Characterization of a serine protease inhibitor from Trichinella spiralis and its participation in larval invasion of host's intestinal epithelial cells

Abstract

Background: Trichinella spiralis serine protease inhibitor (TsSPI) was identified in ES proteins of adult worms (AW), the TsSPI gene was highly expressed at enteral stage worms (AW and newborn larvae), distributed mainly in the cuticle and stichosome of this nematode. Vaccination of mice with rTsSPI exhibited a 62.2% reduction of intestinal AW and a 57.25% reduction of muscle larvae after larval challenge. The aim of this study was to investigate the biological characteristics of TsSPI and its roles in the process of T. spiralis invasion of host's intestinal epithelium cells (IECs).

Methods: The rTsSPI inhibition on trypsin enzymatic activity was detected by SDS-PAGE and spectrophotometry. The binding of rTsSPI with intestinal epithelium from normal mice and the primary cultured mouse intestinal epithelium cells (IECs) was examined by indirect immunofluorescent (IIF), the cellular localization of rTsSPI binding to IECs was observed by confocal microscopy. The inhibition of anti-rTsSPI serum on T. spiralis invasion of IECs was determined by an in vitro invasion assay. Anti-rTsSPI antibody cytotoxicity on the newborn larvae (NBL) was also determined.

Results: The rTsSPI had the inhibitory activity against porcine trypsin. The rTsSPI specifically bound to the intestinal epithelium from normal mice and primary cultured mouse IECs, and the binding sites were located in IEC membrane and cytoplasm. Anti-rTsSPI antibodies depressed the larval invasion of IECs with a dose-dependent mode. Anti-rTsSPI antibodies also participated in the destruction of T. spiralis NBL via an ADCC-mediated manner.

Conclusions: TsSPI might participate in the T. spiralis larval invasion of IECs and it is likely the potential vaccine target against T. spiralis enteral stages.

Keywords: Inhibitory activity; Larval invasion; Serine protease inhibitor; Trichinella spiralis; Trypsin.

Fig. 1

SDS-PAGE analysis of rTsSPI activity for inhibiting trypsin hydrolysis of BSA. a Different quantity of BSA were hydrolyzed by trypsin. Lane M: protein marker; Lane 1: trypsin; Lane 2: BSA; Lanes 3–8: trypsin + BSA (0.75, 1.50, 2.25, 3.00, 3.75 and 4.50 μg, respectively). b Different quantity of rTsSPI were hydrolyzed by trypsin. Lane M: protein marker; Lane 1: trypsin; Lane 2: rTsSPI; Lanes 3–8: trypsin + rTsSPI (3.00, 3.75, 4.50, 5.25, 6.00 and 6.75 μg, respectively). c The trypsin hydrolysis of BSA was inhibited by different quantity of rTsSPI. Lane M: protein marker; Lane 1: trypsin; Lane 2: rTsSPI; Lane 3: BSA; Lanes 4–9: trypsin + BSA + rTsSPI (3.00, 3.75, 4.50, 5.25, 6.00 and 6.75 μg, respectively). d The trypsin hydrolysis of BSA was inhibited by rTsSPI and natural inhibitor PMSF. Lane M: protein marker; Lane 1: TsSPI; Lane 2: BSA; Lane 3: trypsin; Lane 4: trypsin + rTsSPI; Lane 5: trypsin+BSA; Lane 6: rTsSPI+trypsin+BSA; Lane 7: PMSF+trypsin+BSA

Fig. 2

The titration curve graph of trypsin activity inhibited by rTsSPI. rTsSPI of different quantity was added to a fixed quantity of trypsin (1.25 μg). The absorbance at 253 nm was measured by spectrophotometry with the substrate BAEE, and the inhibition rate of trypsin enzymatic activity was calculated as described in method section. Each point is the mean of triplicates

Fig. 3

Stability of rTsSPI activity for inhibiting trypsin after incubation at 37 °C for 30 min. a Stability of rTsSPI inhibiting activity at different temperature. b Stability of rTsSPI inhibiting activity at different pH. The residual enzymatic activity of trypsin was determined with BAEE as a substrate. The experiment was performed in triplicate and the data are the mean ± SD of three tests

