Vol. 58, No. 11
INFECTION AND IMMUNITY, Nov. 1990, p. 3810-3812 0019-9567/90/113810-03$02.00/0 Copyright C) 1990, American Society for Microbiology
Fab Fragments from a Monoclonal Antibody against a Germ Tube Mannoprotein Block the Yeast-to-Mycelium Transition in Candida albicans MANUEL CASANOVA,1 JOSE P. MARTINEZ,1 AND W. LAJEAN CHAFFIN2* Departmento de Microbiolog(a, Facultad de Farmacia, Universitat de Valencia, 4F6010-Valncia, Spain,' and Department of Microbiology, Texas Tech University Health Sciences Center, Lubbock, Texas 794302 Received 13 June 1990/Accepted 24 August 1990
Fab fragments prepared from the immunoglobulin G monoclonal antibody (MAb) 4C12, which reacts with a determinant expressed on the hyphal extension of germ tubes of Candida albicans, inhibited germ tube formation, but intact MAb 4C12 did not. Indirect immunofluorescence showed a punctate binding pattern on cells incubated with Fab fragments but a confluent binding on cells incubated with intact MAb 4C12. ments of human IgG antibodies were obtained from Organon-Teknika Cappel (Malvern, Pa.). Protein was determined
Candida albicans is a dimorphic fungus which grows either by budding or by production of germ tubes, depending on different external factors such as composition of the culture medium, pH, and temperature (15). Although factors which influence the form of growth in vivo are not defined, the yeast form of the organism is associated with the presence of the organism as a commensal in the normal host, while the hyphal form is additionally observed in infections (15). The cell wall as the structure which maintains these morphologic shapes has received considerable attention in regard to composition, function, synthesis, and assembly of its components (3; R. Sentandreu, J. P. Martinez, M. V. Elorza, and S. Mormeneo, in R. Prasad, ed., Candida albicans: Cellular and Molecular Biology, in press). During germ tube formation, several new components determined by antigenicity, function, or chemical nature have been observed in the wall and expressed at the surface (2, 3, 5, 16, 17, 21). Fab fragments from a monoclonal antibody (MAb) directed against the peptide portion of a cell surface-specific glycoprotein of Dictyostelium discoideum inhibit the initial stages of the cell differentiation process in this slime mold (18). It should be stressed that Fab fragments do not cause cross-linking of the cell surface antigens which will result in cell agglutination (1). In this study, we have examined the effect of a MAb and Fab fragments produced from it on induction of germ tubes from yeast-phase cells of C. albicans. MAb 4C12 is an immunoglobulin G1 (IgGl)-class MAb which recognizes the polypeptide moiety of a 260-kDa mycelium-specific cell wall mannoprotein (HMWM-260) (2). Fab fragments of MAb 4C12 were obtained basically as described by Harlow and Lane (9). Immunoglobulin molecules recovered from ascitic fluid by ammonium sulfate (50%, wt/vol) precipitation were treated with papain (10 mg/mg of protein) for 18 h at 37°C. The reaction was terminated with iodoacetamide, and the material was chromatographed on protein A-Sepharose (Sigma Chemical Co., St. Louis, Mo.). Fractions of unbound material eluting from the column were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10) on a 5 to 15% linear gradient slab gel followed by staining with Coomassie blue (Fig. 1). Fab-containing fractions were combined. Fab frag*
colorimetrically (12). Yeast-phase cells of C. albicans 3153A grown to stationary phase at 22°C in defined medium (11) were harvested and resuspended at 5 x 107 cells per ml in 0.5 ml of the same medium containing the various antibody samples tested (200 ,ug of protein). After incubation for 2 h at 37°C to induce germ tube formation, an additional 200 ,ug (0.1 ml) of the corresponding antibody preparation was added and the incubation was continued for 3 h. Cells were fixed for 30 min on ice with 0.5% (vol/vol) Formalin in 0.01 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride (PBS), harvested by centrifugation, and washed three times with PBS. Pelleted cells were resuspended in PBS, and triplicate portions were treated with 0.2 N acetic acid-10% f-mercap-
mm
200
116 97 66 lop
*w'pw
1 2 3 4 5 6
42 31 21
`14
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purification of Fab fragments from ascitic fluid containing MAb 4C12. Material in MAb 4C12 ascitic fluid precipitated by ammonium sulfate was treated with papain and chromatographed on protein A-Sepharose, and 5 pLg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described in the text. Lanes: 1, precipitated untreated material; 2 through 5, fractions progressively eluted from protein A-Sepharose; 6, molecular mass (mm) markers (in kilodaltons). The open arrowhead indicates the electrophoretic mobility of the intact MAb 4C12 IgG, and the solid arrowhead indicates the electrophoretic mobility of Fab fragments.
