Molecular and Biochemical Parasitology, 43 (1990) 97-106 Elsevier
97
MOLBIO 01407
Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii Jeffrey B. Prince 1, Kelly L. A u e r 1., Jayne Huskinson 1, Stephen F. P a r m l e y 1'2, Fausto G. Araujo 1 and Jack S. R e m i n g t o n 1'2 IDepartment of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, CA, U.S.A., and 2Department of Medicine, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, CA, U.S.A. (Received 3 April 1990; accepted 4 June 1990)
Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monocional antibodies to screen a cDNA expression library in Agtl 1, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18 959, As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate. Key words: Toxoplasma gondii; Antigen; Cloning; Sequence
Introduction
Cell surface antigens (Ag) of protozoan parasites have been widely studied for their dual poCorrespondence address: Jack S. Remington, M.D., Department of Immunology and Infectious Diseases, Palo Alto Medical Foundation, 860 Bryant Street, Palo Alto, CA 94301, U.S.A. *Present address: Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, U.S.A. Abbreviations: Ag, antigen; DS-IgM-ELISA, double-sandwich IgM enzyme-linked immunosorbent assay; DT, Sabin-Feldman dye test; ELISA, enzyme-linked immunosorbent assay; HRPO, horseradish peroxidase; mAb, monoclonal antibody; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankTM data base with the accession number M33572.
tential as diagnostic reagents and as immunogens for vaccines. In early studies on the antigenic structure of Toxoplasma gondii, a significant protozoan pathogen of humans and domesticated animals, the invasive tachyzoite form was found to have four major surface proteins accessible to radioiodination [1]. Subsequent reports [2-4] have confirmed the presence of these proteins and other minor radioiodination products on the tachyzoite outer membrane and, although there is still some confusion as to their precise molecular weight under reducing conditions, the four major species have come to be designated P22, P30, P35 and P43. Recent studies using monoclonal antibodies (mAb) directed to these proteins have demonstrated that all four are attached to the membrane by a glycophospholipid anchor [5,6] and that they undergo redistribution following host cell invasion to form a membranous network within the phagocytic vacuole [7]. Furthermore, P22 and P30 ap-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
98 pear to be specifically expressed on the surface of the invasive (tachyzoite) stage of the parasite, but not the sporozoite or bradyzoite forms [8,9]. Of these four major surface Ag, only P30 has been completely sequenced by molecular cloning [10]. Owing to its surface location and its apparent widespread recognition by sera of humans infected with T. gondii [1,11-14], we have undertaken an analysis of protein P22 for its utility in the diagnosis of and vaccination against toxoplasmosis. In the present report we describe the cloning of cDNAs for P22 and the deduced amino acid sequence of the primary translation product. Additionally, we demonstrate that recombinant fusion proteins containing portions of P22 are recognized by sera of humans with acute and chronic (latent) T. gondii infection. Materials and Methods
Reagents. Restriction endonucleases were from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA), or Pharmacia (Piscataway, N J). T4 polynucleotide kinase, hexadeoxyribonucleotides, and T4 DNA ligase were purchased from Pharmacia. Bacterial alkaline phosphatase, M I3 RF DNA, and RNA and DNA m.w. markers were from Bethesda Research Laboratories. Competent JM101 were obtained from Stratagene (La Jolla, CA). Sequenase TM DNA sequencing kit was from United States Biochemical Corporation (Cleveland, OH). Radionuclides were purchased from New England Nuclear (Boston, MA). Horseradish peroxidase (HRPO)-conjugated goat antihuman IgG and anti-mouse IgG were from Caltag Laboratories (South San Francisco, CA). Mouse mAb sub-isotyping kit was obtained from HyClone Laboratories (Logan, UT). Protein m.w. markers were obtained from Bio-Rad (Richmond, CA).
