Proteases secreted by Trichinella spiralis intestinal infective larvae damage the junctions of the intestinal epithelial cell monolayer and mediate larval invasion

Abstract

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

Oral vaccination of mice with attenuated Salmonella encoding Trichinella spiralis calreticulin and serine protease 1.1 confers protective immunity in BALB/c mice

“…The ML was obtained from remaining ten mice from each group at 35 dpi by artificial digestion of infected murine skeletal muscles [ 25 , 51 ]. The immune protective effect induced by TsCRT+TsSP1.1, TsCRT and TsSP1.1 immunization was ascertained as the worm burden reduction of intestinal AWs and muscle larvae per gram (LPG) of muscle tissues from immunized mice compared to the PBS group [ 14 , 52 ]. Additionally, the female fecundity was also ascertained in immunized mice and control groups [ 53 ].…”

“…After being mated, the pregnant female adults produce the newborn larvae (NBL) which pass into blood circulation and migrate to skeletal muscles, where they encapsulate to complete the life cycle [12]. Gut mucosal epithelium is the first native physical barrier to defense the IIL larval invasion, and it is also a principal interaction site between intestinal nematode and the host [13,14]. Gut mucosal immune response is crucial for developing anti-Trichinella vaccines to block larval invasion of gut mucosa, to interrupt IIL development to the AW stage and to expel residual IIL and AW from the gut [15,16].…”

A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells

“…For further analysis of whether mannose could inhibit rTsCTL facilitative on larval invasion of IEC, 10 μg/mL of rTsCTL was first incubated with various doses (0–400 mM) of mannose for 2 h, and the mixture containing rTsCTL, mannose and IILs was added onto the cell monolayer. After culture at 5% CO 2 at 37 °C for 2 h, larval intrusion of IECs was examined under microscopy [ 63 , 64 ].…”

Characterization of a novel pyruvate kinase from Trichinella spiralis and its participation in sugar metabolism, larval molting and development

“…Therefore, ethyl pyruvate was also used as the enzyme inhibitor of TsPKM in the current study. Different concentrations of inhibitors (tannin, ethyl pyruvate) and common protease inhibitors 1,10-phenanthroline (1 mM), E64 (5 μM), EDTA (10 mM) and PMSF (1 mM) were added to the reaction system to determine the effects of different inhibitors on rTsPKM enzyme activity [ 57 ]. Under the optimum catalytic conditions of rTsPKM enzyme activity, the corresponding enzyme kinetic parameters were determined by detecting the reaction of ADP with various concentrations of PEP, and the reaction of PEP with different concentrations of ADP [ 28 ].…”