The Study Of Key Cercarial Proteases Of Schistosoma Japonicum In Host Skin Invasion

Schistosomiasis is a parasitic zoonosis which caused by schistosomes and poses great threat to the health of human and other mammals. Schistosoma japonicum is the major pathogenic organism of schitosomiasis in China. Nowadays, due to the absence of vaccines, the treatment of schistosomiasis still depends almost exclusively on praziquantel, but studies have exhibited potential anti-schistosomal properties for this drug in the laboratory. Thus, it is urgent to explore new ways to prevent and control schitosomiasis.Schistosome cercariae take skin penetration as an initial step to successfully infect the definitive host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the process. In this study, we proceeded depth researches on the function of identified S. japonucum cecarial excretive proteases.[Methods] Firstly, the soluble extracted proteins and cecarial excretive proteases of S. japonucum were identified using LC/MS and those cercarial proeases associated with skin invasion were analyesed and screened. Then, we cloned, expressed and purified seven cercarial proteases of S. japonicum, including cathepsin B2 (SjCB2), elastase (SjCE2b), calpain (Sjcalpain), metallo endopeptidase (SjME), M17 family aminopeptidase (SjM17), mitochondrial metallo peptidase M16 (SjM16) and proteasome beta type 7 subunit (SjP7); The rabbit polyclonal antibodies were prepared with purified recombinant protein by immunizing New Zealand white rabbits. ELISA was utilized to detect titer of prepared rabbit antiserum. Immunolocalization was used to observe the distribution of SjCB2, SjCE2b and Sjcalpain in cercariae tissue; Soluble recombinant SjCB2 and SjCE2b proteins were expressed by wheat germ cell-free system (WGCF). Detection of the enzymatic activity of SjCB2 and SjCE2b were performed in soluble cercariae extractions, secretions and products of WGCF system; In addition, we further explored the function of SjCB2, SjCE2b and Sjcalpain with specific inhibitors in infected mice by S. japonicum.[Results] 685 proteins and 59 proteins were identified in soluble cercariae extractions and cercarial secretions by LC/MS, respectively; We successfully expressed SjCB2, SjCE2b, Sjcalpain catalytic site, Ca2+ binding site, SjME, SjM17, SjMl6 and SjP7 and relative molecular mass (Mr) are around 65 KDa, 31 KDa,43 KDa,39 KDa,50 KDa,60 KDa,51 KDa and 34 KDa respectively. Eight recombinant proteins with high purification were obtained via Ni-NTA resin and results of ELISA showed that the titer of prepared antiserum was higher than 1:80000. Immunolocalization demonstrated that SjCB1, SjCE2b and Sjcalpain mainly expressed in the head of cercariae. Besides, high enzyme activity of SjCB2 and SjCEZb were detected in cercariae extractions, secretions and productions of WGCF system; In inhibitor assys, after incubating with specific inhibitor of SjCB2, SjCE2b and Sjcalpain, the average worm reduction in mice was 48.0%,33.3% and 17.4%, respectively. [Conclusion]SjCB2, SjCE2b and Sjcalpain were abundant in cercariae and mainly expressed in the head of cercariae. Inhibitor assasy suggested that SjCB2, SjCE2b and Sjcalpain were involved in host skin invasion by S. japonicum.In summary, this study utilized enzymology, molecular biology and immunology to deeply investigate the function of main secreted proteins of S. japonicum cercariae. The results of this research may further reveal the key molecules and the molecular mechanism of the S. japonicum cercarial invasion, and may provide new clues for the development of vaccine and drug against schistosomes.