Characterization Of A Cryptosporidium Parvum-specific Insulinase-Like Protease INS20-19 And Preparation Of Antibodies For Subtyping

Cryptosporidium is a water-borne zoonotic protozoan,a significant cause of water-borne disease,and one of the essential microorganisms in water quality index in China.Genotypes or subtypes of Cryptosporidium differ in host range and have different routes of transmission.Thus,the genetic mechanisms of invasion and host adaptation need to be clarified.Results of a comparative genomic analysis indicated that two genes,cgd6₅510 and cgd6₅520,encoding Cryptosporidium parvum-specific insulinase-like proteases,are lost in many Cryptosporidium species,and could potentially contribute to the broad host range of C.parvum.The Ⅱa and Ⅱd subtype families of C.parvum have a worldwide distribution and can cause moderate to severe diarrhea in humans and various mammals.Therefore,a rapid subtyping method is required to assess the risk of disease transmission,for prevention and control of environmental pathogen.1)The sequencing results of the intergenic region indicated that cgd6₅510 and cgd6₅520 are fragments of a full gene ’cgd6₅520-5510’ encoding one insulinase-like protease ’INS20-19’ that is similar in structure to classic insulinases.Then,the relative expression level of the cgd6₅520-5510 gene over a 72-h time course in C.parvum-infected HCT-8 cells was assessed by qPCR.The specific fragment of INS20-19 was expressed in Escherichia coli for antiserum preparation.An indirect immunofluorescence assay was used to examine the expression of INS20-19,and an in vitro neutralization assay was used to evaluate the effect of the polyclonal antiserum on C.parvum infection.Results showed that the expression of the cgd6₅520-5510 gene was the highest at 2 h post-infection and declined thereafter.INS20-19 is likely a protein with high expression in the apical region of sporozoites,and neutralization of the protein led to a partial reduction of parasite load in HCT-8 and MDBK cell cultures.Taken together,our findings support the involvement of INS20-19 in the invasion or early developmental stages of C.parvum.2)The cgd6₅520-5510 sequence of Ⅱa subtype family obtained by PCR or TA cloning was aligned with the reference sequence,and the copy-specific primers were designed based on the nucleotide differences.Then,divergent copies of cgd6₅520-5510 were obtained from different subtype families by using these specific primers and aligned with other whole shotgun sequences.Results showed the presence of a second copy of the cgd6₅520-5510 gene in some C.parvum subtype families with a broad host range,and showed the sequence differences among them.Genetic recombination appeared to have played a role in generating divergent nucleotide sequences between copies and among subtype families.Recently,genetic recombination has been identified as a mechanism for generating host-adapted and virulent Cryptosporidium hominis and C.parvum subtype families.These data support the previous conclusion on the potential involvement of the insulinase-like protease encoded by the subtelomeric cgd6₅520-5510 gene in the broad host range of C.parvum Ⅱa and Ⅱd subtypes.3)Two gp60 proteins（highly homologous）of C.parvum Ⅱa and Ⅱd subtype families,which are commonly found in humans and animals,were expressed for antibody preparation.There are different cross-reactivities among the gp60 antigens and their polyclonal antibodies,suggesting these antigens have different epitopes.We obtained the rabbit polyclonal antibody and the mouse monoclonal antibodies that against gp60 of the IIa subtype family,as well as antibodies against gp60 of the Ⅱd subtype family.These antibodies can be used for coating or detection in C.parvum sandwich ELISA-based subtyping tools.Taken together,our findings support the involvement of INS20-19 in the invasion or early developmental process of C.parvum,and the potential involvement of this protein in the broad host range of C.parvum Ⅱa and Ⅱd subtypes.This study also provides clues for the development of therapeutics for cryptosporidiosis,and provides methods for the study of other insulinase-like proteases of Cryptosporidium.Furthermore,this study laid the foundation for the development of C.parvum sandwich ELISA-based subtyping tools.