Polymorphic Mucin Antigens CpMuc4 and CpMuc5 Are Integral toCryptosporidium parvumInfection In Vitro

Abstract

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 mucinlike 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.

Systemic Antibody Responses to the Immunodominant p23 Antigen and p23 Polymorphisms in Children with Cryptosporidiosis in Bangladesh

“…The finding that these sequences clustered separately from the other C. parvum sequences is interesting and consistent with those of previous studies, which showed that they are divergent at other loci including that of the gp40/15 (also called gp60) 41 and Muc4 genes. 44 Although most children in our study were infected with different C. hominis subtype families, 47 the finding that they developed significant antibody responses to the C. parvum antigen is consistent with that reported by Priest and others. 31 The results from both studies suggest that these responses are directed at epitopes conserved among species and different subtype families.…”

“…41 Most human infections, particularly in resource-poor countries, are caused by C. hominis. Genes encoding other surface-associated Cryptosporidium proteins including gp40 42,43 and Muc4 and 5 44 are highly polymorphic among clinical isolates of different species and subtypes. If p23 is to be considered as a vaccine candidate, it is essential to investigate polymorphisms in the gene that encodes it among clinical isolates in different geographic areas.…”

Cryptosporidium Pathogenicity and Virulence

“…gp900, gp40, gp15, Cpa135, Cp2, P23, and TRAP-C1 (Table 2) have or are predicted to have mucin-type O-glycosylation, suggesting that this type of posttranslational modification is common in proteins involved in attachment and invasion (133). Because mucin-like proteins were shown to be important for host-parasite interactions for Cryptosporidium, O'Connor and colleagues (193) undertook data mining of the Cryptosporidium genome databases to identify other mucin-like genes. They discovered a single locus of seven small mucin sequences (CpMuc1 to CpMuc7), which were expressed throughout the intracellular development stages.…”

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

“…Third, we may only be detecting the most abundant C. parvum glycoproteins, which include gp900 and gp40/gp15 (30,32). Indeed other recently characterized C. parvum mucins (C. parvum mucin 4 [CpMuc4] and CpMuc5), which are important for infectivity, are absent from the MPA affinity-purified proteins (25).…”

“…C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Serand Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4,6,7,25,26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42).…”