Trichinella spiralis serine protease mediates larval invasion of gut epithelium via binding to CK8 and activating RhoA/ROCK1 pathway.

BACKGROUND: Trichinella spiralis adult worms and larvae parasitize respectively in small intestine and skeletal muscles of the same host, and larval invasion of gut mucosa is the pivotal step for T. spiralis infection. A T. spiralis serine proteinase (TsSPc) was identified in its intestinal infective larva (IIL) ES antigens. TsSPc is involved in larval invasion of gut epithelium, but the mechanism is not completely elucidated. The purpose of this study was to investigate the mechanism of TsSPc action in larval invasion of gut mucosa. METHODOLOGY/PRINCIPAL FINDING: The results of molecular docking, immunofluorescence assay (IFA), GST pull-down and co-immunoprecipitation (Co-IP) showed that rTsSPc specifically bound to cytokeratin 8 (CK8) receptor in Caco-2 cells and activated RhoA/ROCK1 signaling pathway, as demonstrated by the evidently increased expression levels of CK8, RhoA and ROCK1. The results of qPCR and Western blot analysis revealed that binding of rTsSPc with CK8 and activation of RhoA/ROCK1 pathway significantly decreased the expression levels of gut epithelial tight junctions (TJs, E-cad, Occludin and Claudin-1), and increased the paracellular permeability. Knocking down CK8 in Caco-2 cells and ROCK1 pathway inhibitor Y27632 obviously inhibited the activation of RhoA/ROCK1 pathway, abolished rTsSPc-decreased TJs expression, rTsSPc-increased paracellular permeability, and inhibited larval invasion of Caco-2 monolayers in vitro. When the mice were pretreated with CK8 inhibitor Dasatinib and ROCK1 pathway inhibitor Y27632, and then orally infected with T. spiralis larvae, the activation of CK8 and RhoA/ROCK1 was significantly suppressed, intestinal permeability and adult worm burdens were obviously decreased, and intestinal inflammation was also distinctly alleviated. The number of intestinal goblet cells, expression of mucins (Muc2 and Muc5ac) and inflammatory cytokines (IL-1β, TNF-α, IL-10 and TGF-β) were significantly reduced. CONCLUSIONS: TsSPc binding to CK8 receptor in gut epithelium activated RhoA/ROCK1 pathway, reduced TJs expression and disrupted gut epithelial integrity, therefore mediated larval invasion of host gut mucosa. TsSPc might be considered as a promising vaccine molecular target for intercepting T. spiralis invasion and infection.