Preliminary Study On The Function Of Cryptosporidium Parvum Rhoptry Protein Candidates Cgd4₂420 And Cgd8₂530

The apicomplexan parasite Cryptosporidium parvum is a waterborne enteric protozoan of significant medical and veterinary importance. In immunocompetent individuals C.parvum determines a self-limiting watery diarrhea, whereas in AIDS patients and other immunocompromised subjects cryptosporidiosis is a chronic and potentially life-threatening disease still lacking effective therapies. Although the molecular mechanisms underlying this peculiar invasion process are poorly understood, there is evidence that C.parvum also relies for invasion on the regulated secretion of apical organelles, i.e., micronemes and rhoptries.The implication of this organelle in C.parvum invasion is supported by ultrastructural observations While a few microneme proteins were described in C.parvum, the repertoire of molecules contained in the single parasite rhoptry is largely unknown. Through bioinformatic analysis, which rhoptry protein candidates cgd4₂420 and cgd8₂530 are found to have conserved domain with T.gondii RON9 and RON10, including N-terminal signal peptide, C-terminus transmembrane region, ankyrin repeat domains and a sushi domain. They are supposed to be rhoptry proteins and may be involved in the invasion process.Firstly, expression of cgd4₂420 and cgd8₂530 were evaluated by realtime PCR in infected HCT-8 cell monolayers following infection by Cryptosporidium parvum.The results showed that both of them have a upward trend of transcription gradually post-infection. It peaked at 24 h and then decreased. Seconly, fragments of cgd4₂420 and cgd8₂530 were amplified by PCR, then cloned into p ET-28 a and p GEX-4T-1 plasmid for expression. The recombinant proteins were purified through affinity chromatography. The polyclonal antibody against these recombinant proteins were prepared in rabbits. Thirdly, the expression of cgd4₂420 and cgd8₂530 in Cryptosporidium parvum were verified by Western blot and IFA. The results showed that both on them were expreesed in the lysate of the occysts and located on the sporozoites. Finally, the binding of GST-tag recombinant proteins to HCT-8 cells was assessed by Western blot and IFA. The results showed that Cp cgd4₂420 could bind to HCT-8 cells while cgd8₂530 could not. It indicated that Cp cgd4₂420 was associated with the invasion of host cells in C.parvum.As the importance of rhoptries in other Apicomplexan invasion, reproduction and virulence-related processes, the study on the biological function of Cryptosporidium parvum putative rhoptry protein is very essential. This study was the foundation for studying on the feature and function of cgd4₂420 and cgd8₂530, revealling the rhoptry proteins family of Cryptosporidium parvum.