Corresponding author. 3810
NOTES
VOL. 58, 1990
toethanol for 20 min at room temperature to reduce clumping of cells. The extent of germ tube formation was determined microscopically by counting 300 organisms in triplicate samples. For a given treatment, samples differed in the extent of germination by
INFECTION AND IMMUNITY, Nov. 1990, p. 3810-3812 0019-9567/90/113810-03$02.00/0 Copyright C) 1990, American Society for Microbiology
Fab Fragments from a Monoclonal Antibody against a Germ Tube Mannoprotein Block the Yeast-to-Mycelium Transition in Candida albicans MANUEL CASANOVA,1 JOSE P. MARTINEZ,1 AND W. LAJEAN CHAFFIN2* Departmento de Microbiolog(a, Facultad de Farmacia, Universitat de Valencia, 4F6010-Valncia, Spain,' and Department of Microbiology, Texas Tech University Health Sciences Center, Lubbock, Texas 794302 Received 13 June 1990/Accepted 24 August 1990
Fab fragments prepared from the immunoglobulin G monoclonal antibody (MAb) 4C12, which reacts with a determinant expressed on the hyphal extension of germ tubes of Candida albicans, inhibited germ tube formation, but intact MAb 4C12 did not. Indirect immunofluorescence showed a punctate binding pattern on cells incubated with Fab fragments but a confluent binding on cells incubated with intact MAb 4C12. ments of human IgG antibodies were obtained from Organon-Teknika Cappel (Malvern, Pa.). Protein was determined
Candida albicans is a dimorphic fungus which grows either by budding or by production of germ tubes, depending on different external factors such as composition of the culture medium, pH, and temperature (15). Although factors which influence the form of growth in vivo are not defined, the yeast form of the organism is associated with the presence of the organism as a commensal in the normal host, while the hyphal form is additionally observed in infections (15). The cell wall as the structure which maintains these morphologic shapes has received considerable attention in regard to composition, function, synthesis, and assembly of its components (3; R. Sentandreu, J. P. Martinez, M. V. Elorza, and S. Mormeneo, in R. Prasad, ed., Candida albicans: Cellular and Molecular Biology, in press). During germ tube formation, several new components determined by antigenicity, function, or chemical nature have been observed in the wall and expressed at the surface (2, 3, 5, 16, 17, 21). Fab fragments from a monoclonal antibody (MAb) directed against the peptide portion of a cell surface-specific glycoprotein of Dictyostelium discoideum inhibit the initial stages of the cell differentiation process in this slime mold (18). It should be stressed that Fab fragments do not cause cross-linking of the cell surface antigens which will result in cell agglutination (1). In this study, we have examined the effect of a MAb and Fab fragments produced from it on induction of germ tubes from yeast-phase cells of C. albicans. MAb 4C12 is an immunoglobulin G1 (IgGl)-class MAb which recognizes the polypeptide moiety of a 260-kDa mycelium-specific cell wall mannoprotein (HMWM-260) (2). Fab fragments of MAb 4C12 were obtained basically as described by Harlow and Lane (9). Immunoglobulin molecules recovered from ascitic fluid by ammonium sulfate (50%, wt/vol) precipitation were treated with papain (10 mg/mg of protein) for 18 h at 37°C. The reaction was terminated with iodoacetamide, and the material was chromatographed on protein A-Sepharose (Sigma Chemical Co., St. Louis, Mo.). Fractions of unbound material eluting from the column were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10) on a 5 to 15% linear gradient slab gel followed by staining with Coomassie blue (Fig. 1). Fab-containing fractions were combined. Fab frag*
colorimetrically (12). Yeast-phase cells of C. albicans 3153A grown to stationary phase at 22°C in defined medium (11) were harvested and resuspended at 5 x 107 cells per ml in 0.5 ml of the same medium containing the various antibody samples tested (200 ,ug of protein). After incubation for 2 h at 37°C to induce germ tube formation, an additional 200 ,ug (0.1 ml) of the corresponding antibody preparation was added and the incubation was continued for 3 h. Cells were fixed for 30 min on ice with 0.5% (vol/vol) Formalin in 0.01 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride (PBS), harvested by centrifugation, and washed three times with PBS. Pelleted cells were resuspended in PBS, and triplicate portions were treated with 0.2 N acetic acid-10% f-mercap-
mm
200
116 97 66 lop
*w'pw
1 2 3 4 5 6
42 31 21
`14
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purification of Fab fragments from ascitic fluid containing MAb 4C12. Material in MAb 4C12 ascitic fluid precipitated by ammonium sulfate was treated with papain and chromatographed on protein A-Sepharose, and 5 pLg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described in the text. Lanes: 1, precipitated untreated material; 2 through 5, fractions progressively eluted from protein A-Sepharose; 6, molecular mass (mm) markers (in kilodaltons). The open arrowhead indicates the electrophoretic mobility of the intact MAb 4C12 IgG, and the solid arrowhead indicates the electrophoretic mobility of Fab fragments.
Corresponding author. 3810
NOTES
VOL. 58, 1990
toethanol for 20 min at room temperature to reduce clumping of cells. The extent of germ tube formation was determined microscopically by counting 300 organisms in triplicate samples. For a given treatment, samples differed in the extent of germination by