Fig. 4

Far Western analysis of rTsSPI binding to IEC proteins. The IEC proteins were analyzed by SDS-PAGE, subsequently the IEC protein binding with rTsSPI was detected in a Far Western analysis. a SDS-PAGE analysis of IEC proteins. Lane M: protein marker; Lane 1: IEC lysates; Lane 2: C2C12 lysates. b Far-Western analysis of IEC protein binding to rTsSPI. The IEC protein was first incubated using rTsSPI (Lanes 1–3), IL1 ES proteins (Lanes 4–6) or BSA (Lanes 7–9), subsequently recognized by anti-rTsSPI serum (Lanes 1, 4 and 7), infection serum (Lanes 2, 5 and 8), and pre-immune normal serum (Lanes 3, 6 and 9). c Far Western analysis of C2C12 protein binding to rTsSPI. The C2C12 protein (Lanes 1–3) was first incubated with rTsSPI, and subsequently incubated with anti-rTsSPI serum (Lane 1), infection serum (Lane 2) or pre-immune serum (Lane 3). There was no binding between rTsSPI and the C2C12 protein

Fig. 5

IIF analysis of binding between rTsSPI and IECs (× 200). rTsSPI, IL1 ES antigens or PBS were used for pre-incubating with IEC for 2 h at 37 °C. rTsSPI was also pre-incubated with C2C12 for 2 h at 37 °C. After washes, the pre-incubated IEC and C2C12 was probed by anti-rTsSPI serum, infection serum or pre-immune serum, subsequently colored with goat anti-mouse IgG-FITC conjugate. Propidium iodide (PI) dyed cell nuclei in red

Fig. 6

Cellular localization of rTsSPI binding to IEC by confocal microscopy (× 1000). The IEC was pre-incubated by rTsSPI, and then by anti-rTsSPI serum, infection serum or pre-immune serum, and stained using anti-mouse IgG-FITC conjugate. Propidium iodide (PI) dyed cell nuclei in red. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide

Fig. 7

IIF analysis of rTsSPI binding with mouse intestinal epithelium (100×). Tissue sections of the intestines (a-c) and livers (e-f) from uninfected mice were incubated with rTsSPI for 2 h at 37 °C. After washing, the sections were probed for 1 h at 37 °C with anti-rTsSPI serum (a, d), infection serum (b, e) or pre-immune serum (c, f), and then with anti-mouse IgG-FITC conjugate. Propidium iodide (PI) dyed cell nuclei in red. These sections were examined by fluorescent microscopy

Fig. 8

The in vitro inhibition of T. spiralis invasion of IEC by anti-rTsSPI serum. a When IEC monolayer was covered and cultured with the semisolid media containing the larvae at 37 °C for 2 h, the invaded (left) and non-invaded larvae (right) in the IEC monolayer (× 200). b Inhibition of larval invasion of IEC by different dilutions of anti-rTsSPI serum. The 1:100 dilutions of infection serum (IS) and pre-immune serum (PI) were utilized as control sera. The results are shown as the percent of the larvae invaded the monolayer out of all larvae added into the media. Asterisks indicate statistically significant differences (P < 0.001) in comparison with the pre-immune serum group

Fig. 9

ADCC killing T. spiralis NBL. a-f Morphology of T. spiralis NBL recovered after ADCC test with different cultivation time periods. The NBL were cultured using anti-rTsSPI serum and 1 × 10⁵ mouse peritoneal exudate cells (PECs) at 37 °C for different time periods. a, b No PECs were adhered to NBL 12 h and 24 h of cultivation and the NBL was wriggling. c A few of PECs adhering to NBL 48 h of cultivation and the NBL was limp with weak activity. d A dead NBL overlaid with PECs 72 h of cultivation. Infection serum (e) and pre-immune serum (f) were utilized as control sera. g-h ADCC killing T. spiralis NBL is anti-rTsSPI antibody dose-dependent and associated with culture time. g The NBL were incubated with same dilution (1:100) of various sera for 12–72 h of incubation; the cytotoxicity had an increasing trend with the culture time prolongation. h The NBL were incubated with different sera diluted at 1:50 to 1:800 for 72 h; the cytotoxicity exhibited an anti-rTsSPI antibody dose-dependent pattern. Asterisks indicate that the cytotoxicity of the anti-rTsSPI serum exhibited a significant difference (P = 0.005) compared with that of the pre-immune serum