Adhesion Characteristics Of The Integrin ? Domain Containing Protein(CpITGA) In Cryptosporidium Parvum

Cryptosporidium belongs to the water-borne apicomplexa parasite and is an important intracellular parasite.However,at present,there is no effective prevention and treatment for cryptosporidiosis.Nitazoxanide,the only drug approved for cryptosporidiosis which is approved by the Federal Drug Administration(FDA)to treat cryptosporidial infections in immunocompetent individuals,but it is ineffective in AIDS patients.Therefore,elucidating the mechanism of adhesion and invasion of Cryptosporidium parvum to host cells is essential for the prevention and control of cryptosporidiosis.Integrins are transmembrance receptors that facilitate cell-extracellular matrix(ECM)adhesion,and integrins activate signal transduction pathways that mediate cellular signals such as regulation of cell cycle,organization of the intracellular cytoskeleton,and movement of new receptors to the cell membrance.Recently,integrin-like proteins have been found in many apicomplexa parasite such as Plasmodium and Toxoplasma,which mediate the adhension of parasites to host cells by interacting with heparin sulfate on the surface of host cells.And related studies have shown that heparin and heparan sulfate can effectively inhibit the adhesion of sporozoites to host cells.However,the research on integrin-like proteins of Cryptosporidium parvum has not yet been reported.In this study,the protein containing the integrin alpha domain of Cryptosporidium parvum was selected,and its subcellular localization and adhesion charcteristicswere studied.First,the sequence of integrin-like protein(CpITGA)of Cryptosporidium parvum was analyzed by bioinformatics.The recombinant proteins of CpITGA were expressed and purified based on the location of the integrin alpha domain.Polyclonal antibodies were obtained by immunizing rabbits with HIS-tag(47 – 263 aa)recombinant protein,followed by Western blot to verify the specificity of polyclonal antibodies.Subcellular localization of CpITGA was detected by indirect immunofluorescence.Total RNA from different stages of C.parvum were extracted and qRT-PCR was used to analyze the transcription level of CpITGA.To investigate whether the protein plays a role in the invasion of host cells,GST-tag recombinant protein(57 – 503 aa)was used to analyze the binding of GST-CpITGA to host cells by ELISA,indirect immunofluorescence and flow cytometry.In order to determine heparan sulfate on the surface of host cells are the major receptors for binding of CpITGA to host cells,the binding activities of GST-CpITGA to heparin was analyzed by pull-down assay.Finally,the blocking effect of anti-CpITGA polyclonal antibody on the invasion of C.parvum to HCT-8 cells was evaluated by qRT-PCR.Bioinformatics analysis indicated that CpITGA contains four integrin-like domains,an N-terminal signal peptide,a C-terminal transmembrane region,a short cytoplasmic tail region and three glycosaminoglycan binding sequence.qRT-PCR showed that CpITGA gene was transcribed at all stages of the parasite,and the highest transcription level was found at 12 h postinfection.Western blot results showed that anti-CpITGA polyclonal antibody can specifically recognize a 120 kDa native protein.IFA results indicated that CpITGA was located on the surface of the sporozoites and merozoites of C.parvum.GST-CpITGA could bind to HCT-8 cells in a dose-dependent and saturable manner.Also,GST-CpITGA could bind to heparin agarose and the binding could be inhibited by heparin,but not chondroitin sulfate A(CSA).Treatment of HCT-8 cells with heparinase ? reduced attachment by ~50%.In vitro neutralization assay,anti-CpITGA-HIS IgG inhibited the invasion by ~55%.In summary,CpITGA participates in the adhesion process of C.parvum by binding to heparan sulfate on the surface of host cells,and is expected to be a target for the prevention and treatment of cryptosporidiosis.