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

The apicomplexan parasite Cryptosporidium parvum is a major cause of seriou s diarrheal disease in both humans and animals. No efficacious chemo- or im munotherapies have been identified for cryptosporidiosis, but certain antib odies directed against zoite surface antigens and/or proteins shed by glidi ng zoites have been shown to neutralize infectivity in vitro and/or to pass ively protect against, or ameliorate, disease in vivo. We previously used m onoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that n eutralized parasite infectivity for cultured epithelial cells. Here we repo rt 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 hydrop hobic C-terminal peptide specifying attachment of a glycosylphosphatidylino sitol 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 degr ee 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 haplo typing and fingerprinting isolates and for establishing meaningful relation ships between C. parvum genotype and phenotype.