pubmed.ncbi.nlm.nih.gov

Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products - PubMed

  • ️Invalid Date

Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products

W B Strong et al. Infect Immun. 2000 Jul.

Abstract

The apicomplexan parasite Cryptosporidium parvum is a major cause of serious diarrheal disease in both humans and animals. No efficacious chemo- or immunotherapies have been identified for cryptosporidiosis, but certain antibodies directed against zoite surface antigens and/or proteins shed by gliding zoites have been shown to neutralize infectivity in vitro and/or to passively protect against, or ameliorate, disease in vivo. We previously used monoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that neutralized parasite infectivity for cultured epithelial cells. Here we report the cloning and sequence analysis of the gene encoding this 11A5 antigen. Surprisingly, the gene encoded a 330-amino-acid, mucin-like glycoprotein that was predicted to contain an N-terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a glycosylphosphatidylinositol anchor. The single-copy gene lacked introns and was expressed during merogony to produce a 60-kDa precursor which was proteolytically cleaved to 15- and 45-kDa glycoprotein products that both localized to the surface of sporozoites and merozoites. The gp15/45/60 gene displayed a very high degree of sequence diversity among C. parvum isolates, and the numerous single-nucleotide and single-amino-acid polymorphisms defined five to six allelic classes, each characterized by additional intra-allelic sequence variation. The gp15/45/60 single-nucleotide polymorphisms will prove useful for haplotyping and fingerprinting isolates and for establishing meaningful relationships between C. parvum genotype and phenotype.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1

Anti-15-kDa antigen-specific MAbs and the HPA lectin decorate gliding sporozoites and identify shed antigen trails. Sporozoites locomoting on poly-

l

-lysine-coated slides were formaldehyde fixed, blocked, and incubated with MAb 11A5 (A), CrA1 (B), or CrA2 (C) or the biotin-conjugated lectin HPA (D). Reactivity profiles were visualized and documented by epifluorescence photomicroscopy following incubation of the washed slides with biotinylated secondary antibodies and/or Cy3-conjugated streptavidin.

FIG. 2
FIG. 2

MAbs 11A5 and CrA2 recognize the same 15-kDa glycoprotein antigen. Triton X-100-soluble oocyst/sporozoite proteins were either directly fractionated and blotted following SDS-PAGE (A and B, lanes 1) or fractionated and blotted following immunoprecipitation with IgG MAb 11A5 (A and B, lanes 2 and 4) or IgA MAb CrA2 (A and B, lanes 3 and 5). The blots were probed with primary MAb 11A5 (A, lanes 1 to 3) or primary MAb CrA2 (B, lanes 1 to 3) followed by alkaline phosphatase-conjugated secondary anti-IgG (A) or anti-IgA (B) antibodies. The arrow indicates the position of the 15-kDa antigen, the arrowhead points to the 25-kDa antigen, the asterisk denotes the 50-kDa antigen, and lanes 4 and 5 in both panels are secondary antibody controls (lacking primary antibody) which reveal the position of the precipitating MAb 11A5 and CrA2 heavy and light chains.

FIG. 3
FIG. 3

REAs affinity purified on plaque lifts of gDNA clones G4C1A and G7A1A recognize a 15-kDa C. parvum antigen. Oocyst lysates were fractionated by SDS-PAGE, blotted, and probed with REA prepared using gDNA (G) and cDNA (S) clones G9A1A (a), G7A1A (b), S10D1A (c), G4D1A (d), G15A1A (e), S9C1A (f), G12B1A (g), G4C1A (h), G1A1A (i), S5F1A (j), and S5C1A (k). The blots in lanes l and m were probed with REAs prepared on plaque lifts of the wild-type λZAP II and λgt11 phage vectors, respectively. Positive control blots were probed with MAbs 11A5 (n), CrA1 (o), and CrA2 (p); the position of the 15-kDa antigen is indicated by the arrowhead.

FIG. 4
FIG. 4

Nucleotide and deduced amino acid sequence of the NINC1 C. parvum gp15 gene. Locations of the gp15ATG and gp15STOP primers are indicated by arrows above and below the nucleotide sequence. The empirically determined N-terminal amino acid sequences of the HPA-affinity purified 15- and 45-kDa proteins are shown enclosed in boxes. The predicted N-terminal signal peptide cleavage site is indicated by the triangle, and the predicted GPI anchor attachment site is circled. Amino acid residues predicted to be O-glycosylated are subscripted by circles, and the single predicted N-glycosylated asparagine residue (Asn202) is boxed.

FIG. 5
FIG. 5

(A) Time course of gp15 mRNA expression in C. parvum-infected MDCK cell monolayers. Total RNA was isolated from infected MDCK cells at various times postinfection, DNase treated, and used as a template for RT-PCR (lanes 2 to 12) or control PCR (lanes 13 to 23), both primed with the gp15ATG and gp15STOP primers. The arrowhead indicates the position of the 1-kb gp15 amplicon. RNA template was isolated from infected MDCK cells at 0.5 h (lanes 3 and 14), 2 h (lanes 4 and 15), 4 h (lanes 5 and 16), 6 h (lanes 6 and 17), 9 h (lanes 7 and 18), 11 h (lanes 8 and 19), 24 h (lanes 9 and 20), and 48 h (lanes 10 and 21) postinfection; from uninfected MDCK cells (lanes 2 and 13); and directly from purified sporozoites (2 μg; lanes 11 and 22). Additional controls for the RT-PCR and PCRs lacked template nucleic acid (lanes 12 and 23 and 24, respectively). (B) Intracellular expression of gp15 protein occurs late in merogony. C. parvum-infected MDCK monolayers were fixed 6 and 11 h postinfection and incubated with control MAb LOI (B and D), which recognizes a parasitophorous vacuole antigen present throughout intracellular parasite development (14), or with anti-gp15 MAb 11A5 (F and H), CrA1 (J and L) or CrA2 (N and P) followed by biotinylated secondary antibodies and Cy3-conjugated streptavidin. Parasite nuclei in the same microscopic fields were stained with DAPI (lanes A, E, I, M, C, G, K, and O).