Preparation of parasite lysates. T. gondii tachyzoites of the RH strain were grown .in and harvested from the peritoneal cavities of mice as previously described [15]. To prepare membraneenriched Ag for immunization of mice, tachyzoites were disrupted by sonication [ 16], the sonicate centrifuged for 10 min at 10000 x g, the
supernatant of this spin centrifuged for 2 h at 32 000 x g, and the resulting pellet resuspended in phosphate-buffered saline (PBS), pH 7.2. Reduced lysate for immunoblotting was prepared by resuspension of tachyzoites in buffer containing 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, plus protease inhibitors and treatment as previously described [17]. To prepare unreduced lysate for immunoblots, tachyzoites were suspended in PBS containing 0.5% NP-40, incubated at room temperature for 30 min, and the lysate clarified by centrifugation for 10 min at 210 x g and applied to gels without further heat denaturation.
Production of monoclonal antibody.
Female BALB/c mice were immunized intraperitoneally with membrane-enriched tachyzoite Ag (100 #g protein in PBS, prepared as described above) mixed with incomplete Freund's adjuvant, given 3 weekly boosts with 100 #g Ag only, and the splenocytes harvested 3 days after the final boost. Cell fusions were performed with Sp2/0Agl4 myeloma cells (ATCC CRL 1581) according to the method of Krhler and Milstein [ 18], as modified by Oi, et al. [19]. Hybridoma cultures were screened for reaction with T. gondii Ag by enzyme-linked immunosorbent assay (ELISA) using 3 izg of tachyzoite sonicate in PBS per well of a microtiter plate [14], and positive cultures were cloned by limiting dilution to yield mAb 8721-5A6 and 87-21-6D10. The development and preparation of mAb 1El 1 and 2G11 have been previously described [20].
Serological assays. Hybridoma culture
supernatants were assayed by the Sabin-Feldman dye test (DT), direct agglutination test, and indirect fluorescent antibody test using living and formalin-fixed tachyzoites essentially as previously described [20]. Prior to use in immunoblots of recombinant fusion protein, human sera were preadsorbed to wild-type Agtll lysogen lysate to remove antibodies against fl-galactosidase and other Escherichia coli Ag [21]. Sera employed for immunoblots were assayed by DT (titer
97
MOLBIO 01407
Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii Jeffrey B. Prince 1, Kelly L. A u e r 1., Jayne Huskinson 1, Stephen F. P a r m l e y 1'2, Fausto G. Araujo 1 and Jack S. R e m i n g t o n 1'2 IDepartment of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, CA, U.S.A., and 2Department of Medicine, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, CA, U.S.A. (Received 3 April 1990; accepted 4 June 1990)
Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monocional antibodies to screen a cDNA expression library in Agtl 1, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18 959, As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate. Key words: Toxoplasma gondii; Antigen; Cloning; Sequence
Introduction
Cell surface antigens (Ag) of protozoan parasites have been widely studied for their dual poCorrespondence address: Jack S. Remington, M.D., Department of Immunology and Infectious Diseases, Palo Alto Medical Foundation, 860 Bryant Street, Palo Alto, CA 94301, U.S.A. *Present address: Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, U.S.A. Abbreviations: Ag, antigen; DS-IgM-ELISA, double-sandwich IgM enzyme-linked immunosorbent assay; DT, Sabin-Feldman dye test; ELISA, enzyme-linked immunosorbent assay; HRPO, horseradish peroxidase; mAb, monoclonal antibody; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankTM data base with the accession number M33572.
