C. immitis, as assessed by the lack of reactivity of MAb in immunoblot and enzymelinked immunosorbent assays of histoplasmin and blastomycin (13). When the anti-TP MAb was used to probe yeast cells of H. capsulatum and B. dennatitidis, only an occasional gold particle was detected (Fig. 3A and B, respectively). The sparse deposition of label was judged to be nonspecific, since it was also observed in sections reacted with the irrelevant MAb or with the colloidal gold-conjugated rabbit anti-mouse IgG alone.
DISCUSSION Immunoelectron microscopic studies with murine MAbs directed against the TP and CF antigens of C. immitis established differences in the cellular localizations of these two antigens. The TP antigen is shown to be localized predominantly to the inner cell wall layer of saprobic- and parasitic-phase cells and present at a low concentration within cytoplasmic vacuoles. By contrast, the CF antigen is a predominantly cytoplasmic component and is demonstrable at a low concentration within the cell walls. The immunolocalization of the TP antigen has been evaluated in a previous study by Cole et al. (4) using human anti-TP antibodies. The antibodies were isolated from patients' sera by immunoadsorption on 3-O-methylmannose, a carbohydrate which has been shown to react with anti-TP antibodies (4, 5). In immunoelectron microscopic analyses of presegmented spherules, the investigators reported that the TP antigen was present within the inner cell wall, being detected at the highest concentration along the plasmalemma (4). They also observed immunolabel within cytoplasmic vesicles. This pattern of reactivity is similar to that which we observed with the murine anti-TP MAb. Since the human anti-TP had been purified by using 3-O-methylmannose as a ligand, we conducted experiments to determine whether the MAb might also recognize this methylated carbohydrate. Preincubation of the anti-TP MAb with 3-O-methylmannose, but not with sterically related carbohydrates, inhibited its reactivity with the TP antigen (11). These results, taken together, provide evidence that both the MAb and the human anti-TP recognize an epitope containing 3-0-methylmannose. In an earlier study, we reported that the anti-TP MAb was weakly reactive with a second coccidioidin component which has been designated antigen 2 (Ag2) (13). This reactivity was demonstrable by the binding of radiolabelled
anti-TP MAb with the cathodal peak of the Ag2 polymer in two-dimensional immunoelectrophoresis. We do not know whether this binding is attributed to the presence of the epitope on Ag2 or, alternatively, to the association of the TP antigen with Ag2. Until this issue is resolved, the possibility that the MAb detected both the TP antigen and Ag2 cannot be excluded. In contrast to the reactivity of the anti-TP MAb prepon-
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FIG. 1. Immunoelectron micrographs of saprobic-phase mycelium (A) and arthroconidium (B) of C. immitis reacted with a murine anti-TP MAb (0.1 p.g/ml) and then with colloidal-gold-labelled rabbit anti-mouse IgG. Bar = 1.0 ,um. 3317
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