Polymorphic mucin antigens CpMuc4 and CpMuc5 are integral to Cryptosporidium parvum infection in vitro

Cryptosporidium, a waterborne enteric parasite, is a frequent cause of diarrheal disease outbreaks worldwide. Thus far, the few antigens shown to be important for attachment to and invasion of the host cell by Cryptosporidium are all mucin-like glycoproteins. In order to investigate other antigens that could be important for Cryptosporidium host-parasite interactions, the Cryptosporidium genome databases were mined for other mucin-like genes. A single locus of seven small mucin sequences was identified on chromosome 2 (CpMuc1 to -7). Reverse transcriptase PCR analysis demonstrated that all seven CpMucs were expressed throughout intracellular development. CpMuc4 and CpMuc5 were selected for further investigation because of the significant sequence divergence between Cryptosporidium parvum and C. hominis alleles. Rabbit anti-CpMuc5 and -CpMuc4 antibodies identified several polypeptides in C. parvum lysates, suggestive of proteolytic processing of the mucins. All polypeptides were larger than the predicted molecular weight, which is suggestive of posttranslational processing, most likely O-glycosylation. In immunofluorescence assays, both anti-CpMuc4 and -CpMuc5 antibodies reacted with the apical region of sporozoites and revealed surface-exposed epitopes. The antigens were not shed during excystation but did partition into the aqueous phase of Triton X-114 extractions. Consistent with a role in attachment and invasion, CpMuc4 and CpMuc5 could be detected binding to fixed Caco-2A cells, and anti-CpMuc4 peptide antibodies inhibited Cryptosporidium infection in vitro. Sequencing of CpMuc4 and CpMuc5 from C. hominis clinical isolates identified several polymorphic alleles. The data suggest that these antigens are integral for Cryptosporidium infection in vitro and may be potential vaccine candidates.