tential as diagnostic reagents and as immunogens for vaccines. In early studies on the antigenic structure of Toxoplasma gondii, a significant protozoan pathogen of humans and domesticated animals, the invasive tachyzoite form was found to have four major surface proteins accessible to radioiodination [1]. Subsequent reports [2-4] have confirmed the presence of these proteins and other minor radioiodination products on the tachyzoite outer membrane and, although there is still some confusion as to their precise molecular weight under reducing conditions, the four major species have come to be designated P22, P30, P35 and P43. Recent studies using monoclonal antibodies (mAb) directed to these proteins have demonstrated that all four are attached to the membrane by a glycophospholipid anchor [5,6] and that they undergo redistribution following host cell invasion to form a membranous network within the phagocytic vacuole [7]. Furthermore, P22 and P30 ap-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
98 pear to be specifically expressed on the surface of the invasive (tachyzoite) stage of the parasite, but not the sporozoite or bradyzoite forms [8,9]. Of these four major surface Ag, only P30 has been completely sequenced by molecular cloning [10]. Owing to its surface location and its apparent widespread recognition by sera of humans infected with T. gondii [1,11-14], we have undertaken an analysis of protein P22 for its utility in the diagnosis of and vaccination against toxoplasmosis. In the present report we describe the cloning of cDNAs for P22 and the deduced amino acid sequence of the primary translation product. Additionally, we demonstrate that recombinant fusion proteins containing portions of P22 are recognized by sera of humans with acute and chronic (latent) T. gondii infection. Materials and Methods
Reagents. Restriction endonucleases were from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA), or Pharmacia (Piscataway, N J). T4 polynucleotide kinase, hexadeoxyribonucleotides, and T4 DNA ligase were purchased from Pharmacia. Bacterial alkaline phosphatase, M I3 RF DNA, and RNA and DNA m.w. markers were from Bethesda Research Laboratories. Competent JM101 were obtained from Stratagene (La Jolla, CA). Sequenase TM DNA sequencing kit was from United States Biochemical Corporation (Cleveland, OH). Radionuclides were purchased from New England Nuclear (Boston, MA). Horseradish peroxidase (HRPO)-conjugated goat antihuman IgG and anti-mouse IgG were from Caltag Laboratories (South San Francisco, CA). Mouse mAb sub-isotyping kit was obtained from HyClone Laboratories (Logan, UT). Protein m.w. markers were obtained from Bio-Rad (Richmond, CA).
Preparation of parasite lysates. T. gondii tachyzoites of the RH strain were grown .in and harvested from the peritoneal cavities of mice as previously described [15]. To prepare membraneenriched Ag for immunization of mice, tachyzoites were disrupted by sonication [ 16], the sonicate centrifuged for 10 min at 10000 x g, the
supernatant of this spin centrifuged for 2 h at 32 000 x g, and the resulting pellet resuspended in phosphate-buffered saline (PBS), pH 7.2. Reduced lysate for immunoblotting was prepared by resuspension of tachyzoites in buffer containing 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, plus protease inhibitors and treatment as previously described [17]. To prepare unreduced lysate for immunoblots, tachyzoites were suspended in PBS containing 0.5% NP-40, incubated at room temperature for 30 min, and the lysate clarified by centrifugation for 10 min at 210 x g and applied to gels without further heat denaturation.
Production of monoclonal antibody.
Female BALB/c mice were immunized intraperitoneally with membrane-enriched tachyzoite Ag (100 #g protein in PBS, prepared as described above) mixed with incomplete Freund's adjuvant, given 3 weekly boosts with 100 #g Ag only, and the splenocytes harvested 3 days after the final boost. Cell fusions were performed with Sp2/0Agl4 myeloma cells (ATCC CRL 1581) according to the method of Krhler and Milstein [ 18], as modified by Oi, et al. [19]. Hybridoma cultures were screened for reaction with T. gondii Ag by enzyme-linked immunosorbent assay (ELISA) using 3 izg of tachyzoite sonicate in PBS per well of a microtiter plate [14], and positive cultures were cloned by limiting dilution to yield mAb 8721-5A6 and 87-21-6D10. The development and preparation of mAb 1El 1 and 2G11 have been previously described [20].
Serological assays. Hybridoma culture
supernatants were assayed by the Sabin-Feldman dye test (DT), direct agglutination test, and indirect fluorescent antibody test using living and formalin-fixed tachyzoites essentially as previously described [20]. Prior to use in immunoblots of recombinant fusion protein, human sera were preadsorbed to wild-type Agtll lysogen lysate to remove antibodies against fl-galactosidase and other Escherichia coli Ag [21]. Sera employed for immunoblots were assayed by DT (titer
