|Jenkins, Mark - US Department Of Agriculture (USDA)|
|Widmer, Giovanni - Tufts University|
|O'brien, Celia - US Department Of Agriculture (USDA)|
|Bauchan, Gary - US Department Of Agriculture (USDA)|
|Murphy, Charles - US Department Of Agriculture (USDA)|
Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2014
Publication Date: 4/1/2014
Citation: Jenkins, M., Widmer, G., O'Brien, C., Bauchan, G., Murphy, C., Santin, M., Fayer, R. 2014. A highly divergent 33 kDa Cryptosporidium parvum antigen. Journal of Parasitology. 100:527-531.
Interpretive Summary: Cryptosporidiosis is an intestinal parasitic disease of humans and animals that generally occurs after ingestion of water contaminated with Cryptosporidium oocysts. The two most important species affecting man are C. parvum and C. hominis. The former is transmitted between animals and humans, while the latter appears to only be transmitted between humans. Sequencing of the entire genome of C. parvum and C. hominis, and comparison of these sequences has identified a number of divergent gene sequences, which may code for proteins that are responsible for host specificity. In the present study, one of these gene sequences was cloned and expressed as a recombinant antigen in Escherichia coli. Antisera prepared against the recombinant protein identified a 33 kDa protein in C. parvum, but failed to identify this protein in C. hominis. The protein was localized to the antierior and posterior ends of the infectious stage of the parasite, and the gene is turned on during a process called excystation that prepares the parasite for invading host cells. This study provided support for the hypothesis that Cp33 plays some role in C. parvum development, and may represent a target to prevent cryptosporidiosis in humans and animals. this information will be of interest to other scientists and to the Veterinary Pharmaceutical industry.
Technical Abstract: Previous studies comparing the genome sequences of Cryptosporidium parvum with C. hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, a C. parvum sequence, namely cgd8-5370, whose amino acid sequence differs appreciably from its homologue in C. hominis, was cloned by PCR and expressed as a recombinant protein in Escherichia coli. Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. Anti-Cp33 sera failed to recognize the 33 kDa homologue in C. hominis. In immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior and posterior ends of air-dried C. parvum sporozoites, but failed to bind C. hominis sporozoites, possibly reflecting its specificity for C. parvum. IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind the parasite, suggesting that the CP33 is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of RNA from resting or excysting C. parvum oocysts or from C. parvum-infected cell cultures revealed mRNA transcription was detectable only during sporozoite excystation. However, antibodies against Cp33 failed to inhibit invasion of cultured cells suggesting that the protein is not involved in host cell penetration.