Vol. 62, No. 4
INFECrION AND IMMUNITY, Apr. 1994, p. 1213-1218 0019-9567/94/$04.00+0 Copyright ( 1994, American Society for Microbiology
Size Heterogeneity among Antigenically Related Giardia lamblia Variant-Specific Surface Proteins Is Due to Differences in Tandem Repeat Copy Number MICHAEL R. MOWATF,l* BACH-YEN T. NGUYEN,'t JOHN T. CONRAD,' RODNEY D. ADAM,2 AND THEODORE E. NASH1 Laboratory of Parasitic Diseases, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 20892, and Department of Internal Medicine and Department of Microbiology/Immunology, University of Arizona Health Sciences Center, Tucson, Arizona 857242 Received 27 September 1993/Returned for modification 19 November 1993/Accepted 5 January 1994
Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units.
considerable size variation among the 6E7 epitope-bearing VSPs. In this report, we describe the characterization of the MAb 6E7 epitope and compare VSP A6 to the VSPs expressed by the Be-2 and G3M cloned lines. Our results suggest that size heterogeneity among VSPs bearing the 6E7 epitope is due to differences in the number of repetitive units among polypeptides that are otherwise nearly identical.
At a given point in time, an individual Giardia lamblia trophozoite is entirely covered by one member of a family of antigenically diverse molecules called the variant-specific surface proteins (VSPs). By a mechanism as yet unclear, one VSP may be replaced spontaneously by another antigenically distinct VSP (17). Antigenic variation has been demonstrated in experimental infections of animals (4, 10) and humans (21) as well as in vitro, where switching frequencies have been estimated at once per 6 to 13 generations (19). Although no link between antigenic variation and the pathophysiology of Giardia infection or Giardia virulence has been established formally, the cell surface location and abundance of VSPs imply a significant role for these molecules in the interaction between parasite and the host intestinal milieu. VSPs constitute a unique family of cysteine-rich proteins that vary in molecular mass from 33 to 200 kDa and contain multiple Cys-X-X-Cys motifs, a conserved carboxy-terminal region, and a unique zinc finger-like motif (17). The first defined VSP, VSP A6 or CRP170, is a 170-kDa antigen expressed by the A6 clone of the WB isolate and is recognized by monoclonal antibody (MAb) 6E7 (2). A 1-kb DNA fragment (M2-1) that encoded a polypeptide bearing the 6E7 epitope was cloned from a Agtll genomic expression library by using MAb 6E7. The M2-1 clone contained 2.6 tandemly repeated copies of a 195-bp unit followed by a nonrepetitive 3' portion (483 bp). Subsequent analysis of the VSP A6 cDNA and its corresponding expressed genomic copy revealed 21 tandemly arranged copies of the 195-bp repeat followed by the same nonrepetitive coding sequence (3). The MAb 6E7 epitope is conserved among some VSPs of several independent Giardia isolates; that is, its expression is not restricted to the WB isolate. 6E7-reactive cloned trophozoite lines have been derived from the Be-2, CAT-1, and G3M isolates (20). Unexpectedly, comparison of these lines by both surface radioiodination and immunoblot analysis indicated
MATERUALS AND METHODS Giardia isolates and clones. Trophozoites were maintained in culture and harvested as described previously (18). MAb 6E7-reactive clones from three isolates (G3M, Be-2, and WB) were used. G3M-B expresses a 66-kDa surface protein (VSP G3M-B), while Be-2-A and Be-2-B express the 115- and 73-kDa surface proteins, VSP Be-2A and VSP Be-2B, respectively (20). The WB/A6 cloned line (18) and the GS/H7 cloned line, which fails to react with MAb 6E7 and lacks a VSP A6-like gene (20), were used as positive and negative controls, respectively. The production, specificity, and reactivity of MAb 6E7 have been reported elsewhere (18). Construction, screening, and analysis of an M2-1 random subclone library in Xgtll. The M2-1 insert was sonicated (8), and the resultant fragments were cloned in Xgtl 1 after addition of XmnI-EcoRI adapters (New England BioLabs, Beverly, Mass.). Clones were selected for 6E7 reactivity and plaque purified by standard methods. Selected clones were characterized in two ways. First, the inserts were amplified in a PCR that employed primers flanking the EcoRI cloning site of Xgt 1 (23) and subsequently sized by agarose gel electrophoresis. In addition, the open reading frame of each insert was verified, and its 5' and 3' limits, relative to the M2-1 sequence, were established by direct DNA sequence analysis of products derived by asymmetric amplification of the insert from the recombinant clone (see below). Exonuclease III deletion analysis of the M2-1 insert. The M2-1 insert was subcloned in the EcoRI site of the expression plasmid pGEMEX-1 (Promega, Madison, Wis.), and the resulting plasmid was called pBY-1. Progressive deletions from
* Corresponding author. Phone: 301-496-6920. Fax: 301-402-2201. Electronic mail address:
[email protected]. t Present address: Division of Cancer Treatment, National Cancer Institute, Bethesda, MD 20892.
