Molecular characterization of zinc metalloproteinase Nas-14 from Trichinella spiralis and its participation in intestinal invasion

Astacins, a family of zinc metalloproteinases, are involved in invasion and tissue migration processes in a variety of parasites. An astacin-like proteinases have been detected in the excretory-secretory products (ESPs) of Trichinella spiralis (T. spiralis), zinc metalloproteinase Nas-14 (TsNas14), but its function in T. spiralis remains unclear. The primary objective of this research was to delineate the molecular characterization of TsNas14 and explore its potential to compromise the integrity of the intestinal barrier. Results showed that TsNas14 contains an Astacin domain and two ShK domains. It is highly conserved and has a consistent transcriptional expression pattern in the Trichinella genus. Quantitative results showed that the TsNas14 is transcribed and expressed during the whole life cycle, but that the expression level was highest in the adult worm (AW)stage. In the 3d AW stage, TsNas14 is mainly distributed on the stichosome, ovary, cuticle, and hypodermis, while in the 6d AW stage, it is only present on the cuticle. Gelatin zymography showed that the oligomerized rTsNas14 had the enzyme activity to degrade gelatin, and could be effectively inhibited by 1,10-Phenanthroline, indicating that it had the natural activity of metalloproteinases. In vitro experiments showed that rTsNas14 can down-regulate the expression of occludin and claudin-1 proteins of human colorectal adenocarcinoma (Caco-2) cells and improve the permeability of an intestinal barrier model. In addition, the direct incubation of rTsNas14 with claduin-1 showed that rTsNas14 could significantly degrade claudin-1. In vivo studies have demonstrated that inhibition of TsNas14 expression significantly impairs the infectivity of T. spiralis in mice, resulting in a decreased AW and muscle larval burden. These findings suggest that TsNas14 plays a crucial role in T. spiralis intestinal invasion and may serve as a novel potential target for Trichinella vaccines or therapeutic interventions.