Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

Background: Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. Methodology/principal findings: RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. Conclusions: rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion. Author summary: Trichinosis is a severe zoonotic disease that affects humans by eating raw or semi-raw meat products containing T. spiralis larvae. The intestinal mucosal barrier is the initial defense against T. spiralis infection and the main point of contact between the host and the parasite. Beyond the mechanical action, the protease released by the worm are essential for T. spiralis invasion of the host small intestine. Cathepsins play a crucial role in the migration of Fasciola hepatica and the invasion of Schistosoma japonicum, essential for their parasitism in the host. Nevertheless, the specific functions of T. spiralis cathepsin L during the worm's invasion of the intestinal mucosa remain unexplored. In this study, the TsCatL gene was silenced using siRNA, which inhibited worm invasion of Caco-2 cells, impaired worm development, and decreased female fertility. Incubation of rTsCatL2 with Caco-2 cells resulted in the translocation of cell tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin) into the cytoplasm or the nucleus, a reduction in claudin 1 expression and cellular autophagy, leading to disruption of the intestinal epithelial barrier. This study provides a novel target for developing anti-trichinosis vaccines and drugs. [ABSTRACT FROM AUTHOR]