Expression And Functional Verification Of The MEDLE Family And Insulin-degrading Enzyme Family Member Proteins Of Cryptosporidium Parvum

Cryptosporidium spp.are important diarrhea-causing pathogens in humans and animals,mainly infecting host small intestine epithelial cells.Comparative genomic analysis indicated that Cryptosporidium-specific MEDLE and Insulin-degrading enzyme family proteins may contribute to host adaptation of Cryptosporidium spp.,and a recent study of one member of MEDLE family in Cryptosporidium parvum,CpMEDLE-2 encoded by cgd5₄590,has provided evidence supporting the potential involvement of MEDLE proteins in the Cryptosporidium invasion of host cells.However,the biological roles of other MEDLE family proteins and Insulin-degrading enzyme family proteins in Cryptosporidium invasion remain to be further studied.In this study,another member of MEDLE family,CpMEDLE-1 of C.parvum encoded by cgd5₄580,was cloned,expressed and characterized to understand its functions.The protein was expressed in Escherichia coli and polyclonal antibodies against the recombinant protein were prepared in rabbits.Quantitative real-time PCR was used to analyze the expression profile of target gene in C.parvum culture.Immunofluorescence staining was used to locate the protein expression in C.parvum lifecycle stages,and in vitro neutralization assay with antibodies was adopted to assess the role of the protein in C.parvum invasion.The results indicated that cgd5₄580 had a peak expression at 2 h of C.parvum culture.CpMEDLE-1 was located in the mid-anterior region of sporozoites,probably within the dense granules,and the neutralization efficiency of anti-CpMEDLE-1 antibodies was approximately 40%.These results indicated that CpMEDLE-1 may play an important role in the early development of C.parvum.The differences in protein and gene expression profiles between CpMEDLE-1 and CpMEDLE-2 suggest that MEDLE proteins have different locations,are developmentally regulated and may expert their functions in different stages of the growth process.In addition,a member of Insulindegrading enzyme family,INS-16 of C.parvum encoded by cgd3₄270,was expressed in vitro as well,while the expression was not ideal,with low expression level of recombinant INS-16 and multiple bands.Thus,future studies are still needed to optimize the expression condition of INS-16.The results above have laid the foundation for understanding the biological roles and molecular mechanism of Cryptosporidium-specific MEDLE and Insulin-degrading enzyme family proteins,which may facilitate the clarification of Cryptosporidium invasion mechanism,so as to provide a theoretical basis for formulating new immunotherapy strategies.