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 sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy Cryptosporidium parvum gene Cpgp40/15. This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the Cpgp40/15 alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in C. parvum to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the Cpgp40/15 locus of 20 C. parvum isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of Cryptosporidium and Cryptosporidium oocyst wall protein loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the Cpgp40/15 locus, raising the possibility of sexual recombination within and between prototypal parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.

The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion-and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains ␣-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and ␣-N-acetylgalactosaminespecific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.Cryptosporidium parvum, an intestinal Apicomplexan parasite, is a significant cause of diarrheal disease worldwide (15,17). In immunocompetent individuals, the disease is usually self-limiting, but it may be chronic and life threatening in immunocompromised patients such as those with AIDS. Recently, the parasite has gained notoriety as the causative agent of numerous outbreaks of waterborne diarrheal disease. There is currently no effective specific therapy approved for disease caused by this parasite.Infection is initiated by ingestion of oocysts, which undergo excystation to release sporozoites. Attachment of sporozoites to epithelial cells and subsequent invasion of the host cell membrane are crucial primary steps in the pathogenesis of cryptosporidiosis. The ultrastructural aspects of attachment and invasion have been characterized in detail (10,24,33,34). Sporozoites attach to host cells by their anterior pole. Attachment is followed by invagination of the host cell plasma membrane, which extends along the surface of the sporozoite and eventually completely surrounds it, leading to formation of a parasitophorus vacuole where the parasite undergoes further development in a unique intracellular but extracytoplasmic location.Using in vitro models of sporozoite attachment to epithelial cells, we previously showed that attachment was dose and time dependent and was influenced by pH, divalent cations, and the degree of differentiation of host cells (20,22). Further, attachment could be inhibited by polyclo...