Stefin of Eudiplozoon nipponicum (Monogenea): immunomodulator or houskeeping protein?
|Year of publication||2017|
|MU Faculty or unit|
|Description||Hematophagous fish parasite Eudiplozoon nipponicum (Monogenea: Diplozoidae) produces cysteine peptidase inhibitor (stefin) which has been detected in excretory-secretory products of the adult worms and thus might play a role in host-parasite interaction. Our study aims to reveal the biological function of the stefin of E. nipponicum (EnStef). Inhibitors of cysteine peptidases are synthetized by wide range of parasitic species. Besides regulation of endogenous processes in parasite bodies they play a substantial role e.g. in manipulation of the host immune system and/or blood digestion. We prepared recombinant form of EnStef (rEnStef) in E. coli BL21 host strain using pET19b expression plasmid vector. By adoption of fluorometric assay we observed efficient inhibition of cysteine peptidases (cathepsins L and B from E. nipponicum and mouse cathepsin L) via its conserved papain-binding domain as well as inhibition of asparaginyl endopeptidase (legumain) probably due to legumain-binding domain, untypical for stefins. rEnStef blocked proteolytic degradation of hemoglobin mediated by cysteine peptidases in the excretory-secretory products, soluble protein extracts from E. nipponicum and by recombinant cathepsins L3 and B of E. nipponicum, which manifests its role in blood meal digestion. In order to assess the immunomodulatory potential of EnStef we tested its effect on activation of complement in carp’s plasma and oxidative burst in full blood using luminol-enhanced chemiluminescence. We performed series of experiments with stimulated porcine alveolar macrophages and rEnStef to examine its effect on cytokine production (IL-1beta, TNF-alfa, IL-6 and IL-10). The immunohistochemistry techniques (using specific rabbit rEnStef antibodies) and in situ RNA hybridization (using DIG-labelled RNA probes) were adopted for localization of EnStef on paraffin sections of E. nipponicum.|