Aspartic protease 2 from Trichinella spiralis excretion/secretion products hydrolyzes tight junctions of intestinal epithelial cells

by Shao Rong Long, Hui Ran Zhang, Jing Jing Wang, Zi Xuan Liao, Qi Xue Fan, Yu Qing Liang, Cheng Yu Gan, Ruo Dan Liu, Jing Cui, Xi Zhang, Zhong Quan Wang, Xin Qi

Trichinella spiralis is a globally distributed foodborne parasitic nematode causing zoonotic infections. The invasion and subsequent development of intestinal infective larvae within the intestinal mucosa are critical steps in T. spiralis infection of the host. Previous studies have demonstrated that aspartic protease 2 from Trichinella spiralis excretion/secretion products (TsASP2) plays a role in facilitating the invasion of host intestinal epithelial cells by the larvae; however, the underlying mechanism remains unclear. Studies have suggested that dysfunction of the intestinal barrier creates a more favorable environment for intestinal helminths to invade intestinal epithelial cells. Here, the role of recombinant TsASP2 (rTsASP2) in disrupting the intestinal barrier by hydrolyzing tight junctions (TJs) was analyzed using both in vitro and in vivo experiments. A Lactococcus lactis- based heterologous protein delivery system was constructed to deliver TsASP2 to intestinal mucosa. The expression and mucosal colonization of rTsASP2 in L. lactis, the integrity of the intestinal epithelium, and the degradation of TJ proteins and hydrolysis sites of rTsASP2 were confirmed with Western blot, immunofluorescence, transepithelial electrical resistance (TEER) detection, paracellular permeability to FITC-dextran, transmission electron microscopy (TEM), dextran sulfate sodium (DSS)-induced colitis model, and High-Performance Liquid Chromatography Coupled to Time-of-Flight Mass Spectrometry (HPLC-TOF/MS). Results revealed TsASP2 delivery system using L. lactis was successfully construction and capable of colonizing the gut and delivering rTsASP2 to the intestinal mucosa. The rTsASP2 could directly degrade the TJ proteins Occludin and Claudin-1, thereby increasing paracellular permeability in the intestinal barrier and exacerbating DSS- induced colitis in mice. HPLC-TOF/MS analysis of the bands generated by rTsASP2- mediated hydrolysis of TJ protein specifically identified the cleavage sites as K-S, R-A, R-T, R-E and R-L. These results elucidated the mechanism by which TsASP2 facilitates T. spiralis invasion through the hydrolysis of tight junction proteins, providing critical experimental evidence for understanding host-parasite interactions.