Abbreviations: ORF, open reading frame; mAb, monoclonal anti- ... One long open reading ..... 652 amino acids in WA-O, and 2124 bp and 708 amino acids.
Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3220-3224, April 1990 Microbiology
Molecular basis for surface antigen size polymorphisms and conservation of a neutralization-sensitive epitope in Anaplasma marginale (tick-borne diseases/rickettsia/gene structure/tandem repeats)
DAVID R. ALLRED*t, TRAVIS C. MCGUIREO, GuY H. PALMERt, STEVE R. LEIBt, TERESA M. HARKINS*§, TERRY F. MCELWAIN*§, AND ANTHONY F. BARBET* *Department of Infectious Diseases, University of Florida, Gainesville, FL 32610; and tDepartment of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164
Communicated by George K. Davis, January 24, 1990
Anaplasmosis is one of several tick-borne disABSTRACT eases severely constraining cattle production and usage in many parts of the world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginae carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates of A. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the msplk gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.
MATERIALS AND METHODS Cloning of the mspla Gene. DNA was isolated from purified A. marginale initial bodies as described (5, 8). Plasmid pAMT1 was constructed by partial Sau3A digestion of Florida isolate (FL) genomic DNA, C-tailing, ligation (11) into G-tailed pUC9 plasmid, and transformation of Escherichia coli JM83 (12). pAMT1, which expresses a 56-kDa product, was isolated by expression screening (13) with mAb Ana22B1 and 125I-labeled protein A (8). To obtain the complete gene, Nco I linkers were added to FL genomic DNA randomsheared by sonication, with ligation into the expression vector pKK233-2. Transformants were screened with mAb Ana22B1 as above, yielding plasmid pKAna420. The insert of pKAna420 was subcloned into the Sma I site of plasmid pGEM-4 after filling-in the Nco I overhangs (11), yielding plasmid pFL10. pVA1 was cloned as a Kpn I fragment, whereas pID6 and pWA1 were cloned as Kpn I-Pst I fragments in pGEM-4. pVA1, pID6, and pWA1 were isolated by colony hybridization screening (14) with 32P-radiolabeled (15) pAMT1 sequences. Immunoblot Analysis ofRecombinants. Recombinants were analyzed by SDS/polyacrylamide gel electrophoresis (16) on 7.5-17% (wt/vol) polyacrylamide gradient gels and by immunoblotting with mAb Ana22B1 and 1251I-labeled protein A (5). Nucleic Acid Analyses. DNAs were isolated, restriction mapped, and compared by Southern blot analysis (11). Plasmid inserts were sequenced as double-stranded DNA (17, 18), using Sequenase (United States Biochemical) as recommended by the manufacturer. SP6 and 17 promoter-specific primers were used in the initial sequencing reactions, and then new oligonucleotide primers were synthesized (19) based on the sequences obtained ("primer-walking"). RNA was isolated from FL initial bodies (20) and sequenced by a modification (21) of the method of Inoue and Cech (22). The primer was the reverse complement of bases 147FL to 166FL (for bases 147 to 166 of the FL isolate sequence; all sequence numbering hereafter is given relative to FL). Computer Analyses of Sequence Data. Sequence homology searches were performed using the FASTN program (23) (Cyborg Database Manager, International Biotechnologies). Probable transcription termination sites, structural characteristics, and hydropathy of AmF105 were predicted as described (24-26).
Anaplasmosis, a hemoparasitic disease of cattle caused by the rickettsia Anaplasma marginale, is devastating to the production, utilization, and movement of cattle. A halfbillion cattle are at risk worldwide, primarily in tropical and subtropical areas, restricting particularly the advancement of lesser-developed countries. Annual losses due to anaplasmosis total more than $100 million in the United States (1), where animal husbandry practices limit the effects of the disease. A. marginale is transmitted through the bite of infected ticks or by contaminated needles or fomites (2, 3) and invades only circulating erythrocytes (4). Antibody-mediated immunity to anaplasmosis is likely to be particularly important (5), due to a lack of parasite stages susceptible to direct cellmediated cytotoxicity. One target of humoral immunity is the immunoprotective (5) major surface protein 1 (MSP-1) (6). A subunit of this heterodimeric protein (5, 7, 8), AmF105, exhibits apparent size polymorphisms of up to 50%o among isolates (6), yet all isolates tested from the United States, Israel, and Kenya carry an epitope sensitive to neutralization by mouse monoclonal antibody Ana22B1 (mAb Ana22B1) (7, 9, 10). To understand the molecular basis for these observations, we cloned and sequenced the gene (mspla)¶ for this subunit from four isolates. The epitope recognized by mAb Ana22B1 was then mapped to determine its involvement in the size variation.
Abbreviations: ORF, open reading frame; mAb, monoclonal antibody; MSP-1, major surface protein 1. tTo whom reprint requests should be addressed. §Present address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164. IThe sequences reported in this paper have been deposited in the GenBank data base (accession nos. M32868-M32872).
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Mapping of the Neutralization-Sensitive Epitope. The epitope recognized by mAb Ana22Bl was mapped by assaying antibody binding to synthetic oligopeptides containing overlapping portions of the AmF105 tandem repeat unit B form. Antibody binding was assayed by a solution-phase inhibition radioimmunoassay using 125I-labeled AmF105 (27) and was confirmed by an ELISA (28) and by immunoblots (8), using peptides linked to the solid phase with glutaraldehyde.
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