Cloning and Sequence Analysis of a Highly PolymorphicCryptosporidium parvumGene Encoding a 60-Kilodalton Glycoprotein and Characterization of Its 15- and 45-Kilodalton Zoite Surface Antigen Products

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 singlecopy 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.Cryptosporidium parvum, a protozoan parasite of the phylum Apicomplexa, is an enteric pathogen that infects humans and many animals and causes an acute diarrheal disease (30, 61). Cryptosporidiosis is self-limiting in immunocompetent individuals (22,25,116) but is often chronic and life-threatening in immunocompromised patients, such as those with AIDS (20,22,72,76,78) or in persons with weakened immune systems, for example, malnourished children and the elderly (18,24,27,46,89). Despite exhaustive attempts at chemotherapy with a wide variety of drugs, including many that are effective against related apicomplexan parasites, no efficacious treatment for cryptosporidiosis has been identified. Passive immunotherapy with hyperimmune bovine colostral immunoglobulin (HBC Ig) has, however, shown some ability to ameliorate the clinical symptoms of disease in humans (29,62,77,88,93,(108)(109)(110). HBC Ig inhibits sporozoite invasion in vitro and neutralizes sporozoite infectivity in animal models of cryptosporidiosis (5, 9, 10, 23, 69-71, 84-86, 107). Although the exact identities of the HBC Ig neutralization antigens are unknown, several candidate molecules have been identified (8,39,69,(84)(85)(86)107). The majority are protein or glycoprotein antigens present on the zoite surface ...

Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15

“…To determine if gp40 associates with the sporozoite membrane by forming a complex with gp15, we performed co-immunoprecipitations using a rat polyclonal antibody raised against native gp40 (a gift from Dr Richard Nelson, University of California, San Francisco, CA [8]). Control immunoprecipitations were run in parallel using normal rat serum.…”

“…One research focus has been to identify the antigens on the invasive zoite stages that allow the parasite to attach to and invade the epithelial cell with the goal of preventing these parasite-host cell interactions. The best characterized of the zoite antigens is Cpgp40/15, (also called Cp17, gp15/45/60 or S60) [6][7][8][9][10], a mucin like glycoprotein antigen that is synthesized as a single precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15 [11]. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol (GPI) moiety [12], while the gp40 glycoprotein does not contain any predicted transmembrane domains or GPI anchors and is predicted to be soluble [7].…”

“…Figure 2A shows that both gp40 (Figure 2A, panel 1, lane 3) and gp15 ( Figure 2A, panel 2, lane 3) were precipitated by the gp40 specific antisera, but not by normal rat serum ( Figure 2A, panels 1 and 2, lane 2). Since the rat anti-gp40 serum does not react with gp15 [8], gp15 was precipitated via its interaction with gp40, suggesting that the two antigens associate on the sporozoite surface to form a heteroprotein complex.…”

“…Lane 1: total lysate, lane 2: precipitate using normal rat serum, lane 3: precipitate using rat anti-gp40 serum. The high molecular weight band recognized by CrA2 in total lysates is elongation factor EF1α [8,21]. B.…”

Molecular characterisation of Cryptosporidium outbreaks in Western and South Australia

“…glycoproteins (gp45 and gp15), both of which are implicated in zoite attachment to and 42 invasion of enterocytes (Strong et al, 2000). 43…”