Molecular Cloning and Expression of a Gene EncodingCryptosporidium parvumGlycoproteins gp40 and gp15

Abstract

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal disease worldwide (7,36). Cryptosporidial infection is asymptomatic or self-limiting in immunocompetent hosts, but it may be chronic and lifethreatening in immunocompromised patients such as those with AIDS. In children in developing countries, C. parvum infection has been reported to be associated with persistent diarrheal disease, which may result in subsequent growth impairment (8). Recently, this parasite has been implicated as the causative agent of numerous outbreaks of waterborne diarrheal disease (6). There is currently no specific therapy approved for the treatment of cryptosporidiosis.Infection with C. parvum is initiated by ingestion of oocysts, which undergo excystation to release sporozoites. Sporozoites attach to and invade intestinal epithelial cells, where the parasite undergoes further intracellular development through asexual as well as sexual cycles. Merozoites released into the lumen during the asexual cycle can attach to and invade adjacent epithelial cells and thus maintain intracellular replication. The molecular mechanisms involved in the pathogenesis of cryptosporidiosis and the specific molecules that mediate C. parvum-host cell interactions are unknown. A number of C. parvum proteins have been implicated in attachment and in...

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

“…Control immunoprecipitations were run in parallel using normal rat serum. Western blots of the captured proteins were probed with the mouse IgM anti-gp40 mAb 4E9 [6], and the mouse IgA anti-gp15 mAb CrA2, (mAb CrA2 was a gift from Dr. Marian Neutra, Harvard Medical School, Boston, MA [7]). Detection with these reagents allowed identification of the precipitated antigens without interference from reactions with the precipitating antibody.…”

“…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]. However, gp40 has been shown to bind intestinal epithelial cells in a dose dependent and saturable manner [7] suggesting that this antigen recognizes a host cell receptor.…”

“…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]. However, gp40 has been shown to bind intestinal epithelial cells in a dose dependent and saturable manner [7] suggesting that this antigen recognizes a host cell receptor. If this interaction is important for zoite attachment and invasion, then gp40 must have some mechanism of associating with the parasite membrane.…”

“…To generate gp40 specific antiserum, recombinant (r)gp40 fusion protein [7] was purified via the His tag with Talon metal affinity resin (Clontech, Mountain View, CA) and resolved on preparative SDS-PAGE gels. Mice were immunized as described [7] and the specificity of the antiserum confirmed by western blot. To determine if gp40 is associated with the sporozoite membrane, we subjected excysted oocysts (C. parvum Iowa strain) to Triton X-114 extraction and phase separation [13] and analyzed aqueous and detergent phase antigens by Western blotting ( Figure 1A).…”

“…Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To generate gp40 specific antiserum, recombinant (r)gp40 fusion protein [7] was purified via the His tag with Talon metal affinity resin (Clontech, Mountain View, CA) and resolved on preparative SDS-PAGE gels. Mice were immunized as described [7] and the specificity of the antiserum confirmed by western blot.…”