Characterization of a novel invasive virulence of chymotrypsin-like elastase family member 2A from Trichinella spiralis.

A novel chymotrypsin-like elastase family member 2A (TsCELA2A) was identified in Trichinella spiralis intestinal infectious larvae (IIL) excretion/secretion protein (ESP), but its properties and function are unclear. The objective of this study was to identify TsCELA2A's biological characteristics and functions at the process of T. spiralis invasion of enteral epithelium. The results revealed that TsCELA2A has a Tryp_SPc conserved functional domain at 30-273aa, and contains three active sites and three substrate binding sites. rTsCELA2A was expressed and purified. On Western blotting, rTsCELA2A was recognized by anti-rTsCELA2A serum and Trichinella-infected murine serum, and native TsCELA2A in crude proteins of diverse stage worms was recognized by anti-TsCELA2A serum. The results of RT-PCR, Western blotting and immunofluorescence test (IFT) showed that TsCELA2A was expressed at diverse worm stages, and principally located in external cuticle, stichosome and intrauterine embryos of the nematode. rTsCELA2A had enzymatic activity of natural serine protease, which could hydrolyze the substrate N-benzoyl-L-arginine-ethylester (BAEE). rTsCELA2A was specifically combined with enteral epithelium cells, and binding location was primarily located in cellular membrane and cytoplasm. rTsCELA2A mediated larva intrusion of gut epithelium; whereas anti-TsCELA2A antibodies obviously impeded the invasion. Silencing of the TsCELA2A gene significantly decreased the expression and activity of TsCELA2A, and the larval invasion and intestinal adult burdens were reduced respectively by 28.71 and 35.78%. The results indicated that TsCELA2A was an invasive virulence factor and participated in T. spiralis larval intrusion of enteral mucosa, and it might be a candidate vaccine molecule against T. spiralis infection.