Molecular characterization of a novel cathepsin L from Trichinella spiralis and its participation in invasion, development and reproduction.

Cathepsin L is one member of cysteine protease superfamily and widely distributed in parasitic organisms, it plays the important roles in worm invasion, migration, nutrient intake, molting and immune evasion. The objective of this study was to investigate the biological characteristics of a novel cathepsin L from Trichinella spiralis (TsCL) and its role in larval invasion, development and reproduction. TsCL has a functional domain of C1 peptidase, which belongs to cathepsin L family. The complete TsCL sequence was cloned and expressed in Escherichia coli BL21. The rTsCL has good immunogenicity. RT-PCR and Western blotting analysis showed that TsCL was transcribed and expressed at different T. spiralis phases (e.g., muscle larvae, intestinal infectious larvae, adult worms and newborn larvae). Immunofluorescence test revealed that TsCL was principally localized in the cuticle, stichosome, midgut and female intrauterine embryos of the nematode. rTsCL has the capacity to specially bind with intestinal epithelial cells (IECs) and the binding sites was located in the cytoplasm. rTsCL promoted larval penetration into IEC, while anti-rTsCL antibodies inhibited the invasion. The silencing of TsCL gene by specific dsRNA significantly reduced the TsCL expression and enzyme activity, and also reduced larval invasive ability, development and female reproduction. The results showed that TsCL is an obligatory protease in T. spiralis lifecycle. TsCL participates in worm invasion, development and reproduction, and may be regarded as a potential candidate vaccine/drug target against T. spiralis infection.

Proteomic analysis of hydrolytic proteases in excretory/secretory proteins from Trichinella spiralis intestinal infective larvae using zymography combined with shotgun LC-MS/MS approach.

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.