Abstract
In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.
Highlights
Trichinella spiralis is an important foodborne nematode that parasitizes over 150 kinds of mammals worldwide [1]
Our results indicated that rTsP immunization elicited mixed Th1/Th2 responses according to the level of specific IgG subclasses and cytokines, suggesting that subcutaneous immunization with rTsP induced both systemic and local enteral mucosal (MLN) cellular responses
In the present study, the complete sequence of the T. spiralis peptidase (TsP) gene was cloned, and the TsP gene was expressed in a prokaryotic expression system
Summary
Trichinella spiralis is an important foodborne nematode that parasitizes over 150 kinds of mammals worldwide [1]. After infected meat is eaten, T. spiralis ML in muscle tissues are released from their collagen capsules with the help of gastric fluid digestion and activated to intestinal infective larvae (IIL) following exposure to the intestinal contents/bile [9]. These IIL larvae invade the intestinal mucosal columnar epithelium and develop to the adult worm (AW) stage after four moults. The intestinal epithelium is the primary native defence against Trichinella invasion and the principal site of interaction between IIL and the host [11, 12]. Characterization of molecules involved in Trichinella invasion will assist in elucidating the mechanism by which T. spiralis and its host interact and developing vaccines to inhibit Trichinella infection in animals [13, 14]
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