The Cryptosporidium Parvum TSP4 Mediates Adhesion Of Intestinal Epithelial Cells Via Interactions With Sulfated Heparin

Cryptosporidium parvum infects both humans and animals,and often causes water-bome outbreaks all over the world.Especially it is harmful to immunodeficient patients and children under five years old.No effective vaccines and drugs against Cryptosporidiosis of human and animals are available.Therefore,it is critical to elucidate the molecular mechanism that related to adhesion and invasion of C.parvum.Previous studies have shown that the thrombospondin protein family(TSP protein family),which containing the TSP1 domain,play an important role in invasion of the apicomplexan parasites and likely to be potential vaccine candidates.The family contains 12 members.The function of these proteins remains to be elucidated.In this study,TSP4 protein was selected as the target,the identification and adhesion function of the protein were evaluated.Through the bioinformatic analysis of TSP4 protein,peptide fragment with high specificity and immunogenicity was designed.Peptide was synthesized and conjugated to KLH and BSA carrier protein by MBS coupling method.TSP4-KLH was used as immunogen,while TSP4-BSA was used as the detection antigen in ELISA to select monoclonal antibody against TSP4 peptide.The subtype of monoclonal antibody was identified.The transcriptional level of TSP4 in oocysts,sporozoites and different stages postinfection was evaluated by RT-q PCR.Native TSP4 protein in the lysate of the oocysts after excystation was identified by Western blot.The distribution of this protein in sporozoites and merozoites were identified by indirect immunofluorescence method.TSP4-GST recombinant protein was expressed and purified.The binding of TSP4-GST to HCT-8 cells was evaluated by western blot,flow cytometry and cell-binding ELISA.Finally the binding properity of TSP4-GST to heparin was evaluated.Through bioinformatics analysis,TSP4 protein sequence has a signal peptide(1-26 amino acids),two TSP1 domain,one Apple domain,but without introns.The monoclonal antibody against TSP4 specific peptide was prepared with highly specificity and sensitivity,and the antibody subtype was Ig G1.RT-q PCR showed that TSP4 was transcribed in all stages,but the transcription level reached the maximum at 12 h postinfection,and then began to decline.By Western blot,a 55 k Da band was identified.TSP4 protein was localized on the surface of sporozoites and merozoites,co-localized with membrane protein GP15.The recombinant protein of TSP4-GST was expressed in E.coli.Western blot and flow cytometry showed that TSP4-GST was able to bind to HCT-8 cells.Cell-binding ELISA assay showed that TSP4-GST bind to HCT-8 cells with dose-dependent manner.Recombiant TSP4-GST protein can bind to heparin but not to chondroitin sulfate A.In summary,The C.parvum TSP4 mediates adhesion of intestinal epithelial cells via interactions with sulfated heparin.The protein may serve as a target for cryptosporidiosis treatment and vaccine antigen candiadate.