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FIG. 1. Immunoblot analysis of 3' deletion mutants of pBY-1. Lysates of bacterial cultures, induced to express different T7 gene 10 M2-1 fusion proteins, or control T7-Tag protein were fractionated by SDS-PAGE, transferred to nitrocellulose, and analyzed for reactivity with MAb 6E7 (A) or anti-T7-Tag antiserum (B). The reactivities of pBY-1.9 and pBY-1.10 fusion proteins with MAb 6E7 appeared consistently weaker than those of pBY-1, -1.1, and -1.11. Asterisks in panel B are located directly below the fusion proteins. Size markers are indicated on the left side of each panel.
the 3' end of the open reading frame were made by the exonuclease III procedure (11), using the Erase-a-Base system (Promega). T7 gene 10 fusion proteins were screened for reactivity to 6E7 by dot blot immunoassay and verified by immunoblot analysis (described below). The insert sizes of select clones were determined by PCR amplification of the recombinant plasmid inserts, and the 3' limits of the inserts, relative to the M2-1 sequence, were established by direct DNA sequencing as described below. Immunoblot analysis. T7 gene 10 fusion proteins were produced from recombinant pGEMEX-1 constructs trans-
TABLE 1. Summary of pBY-1 deletion analysis Plasmid Plasmid
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' Nucleotide numbering is based on M2-1 sequence (GenBank accession number X06741); all inserts start at nt 1. M2-1 repeat 1 begins at nt 1 and ends at nt 111; repeat 2 begins at nt 112 and ends at nt 306; repeat 3 begins at nt 307 and ends at nt 503. b ND, not determined by sequence analysis, but insert size was estimated as 756 bp. c Although pBY-1.10 and pBY-1.9 contain identical inserts, the M2-1 open reading frame in pBY-1.9 terminates later than that of pBY1-1.10 as a result of a translational frameshift in the downstream vector sequences; thus, the fusion proteins have different mobilities (Fig. 1).
fected into Escherichia coli JM109(DE3) as described by the supplier of the plasmid (Promega). MAb 6E7 and rabbit anti-T7-Tag antiserum (Novagen, Madison, Wis.), which reacts with the T7 gene 10 protein product, were used to detect fusion proteins in immunoblots of bacterial lysates by methods described previously (22). Northern (RNA) blots. Polyadenylated RNA was purified, analyzed by electrophoresis through 1.2% agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher & Schuell, Inc., Keene, N.H.), and hybridized to 32P-labelled probes as described previously (13). cDNA library construction and screening. A cDNA library was constructed in Xgt22 from polyadenylated RNA of the G3M-B cloned line by using the Superscript Lambda system (GIBCO-BRL, Gaithersburg, Md.). The library was screened with the 32P-labelled insert gel purified from pBY-1.8, a deletion mutant of pBY-1 that contains a 404-bp insert consisting of repeat sequences only. The inserts of recombinant Xgt22 clones were amplified, sized, and partially sequenced by the methods described above and below for Xgtll clones. DNA and RNA sequence determination. DNA sequences were determined from the products of asymmetric amplification of selected Xgtl1 recombinant clones (12) by using Sequenase version 2.0 (United States Biochemical, Cleveland, Ohio). Xgt22 cDNA clones were sequenced by the affinity strand separation technique (16), using 5'-biotinylated primers for PCR and oMM35 (5'-GACATCATTCTGATCGC-3', antisense corresponding to nucleotides [nt] 590 through 574 of M2-1; see also Fig. 4) or nested primers for sequencing. Primers flanking the EcoRI cloning site of pGEMEX-1 were used to sequence recombinant plasmid templates. DNA sequence ambiguities were resolved by using 7-deaza-GTP (United States Biochemical) in extension reactions. The sequences of VSP transcripts were determined directly from total
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INFECT. IMMUN.
MOWATT ET AL. A. Location of Probes Within VSP A6 DNA 195bp Tandem repeats
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