Characterization of a novel aspartyl protease inhibitor from Haemonchus contortus

Abstract

Background: Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied.

Results: A DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 10³. Results of Western blot showed that the recombinant API could be recognized by serum from goat infected with H. contortus. It was found that API was localized exclusively in the subcutaneous tissue and epithelial cells of the gastrointestinal tract in adult H. contortus. qRT-PCR suggested that the API gene was differentially transcribed in different life-cycle stages, with the lowest level in female adults and the highest in free-living L3 larvae. Enzyme inhibition assay indicated that the recombinant API can inhibit the activity of pepsin significantly, and the optimal reaction pH and temperature were 4.0 and 37-50 °C respectively. In vitro study showed that the recombinant API could induce goat PBMCs to express IFN-γ, IL-4 and IL-10.

Conclusions: A new aspartyl protease inhibitor was cloned from H. contortus and its characteristics were studied for the first time. The results indicate that API may regulate the immune response of the host and play roles in the infection.

Keywords: Aspartyl protease inhibitor; Differential expression; Haemonchus contortus; Induction of cytokines; Inhibitory activity; Localization.

Fig. 1

Alignment of H. contortus API with aspins from other nematodes. The amino acid sequence of H. contortus API is compared with those from C. elegans (AAC46663), T. colubriformis (AY189824) and O. ostertagi (CAD10783). The conserved cysteine residues and cleavage sites are marked by ■ and ★. “RDL” motif is indicated by ▲. Putative signal sequence is overlined

Fig. 2

Purification of recombinant API protein and western blot. Recombinant API from the supernatants (Lane 1) and the inclusion body (Lane 2) of the bacterial cells were purified and separated by SDS-PAGE. Recombinant API was detected by serum from goat infected with H. contortus (Lane 3), and normal goat serum as negative control (Lane 4). API extracted from H. contortus was detected by rat antibody against recombinant API protein (Lane 5), and normal rat serum as negative control (Lane 6). Lane M: pre-stained protein ladder

Fig. 3

Localization of API in adult worms. Cryostal sections of H. contortus were incubated with rat antibody against the recombinant API, following with the second antibody Cy3-labeled Goat Anti-Rat IgG. The red fluorescence was detected in both female (a, d) and male (b, e) adult H. contortus, but no fluorescence was observed in control experiments (c, f). To see the distributions of the API, vertical (a, b, c) and transverse sections (d, e, f) were checked. Images in lines 1 and 2 were taken under UV light and white field; merged images are given in line 3. Scale-bars: 100 μm

Fig. 4

Expression patterns of API in different life stages of H. contortus. Expression levels of API mRNA in egg, L3, xL3, adult male and female were tested by real time PCR. The relative quantities (RQ, compared with female adult worm, female = 1) in egg, L3, xL3 and adult male were 4.3, 43.8, 10.5 and 2.6, respectively. The value of API gene in the female was normalized to 1.0. The relative changes in gene expression ratios of selected genes were normalized to the expression of a single reference gene and calculated as described by the 2^-ΔΔCt method. Error bars indicate SEM from three independent experiments

Fig. 5

Inhibition assay of API on pepsin and trypsin. The products of hemoglobin (BSA) digested by pepsin (trypsin) with (or without) API were analysed by SDS-PAGE. Lane 1: hemoglobin; Lane 2: pepsin; Lane 3: API; Lane 4: pepsin + hemoglobin; Lane 5, pepsin incubated with API for 30 min (at 37 °C, pH 4.0) + hemoglobin; Lane 6: BSA; Lane 7: trypsin; Lane 8: trypsin + BSA; Lane 9: trypsin incubated with API for 30 min (at 37 °C, pH 4.0) + BSA

Fig. 6

Effects of pH value and temperature on the inhibition activities of recombinant API. Recombinant API was dissolved in the buffer with a serial pH value from 2 to 10 for 30 min (a), or treated at temperatures between 37 and 100 °C (b). The result API was added to the reaction with Pepsin and hemoglobin, and the relative inhibitory rate was tested by pepsin assay kit. The inhibition activity was defined as 100 when the reaction was carried out under pH 4.0 at 37 °C

Fig. 7

Levels of multiple cytokines stimulated by the recombinant API. Goat PBMCs were incubated with the recombinant API for 12 h, the mRNAs encoding IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β were quantified by real time PCR. Different letters (a, b and c) above the error bars indicate significant differences (P < 0.05)