The Structures And Functions Of Phenoloxidase Genes In Schistosoma Japonicum

Backgrounds Schistosomiasis impairs the public sanitation, agriculture and animal husbandry. A female adult S. japonicum can produce about2,200eggs daily on average. Most schistosome eggs deposit in the tissues of terminal hosts. The miracidium in eggs excrete soluble egg antigen (SEA) to initiate the host immun responses, subsequently, egg granuloma and fibrosis form. Some eggs released into water with the host feces are incubated into miracidium, which can infect the intermediate host, is the initial step of a new circle. So eggs play an important role in the transmission of schistosomiasis. Theoretically, inhibition of egg production or prevention of egg maturation will contribute to the prevention and controll of schistosomiasis. To date, there are still no effective vaccines against S. japonicum, and currently, the prophylaxis and cure for schistosomiasis is dependent on a chemical drug pyquiton, which can bring with side effects and drug resistance for schistosomes. It’s urgent to develop a new method for treating schistosomiasis. Phenol oxidases (POs) play an important role in the cross-linking process of helminth eggshell protein. POs are copper containing enzymes, which can the catalyze ortho-hydroxylation of monophenols to o-diphenols (momophenol oxidase activity, MPO, EC1.14.18.1), and the oxidation of o-diphenols to o-quinones (diphenol oxidase activity, DPO, EC1.10.3.1). Schistosome genes encode two forms of phenoloxidases. The molecular characteristics of Schistosoma mansoni phenoloxidases were describe by Fitzpatrick (2007). Cai and coworkers had reported the molecular characteristics and expression of two S. japonicum phenol oxidases (SjPO1and SjP02) in2009. But the genomic structures of two SjPO genes had never been reported before, and it remains elusive that whether there are other forms of POs in S. japonicum. The exact functions of SjPO1and SjP02in eggshell formation also need to be clarified.Object The mRNA sequences of both SjPO1and SjP02have been published, and the polypeptides encoded by them have38%identity, consequently, we supposed they have the same function of playing a role in eggshell hardening. It’s essential to deeply study the molecular characteristic of SjPO1and SjP02, their expression at different developmental stages of S. japonicum, as well as their specific tissue localization, and whether they can induce anti-reproduction in females. This study also revealed the functions of SjPOs at genetic level, especially the mechanism of their roles in eggshell hardening.Methods In this study, SjPO1/SjPO2genomic DNA fragments were amplified by PCR according to their mRNA sequences, then the DNA fragments were sequenced and their structures were analyzed; Semi quantitative RT-PCR was used to determine SjPO1/SjPO2gene expression in S. japonicum at different developmental stages; In situ hybridization (ISH) and immunohistochemistry were used to detect the localization of SjPO1/SjPO2in adult worms and eggs; Recombinant SjPO1protein was prepared for immunoprotection; Small interfering (si) RNAs were used to knock down SjPO1/SjPO2genes in S. japonicum adult females, and the effects of knockdown of single SjPO gene and of both SjPO genes were compared; Lentiviral (LV) vectors were used to introduce transgenes into S. japonicum adults, subsequently, siRNAs were produced for silencing SjPO1gene in the transduced cells of worms; The knockdown efficiency of siRNA and LV vector was compared by evaluating the effects on SjPO transcript levels and PO enzyme activities.Results SjPO1and SjPO2might derive from the common ancestor gene, and their genomic structures are composed of3extrons and2introns; Neither of SjTYR genes are transcribed in cercaria, but with the maturation of worms in terminal hosts, their transcript levels increase gradually; SjPO1gene has a more abundant expression level than SjPO2, and the transcript levels of them in females are higher than in males. Both SjPO1and SjPO2are expressed in the vitelline glands of the female adults, tegument of male adults and miracidium in eggs. The recombinant SjPO1protein don’t affect the worm survival, but had some effects on egg decrease, which is21.67%in egg reduction rate; Knockdown of SjPO1/SjPO2gene expression can cause significant reduction in PO activities and lead females to produce irregular eggs including spindle eggs and invaginated eggshells adhereing to each other. When the DPO activity was reduced about more than45%, the autofluorescence of eggshells weakened obviously, meanwhile the backgrounds of female uteri got fluorescenct. Knockdown of both SjPO genes was of better effects than single SjPO gene. The knockdown efficiency of LV vector was higher than siRNA.Conclusions Both SjPO1and SjPO2play an important role in the eggshell hardening of S. japonicum, and the role of SjPO1is major. This study further demonstrate the functions of the SjPO1and SjPO2, and provide a theoretical reference for further studies on anti-reproduction of S. japonicum females.