Cryptosporidium Parvum Cgd7₂530 And Cgd7₄560 Bind To Heparan Sulfate On The Host Cell Surface

Cryptosporidium parvum is a parasitic protozoan.It’s an important intestinal parasite that can cause diarrhea in humans and many mammals,and even lead to death,which is a serious threat to human health and animal husbandry.However,there is currently no effective drug for the treatment of Cryptosporidisis.Therefore,the study on the mechanism of C.parvum invading host cells is very important for the prevention and treatment of Cryptosporidisis.Like other apicomplexan parasites,C.parvum has a typical secretory apical complex in apical end,and the secreating proteins play important roles during invasion to host.Some of the proteins involved in host cell invasion are type I transmembrane proteins that are located at the apical end of C.parvum,such as GP900,CpMIC1,and TRAP-C1.Sulfated heparin is a glycosaminoglycan that binds to apicomplexan,such as Toxoplasma gondii and Plasmodium falciparum,and inhibits their invasion into host cells.Studies have demonstrated that C.parvum protein can also interact with Sulfated heparin on the surface of host cells,and heparin can inhibit the invasion of C.parvum into host cells.In this study,the C.parvum type I transmembrane proteins Cgd7₂530 and Cgd7₄560 were analysed.Bioinformatics analysis was made and specific B-cell epitopes were selected.Peptides were designed and synthesized,followed by coupled to carrier protein KLH or BSA by the MBS coupling method.Polyclonal antibodies against polypeptide were made by immunizing rabbits and purified by affinity chromatography.Western blot was used to identify the expression of native proteins in the lysate of the oocyst after excystation.Subcellular localization of the proteins in sporozoites and type I merozoites were identified by indirect immunofluorescence.Total RNA was extracted from HCT-8 cells after in vitro invasion by C.parvum,and the transcription levels of Cgd7₂530 and Cgd7₄560 were determined at different stages by Real-time PCR.Parts of sequence of Cgd7₂530 and Cgd7₄560 were selected to construct recombinant expression vectors,and the recombinant proteins were expressed and purified.The binding of the recombinant proteins to HCT-8 cells was detected by cell-binding ELISA and flow cytometry,and the binding of the recombinant proteins to heparin and chondroitin sulfate A was evaluated by Western blot.Finally,the glycosaminoglycan binding motifs in the proteins were analyzed.By bioinformatics analysis,the C.parvum type I transmembrane proteins Cgd7₂530 and Cgd7₄560 were selected,which contained the N-terminal signal peptide and the C-terminal transmembrane region.Two peptides Cgd7₂530⁴⁰⁶NSVNITPFQTEYINE-C⁴²⁰ and Cgd7₄560 ²²⁰GFGLSENSRVMEI-C²³² were designed and synthesized,and polyclonal antibodies against them were prepared.Western bloting results showed that native Cgd7₂530 and Cgd7₄560 proteins were260kD and 340kD respectively,which were in line with the predicted molecular weights.Subcellular localization of proteins by indirect immunofluorescence assay revealed that both Cgd7₂530 and Cgd7₄560 were localized in the apical end of the sporozoite and type I merozoites.The transcription level of Cgd7₂530 and Cgd7₄560 were detected by Real-time PCR.The results showed that both of Cgd7₂530 and Cgd7₄560 were expressed at high levels during early stages of infection.TherecombinantexpressionvectorspGEX-Cgd7₂530and pGEX-Cgd7₄560 were constructed and the recombinant proteins Cgd7₂530-GST and Cgd7₄560-GST were expressed and purified.Indirect cell binding ELISA and flow cytometry results showed that both Cgd7₂530-GST and Cgd7₄560-GST could specifically bind to HCT-8 cells in a dose-dependent and a dose-saturable manner.Western Blot results showed that both Cgd7₂530-GST and Cgd7₄560-GST could bind to heparin,which could competitively inhibit by heparin·Na,whereas chondroitin sulfate A had no inhibitory effect.The amino acid sequences analyzed of Cgd7₂530 and Cgd7₄560 found that both proteins contained glycosaminoglycan binding motifs.There are 3 motifs in Cgd7₂530 and 4 motifs in Cgd7₄560.Among them the recombinant proteins Cgd7₂530-GST and Cgd7₂530-GST contained 2 or1 glycosaminoglycan binding motifs,respectively.These results suggest that Cgd7₂530 and Cgd7₄560 were highly expressed in the early stages of infection and were located in the apical complex.Cgd7₂530 and Cgd7₄560 proteins could bind to HCT-8 cells,which might be mediated by Sulfated heparin on the host cell surface.Therefore,these two proteins may play important roles during the invasion of host cells.The study of C.parvum heparin binding protein is helpful to further understand the molecular basis of C.parvum invasion and is of great value for the development of new anti-cryptosporidiosis agents.