Abstract
<title>Abstract</title> <p> Trichinellosis is a globally prevalent zoonotic parasitic disease, and cysteine proteases (CPs) secreted by <italic>Trichinella spiralis</italic> are critical virulence effectors that facilitate larval invasion into the host intestinal epithelium. Our previous study identified autophagy-related protease 4B (ATG4B) as a CP potentially involved in larval invasion. However, its detailed enzymatic characteristics and precise invasive mechanism remain unclear. On this basis, the present study systematically characterized the biochemical properties, substrate hydrolysis spectrum, epithelial cytotoxicity, and intestinal tissue binding capacity of recombinant TsATG4B (rTsATG4B). Bioinformatics analysis confirmed that TsATG4B belongs to the C54 peptidase family with conserved catalytic sites Cys113 and His354. The optimal reaction conditions for rTsATG4B were pH 5.5 and 37℃, with <italic>Km</italic> = 60.06 nM and Vmax = 2.391 nM/min. rTsATG4B effectively degraded IgG, hemoglobin and type I collagen, and its enzymatic activity was markedly suppressed by CP-specific inhibitor E-64. <italic>In vitro</italic> immunofluorescence and cell injury assays showed that rTsATG4B time-dependently destroyed tight junction proteins (E‑cadherin, claudin‑1, occludin) of HT29 epithelial cells and triggered severe morphological damage to primary mouse intestinal epithelial cells. Indirect immunofluorescence test <italic>in vivo</italic> further verified that native TsATG4B specifically bound to intestinal epithelial tissues and aggravated mucosal erosion and villous edema during larval infection. Collectively, these results demonstrate that TsATG4B acts as a functional C54 cysteine protease that promotes <italic>T. spiralis</italic> larval invasion by degrading host structural and immune molecules and disrupting intestinal epithelial barriers. This study elucidates the pathogenic function of TsATG4B and provides experimental evidence for developing novel vaccines or targeted protease inhibitors against trichinellosis. </p>