The Cryptosporidium Parvum TSP7 Mediates Adhesion Of HCT-8 Cells Via Interactions With Sulfated Heparin

The apicomplexan parasite Cryptosporidium parvum is a gastrointestinal parasite that causes severe diarrheal disease worldwide.There are no effective anti-cryptosporidial agents,in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood.Therefore,it is critical to find the parasite molecules related to the adhesion and invasion of host cells.The prerequisite for host invasion of all apicomplaxan parasites is the attachment of the parasite ligands to the host cell receptors.Glycosaminoglycan molecules have been regarded as important receptors for invasion.In this study,TSP7 was selected as the target protein,the identification and related adhesion function were evaluated.A polypeptide fragment,361QISQWTDWSTC371,with high specificity and immunogenicity was designed by the bioinformatic analysis of TSP7 protein.The peptide was conjugated to KLH and BSA,respectively.KLH-peptide was used as immunogen BALB/c mice for prepare the anti-peptide monoclonal antibody.BSA-peptide was used as coating antigen for ELISA.The distribution of Cp TSP7 in sporozoites and merozoites was identified by indirect immunofluorescence method.The naive protein in the lysate sporozoites could be detected by western-blotting.The total RNA of the C.parvum oocysts,sporozoites and intercellular stages were collected for RT-q PCR,and the transcript levels of TSP7 was analyzed.In order to study the related adhesion function of TSP7,the 326-656 aa of Cp TSP7 gene was cloned into PGEX-4T-1 vector and expressed in E.coli BL21(DE3).The binding of the recombinant protein to HCT-8 cells was evaluated by cell binding ELISA assay,indirect immunofluorescence and flow cytometry.To demonstrate that heparin sulfate is one of the major receptors for binding of this recombinant protein to HCT-8,the binding activities of recombinant protein TSP7-GST to heparin was analyzed by Pull-down assays.Finally,the q RT-PCR method was used to analyze the effect of anti-TSP7 Mc Ab on blocking the invasion.Bioinformatics analysis indicated that TSP7 has a N-terminal signal peptide,and a transmembrane region at C-terminal,has three TSP1 domains,has one EGF-like domain and four heparin-binding motifs.Cp TSP7 is expressed on the surface of sporozoites and merozoites by immunofluorescence.It was co-localized with Gp15,a previously identified membrane protein.The 125 KDa band of naive protein in the lysate sporozoites could be identified by Western-blot.RT-q PCR showed that TSP7 was transcribed in all periods,but the transcription level reached the highest value at 12 h.Recombinant TSP7-GST displayed dose-dependent and saturable binding to HCT-8 cells by Western blot,indirect immunofluorescence and flow cytometry.It also showed a relatively strong specific binding to heparin agarose beads by a pull-down assay.Lastly,we assessed the inhibitory effect of the anti-TSP7 antibody on C.parvum invasion of host cells by a RT-q PCR assay,and the result revealed that the antibody had a significant inhibition effect on invasion.The inhibition rate of sporozoite invasion was 48% when the antibody concentration is 100 μg/m L.Taken together,these findings suggest that Cp TSP7 plays a role in C.parvum invasion in vitro and mediates adhesion of intestinal epithelial cells via interactions with heparin sulfate.Cp TSP7 may serve as a promising target or vaccine candidate for the control of cryptosporidiosis.