The cysteine protease ATG4B of Trichinella spiralis promotes larval invasion into the intestine of the host

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.

Figure 1

Analysis of the phylogenetic tree of TsATG4B. Phylogenetic relationship of the peptidase_C54 domain of nematodes and humans using the maximum parsimony method and drawn by MEGA. Bootstrap values of less than 70 are hidden in the branches. The sequence denoted by a solid circle is that of the TsATG4B protein expressed in this study.

Figure 2

Expression, purification and gelatinolytic activity of rTsATG4B. A Expression and purification of rTsATG4B. Lane M: standard marker; lanes 1/2, lysate of BL21 bacteria harbouring pQE-80L/TsATG4B without/with IPTG induction; lane 3: purified recombinant TsATG4B; B Proteolytic activity of refolded TsATG4B as detected by gelatine zymography. Lane M: protein molecular weight marker; lane 1: transparent strips due to the proteolytic activity of refolded TsATG4B in acrylamide-gelatine gels; lane 2: refolded rTsATG4B pre-incubated with the protease inhibitor E-64; C Identification of refolded rTsATG4B by Western blotting. Lane M: protein molecular weight marker; lane 1: refolded rTsATG4B recognized by anti-TsATG4B serum (1:100 dilution); lane 2: denatured rTsATG4B recognized by anti-TsATG4B serum (1:100 dilution).

Figure 3

Identification and expression of TsATG4B. A qPCR analysis of TsATG4B gene transcription levels at various stages of T. spiralis development. 18S rRNA was used as the housekeeping gene control. ***P < 0.001, compared to the level in ML. B Results of TsATG4B analysis by SDS-PAGE. Lane M: protein molecular weight marker; lane 1: somatic proteins of T. spiralis ML; lane 2: ES proteins of ML; lane 3: purified rTsATG4B protein product; C Analysis of rTsATG4B antigenicity by Western blotting. Lanes 1, 4, and 7: soluble proteins from ML; lanes 2, 5, and 8: ES proteins of ML; lanes 3, 6, and 9: purified rTsATG4B. Proteins were probed with infection serum (1:100 dilution, lanes 1, 2, and 3), anti-TsATG4B serum (1:100 dilution, lanes 4, 5, and 6) and normal mouse serum (1:100 dilution, lanes 7, 8, and 9). D Native TsATG4B in soluble proteins of different developmental stages of T. spiralis (lane 1: NBL; lane 2: ML; lane 3: IIL; lane 4: 3 days AW; lane 5: 6 days AW) was recognized by anti-TsATG4B serum.

Figure 4

Identification and expression of TsATG4B in the various developmental stages by immunolocalization. A–H Intact worms were recognized by anti-rTsATG4B mouse serum, as shown by IFA. Bright immunostaining was displayed on the surface of ML A, IIL B, 3 days female AW C, 3 days male AW D, 6 days female AW E, 6 days male AW F, and NBL G. ML H were probed with infection serum as a positive control. i–l Paraffin sections of Trichinella spiralis were recognized by anti-rTsATG4B mouse serum, as shown by IFA. Bright immunostaining was displayed on the cuticles and stichosomes of ML I and IIL J and in the embryos of 3 days female AW K. Paraffin sections of Trichinella spiralis ML L probed with infection serum were used as a positive control. Scale bars: 50 μm.

Figure 5

Immunofluorescence assay of rTsATG4B binding to IECs (200×). IECs and C2C12 cells were pre-incubated with rTsATG4B at 37 °C for 2 h. Then, they were probed with different antibodies (anti-TsATG4B polyclonal antibody, T. spiralis-infected mouse serum and pre-immune mouse serum) and the FITC-conjugated goat anti-mouse IgG secondary antibody and were then counterstained with propidium iodide (PI; the fluorescent dye PI is a nuclear staining reagent that can stain the nucleus through the disrupted cell membrane and emits red fluorescence after embedding in double-stranded DNA).

Figure 6

Interaction of TsATG4B with IECs by confocal microscopy and Far-Western blot analysis. A The subcellular localization of rTsATG4B bound to IECs was observed via confocal microscopy (1000×). IECs were pre-incubated with rTsATG4B for 2 h at 37 °C, and then the same procedure used in the immunofluorescence assay was performed. Images were acquired and analysed with an Olympus FV1200 laser scanning microscope. B Analysis of rTsATG4B binding to IEC proteins by Far-Western blotting. After the IEC lysates (lanes 1–3) and C2C12 lysates (lanes 4–6) were transferred to NC membranes, the NC membranes were incubated with rTsATG4B for 2 h at 37 °C. Then, the membranes were individually probed with anti-TsATG4B serum (lanes 1, 4), infection serum (lanes 2, 5) and pre-immune serum (lanes 3, 6). Binding between rTsATG4B and IEC lysates was detected with anti-TsATG4B serum (lane 1) and infection serum (lane 2).

Figure 7

The process of IIL invasion into the intestinal epithelium in vitro. AT. spiralis IIL were shown to invade the epithelial cell monolayer. Larvae penetrated IEC cells and migrated through them, leaving behind trails of dead cells and damaging the IEC monolayer. B Non-invaded larvae on the IEC monolayer. Nematodes that stayed coiled on the surface of the IEC monolayer or suspended in the medium were counted as non-invaded larvae. The IEC monolayer was intact. C Non-invaded larvae on the C2C12 cell monolayer. The larvae on the C2C12 monolayer were coiled. The C2C12 cell monolayer was not damaged. Scale bars: 100 μm.

Figure 8

Promotive effect of rTsATG4B protein and inhibitory effect of anti-rTsATG4B serum on larval invasion of the IEC monolayer in vitro. A Promotive effect of rTsATG4B on larval invasion. B The promotive effect on larval invasion was diminished when rTsATG4B was pre-incubated with E-64 (10 μM, 1:1); c The promotion rate of rTsATG4B protein on T. spiralis larval invasion of the IEC monolayer in vitro. Data were compared with the PBS group as the control. D The inhibition rate of anti-rTsATG4B serum showed a decreasing trend with increasing dilution and displayed a dose-dependent relationship with anti-rTsATG4B serum Data were compared with the PBS group as the control. *P < 0.01.