FIG. 6
FIG. 6

HPA affinity chromatography identifies 15- and 45-kDa glycoproteins. A Triton X-100-soluble, oocyst-sporozoite lysate was chromatographed on HPA-agarose, and bound proteins were eluted with GalNAc, fractionated by SDS-PAGE, and stained with Coomassie Serva blue G (lane C) or Western blotted (B) and probed with MAb CrA1 (lane 1), CrA2 (lane 2), 11A5 (lane 3), wild-type λgt11 REA (lane 4), G4C1A REA (lane 5), HPA-biotin (lane 6), or rat anti-gp45 polyclonal antibody (lane 7). The arrowheads indicate the positions of gp15 and gp45. Molecular weight markers are shown in lane M. Sizes are indicated in kilodaltons.

FIG. 7
FIG. 7

A 60-kDa precursor glycoprotein is synthesized by intracellular parasites and processed to produce gp15 and gp45 products. GalNAc-containing O-linked glycoproteins were affinity purified on HPA-agarose from uninfected (lane 1) and C. parvum-infected MDCK monolayers 11 h postinfection (lane 2), eluted with GalNAc, fractionated by SDS-PAGE, blotted, and probed with rat anti-gp45 polyclonal antiserum (A) or MAb CrA2 (B). The positions of the gp60 precursor (A and B, asterisk) and the presumptive gp15 (B, arrow) and gp45 (A, arrowhead) proteolytic products are indicated.

FIG. 8
FIG. 8

gp15/45/60 protein sequences are extremely polymorphic. The deduced gp15/45/60 amino acid sequences of 23 human and animal isolates were aligned using the ClustalW algorithm, and the sequence of the NINC1 animal isolate was used as a reference to which all others were aligned. The gp15/45/60 sequences from Iowa, NT009, KSU1, Peru, UCP, and TAMU were identical, and only the Iowa sequence is shown; likewise, only the first sequence from each of the identical pairs, 0542J and 0541L, SFGH4 and SFGH3, and 9877 and NEMC1, is presented. The five allelic groupings are explicitly indicated in the left margin by II, Ia, Ib, Ic, and Id. Hyphens in the alignment indicate identical residues, and filled circles indicate gaps; boxed residues depict SAAPs among sequences within a particular allelic grouping. The eight residues strictly conserved within either the allele I or allele II groupings are vertically highlighted throughout all sequences in black. The hypervariable region present within the gp45 sequence of human isolates (allelic groups Ia to Id) is indicated by shading. The tandemly repeated DGGKE sequence found within this region in all genotype Ia isolates is italicized. The two glutamic acid residues found in the putative protease cleavage site of the gp60 precursor are boxed throughout. The numbering above the sequences includes the gaps introduced for alignment purposes, while the numbers at the end of each line denote the amino acid position within that particular sequence.

FIG. 8
FIG. 8

gp15/45/60 protein sequences are extremely polymorphic. The deduced gp15/45/60 amino acid sequences of 23 human and animal isolates were aligned using the ClustalW algorithm, and the sequence of the NINC1 animal isolate was used as a reference to which all others were aligned. The gp15/45/60 sequences from Iowa, NT009, KSU1, Peru, UCP, and TAMU were identical, and only the Iowa sequence is shown; likewise, only the first sequence from each of the identical pairs, 0542J and 0541L, SFGH4 and SFGH3, and 9877 and NEMC1, is presented. The five allelic groupings are explicitly indicated in the left margin by II, Ia, Ib, Ic, and Id. Hyphens in the alignment indicate identical residues, and filled circles indicate gaps; boxed residues depict SAAPs among sequences within a particular allelic grouping. The eight residues strictly conserved within either the allele I or allele II groupings are vertically highlighted throughout all sequences in black. The hypervariable region present within the gp45 sequence of human isolates (allelic groups Ia to Id) is indicated by shading. The tandemly repeated DGGKE sequence found within this region in all genotype Ia isolates is italicized. The two glutamic acid residues found in the putative protease cleavage site of the gp60 precursor are boxed throughout. The numbering above the sequences includes the gaps introduced for alignment purposes, while the numbers at the end of each line denote the amino acid position within that particular sequence.

Similar articles

Cited by

References

    1. Aiello A E, Xiao L, Limor J R, Liu C, Abrahamsen M S, Lal A A. Microsatellite analysis of the human and bovine genotypes of Cryptosporidium parvum. J Eukaryot Microbiol. 1999;46:46S–47S. - PubMed
    1. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. - PubMed
    1. Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. - PMC - PubMed
    1. Arrowood M. Ph.D. thesis. Tuscon: University of Arizona; 1988.
    1. Arrowood M J, Mead J R, Mahrt J L, Sterling C R. Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice. Infect Immun. 1989;57:2283–2288. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources