Abstract
Herein, we report the biochemical and functional characterization of a novel Ca2+-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.
Highlights
Cryptosporidiosis, caused by the obligate intracellular protozoan Cryptosporidium, is a ubiquitous infectious disease that can cause a persistent and chronic diarrhea [1]
Ethics statement All mice were housed at the vivarium at Virginia Commonwealth University (VCU) in an AAALAC-accredited facility and experimentation followed VCU Institutional Animal Care and Use Committee approved protocols (VCU IACUC Approved Protocol AM10329, Cryptosporidium Reverse Vaccinology)
We describe the cloning, expression and purification of recombinant C. hominis apyrase gene (CApy) derived from C. hominis, designated rCApy
Summary
Cryptosporidiosis, caused by the obligate intracellular protozoan Cryptosporidium, is a ubiquitous infectious disease that can cause a persistent and chronic diarrhea [1]. Cryptosporidium hominis (C. hominis) is the most prevalent cause of endemic disease in humans, Cryptosporidium parvum (C. parvum) can cause serious outbreaks when humans are exposed to contaminated water supplies or are otherwise in close contact with the non-human carriers of this parasite. Interactions between macromolecules of the parasites and host cells are of critical importance for the establishment of the infection and consequent survival of the parasite. Pathogenic factors such as parasite proteins or macromolecules responsible for attachment or invasion, or factors that block host cell responses, are ideal targets for drug and vaccine development
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