Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, PR China.
PLoS Negl Trop Dis. 2023 Dec 4;17(12):e0011816. doi: 10.1371/journal.pntd.0011816. eCollection 2023 Dec.
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.
rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.
组织蛋白酶 L 是一种溶酶体酶，参与多种生理过程。重组旋毛虫组织蛋白酶 L 结构域（rTsCatL2）表现出天然半胱氨酸蛋白酶活性，并在体外水解宿主免疫球蛋白和细胞外基质蛋白，但它在幼虫入侵中的功能尚不清楚。本研究旨在探讨其在旋毛虫幼虫入侵宿主肠道上皮细胞中的作用。
方法/主要发现：用 TsCatL 特异性 siRNA-302 显著抑制了 TsCatL mRNA 和蛋白的表达。当使用抗 TsCatL2 血清和 siRNA-302 时，旋毛虫幼虫对 Caco-2 细胞的入侵分别减少了 39.87%和 38.36%。与 PBS 组相比，用 siRNA-302 处理的肌肉幼虫（ML）感染的小鼠肠道感染性幼虫、成虫和 ML 负荷显著减少，分别减少了 44.37%、47.57%和 57.06%。从感染 siRNA-302 处理的 ML 的小鼠中分离出的雌性的发育和繁殖能力受到显著抑制。在用 rTsCatL2 孵育 Caco-2 细胞后，免疫荧光试验显示 rTsCatL2 逐渐进入细胞，改变了细胞紧密连接蛋白（claudin 1、occludin 和 zo-1）、黏附连接蛋白（e-cadherin）和细胞外基质蛋白（laminin）的定位，细胞间连接丢失。Western blot 显示 rTsCatL2 处理的 Caco-2 细胞中 claudin 1 的表达减少了 58.65%。Co-IP 显示 rTsCatL2 与层粘连蛋白和胶原蛋白 I 相互作用，但与 Claudin 1、E-cadherin、occludin 和纤维连接蛋白不相互作用。此外，rTsCatL2 通过诱导细胞自噬破坏肠道上皮屏障。
rTsCatL2 破坏肠道上皮屏障，促进旋毛虫幼虫入侵。
