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.

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 µM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.