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Abstract
The cysteine proteases of parasites are vital contributors that induce parasite migration to and invasion of host tissue. In this study, we analysed the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) isolated from the soluble proteins of Trichinella spiralis (T. spiralis) adult worms to ascertain its biochemical properties and functions during invasion into the intestine of the host. The 43 kDa recombinant cysteine protease ATG4B protein (rTsATG4B) consists of a conserved peptidase_C54 domain and was expressed in Escherichia coli. Gelatine zymography showed that rTsATG4B could hydrolyse gelatine and that the hydrolytic activity was prevented by the cysteine protease inhibitor E-64 (pH 5.2). Immunofluorescence assays showed that TsATG4B is expressed at different stages and is localized at the cuticles and stichosomes of worms. Far-Western blotting and confocal microscopy revealed that rTsATG4B interacts with intestinal epithelial cells (IECs) and that it was subcellularly localized to the membrane and cytoplasm in IECs. Real‑time quantitative PCR (qPCR) results indicated that the transcription level of the TsATG4B gene was the higher in 6-day-old adult worms (6 days AW) than in any other stage. An in vitro larval invasion assay verified that rTsATG4B promoted larval invasion and that invasion was inhibited when rTsATG4B was pre-incubated with E-64, whereas anti-rTsATG4B serum inhibited larval invasion in a dose-dependent manner. Collectively, these results suggested that the enzymatic activity of TsATG4B significantly influences the hydrolysis process, which is necessary for larval invasion of the host intestinal epithelium.
Highlights
Trichinellosis is a global food-borne parasitic zoonosis that remains an emerging disease that has been threatening public health and affecting economic growth [1,2,3]
The 6 days adult worms (AW) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium for 24 h, and newborn larvae (NBL) were harvested from the culture
Hasnain et al [39] revealed that proteolytic activity in T. muris ESPs was necessary for degrading host intestinal tissues. This group indicated that cysteine proteases exist in ESPs and disrupt the network of polymeric mucin. Based on these preliminary studies, this project was instigated to study the role played by the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) in larval invasion of the intestine of the host, and we found promising results in invasion assays
Summary
Trichinellosis is a global food-borne parasitic zoonosis that remains an emerging disease that has been threatening public health and affecting economic growth [1,2,3]. Infection is initiated when uncooked or raw animal meat that has been contaminated with Trichinella spiralis (T. spiralis) is consumed [4, 5]. After exposure to gastric juice in the gastrointestinal environment, T. spiralis muscle larvae (ML) are released from the capsules in the stomach. The worms grow by relying on intestinal contents, and they develop into intestinal infective larvae (IIL) in the intestine. IIL invade the epithelium of the small intestine, where they undergo 4 moults before developing into adult worms (AW), and they mate and produce newborn larvae (NBL). NBL travel through the blood and lymph from the intestine to striated muscle, where they develop into L1 stage larvae in muscle cells [6, 7]
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