Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases

Abstract

Background: Haemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates.

Methods: In silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex⁺) and non-treated (Dex^-) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex⁺. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions.

Results: Immunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex⁺ animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex⁺ and Dex^- animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86.

Conclusions: These results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.

Keywords: Cathepsin B; Cysteine proteases; Gastrointestinal; Haemonchus contortus; Peripheral blood mononuclear cells.

Fig. 1

In silico analysis for identifying H. contortus cathepsin B-like proteases, Hc-CBP-1 and Hc-CBP-2. H. contortus proteases were selected from NCBI and MEROPS databases and subjected to local BLASTp against clade V parasitic helminths

Fig. 2

Three-dimensional modeling of H. contortus cathepsin B-like proteases Hc-CBP-1 and Hc-CBP-2. The 3D models of Hc-CBP-1 and Hc-CBP-2 were generated using the x-ray crystallographic structure of mature cathepsin B from the genus Rattus using SWISS-MODEL. The catalytic triad residues Cys119 (a) and Cys112 (b) were in the left domain and His289 (a) and His293 (b) and Asn309 (a) and Asn312 (b) in the right domain. On the right panel, the surface-exposed loops specific to H. contortus are highlighted in green. The residues delimitating the loops—Leu73-Pro80, Asn182-Pro193, Arg221-Asp233 for Hc-CBP-1 and between Arg-74-Asp89, Tyr186-Pro194 and Cys221-Thr238 for Hc-CBP-2—are highlighted in green using Microsoft Paint (Microsoft Corporation, Redmond, WA)

Fig. 3

Detection of native Hc-CBP-1 and Hc-CBP-2 in adult Hc-ESP and crude worm extract. ELISA was used to detect Hc-CBP-1 and Hc-CBP-2 in Hc-ESP (a) and adult crude worm extract (b) using anti α-rHc-CBP-1 (a and c), α-rHc-CBP-2 (b and d) and pre-immune serum (1:200 dilution). Data are reported as the mean (M) of triplicates ± SEM captured at Absorbance of 410 nm (A₄₁₀). ****p ≤ 0.0001 indicates statistical significance between pre-immune (PI) and immune (I) serum samples. NS means not significant

Fig. 4

Immunolocalization of native Hc-CBP-1 and Hc-CBP-2 in H. contortus adults. Immunohistochemical staining of sectioned H. contortus adult worms was performed using rabbit anti-α-rHc-CBP-1 (a), α-rHc-CBP-2 (b) antibody and pre-immune serum (c) (1:800 dilution). CUT = cuticle, GUT = gut, REP = reproductive organs, HYP = hypodermis. Magnification = 10× (a); 20× (b, c). Red arrows indicate the positive binding of α-rHc-CBP-1 or α-rHc-CBP-2 antibody to the tissues

Fig. 5

Peptide arrays of Hc-CBP-1 and Hc-CBP-2 using homologous and heterologous antisera. Forty-six 15-amino-acid peptide with 8 amino acid overlaps among adjacent peptides were generated for Hc-CBP-1 (a) and Hc-CBP-2 (b). Antibody binding to each peptide was evaluated by ELISA at 410 nm using rabbit α-rHc-CBP-1 and α-rHc-CBP-2, and sheep H. contortus-infected serum. Percent changes between immune and pre-immune sera (× 100) were plotted. Presence of active site is indicated in brackets. Cys = cysteine, His = histidine, Asn = aspargine. The data are represented as means ± standard error (SE); values where p ≤ 0.05 were considered statistically significant

Fig. 6.

Transcript abundance of Hc-cbp-1 and Hc-cbp-2 in Dex⁺- and Dex⁻-treated H. contortus-infected sheep. Relative transcript abundance for Hc-cbp-1 (a) and Hc-cbp-2 (b) was determined using cDNA synthesized from adult H. contortus total RNA obtained from Dex⁺ and Dex⁻ (control) animals and Hc-cbp-1 and Hc-cbp-2 specific qPCR primers. The values were normalized to adult H. contortus from Dex⁻ animals and GAPDH was used as internal control. Data were analyzed using the ΔΔCt method where *p ≤ 0.05. Data are reported as mean (M) of three replicates ± SEM

Fig. 7

Peptide arrays of Hc-CBP-1 and Hc-CBP-2 using sera from Dex⁺ and Dex⁻ H. contortus-infected sheep. Hc-CBP-1 (a) or Hc-CBP-2 (b) specific peptides (1-46) were incubated with serum obtained from Dex⁺ and Dex⁻ animals. Percent change between drug treated and non-drug treated (×100) were plotted. The antibody binding percent signal intensities between animals were normalized by averaging the absorbance intensities of two animals/group using the formula: absorbance/mean absorbance of two animals × 100 ± standard error (SE); values where p ≤ 0.05 are considered statistically significant

Fig. 8

Activity of rHc-CBP-1 on bovine peripheral blood mononuclear cells. Relative transcript abundance of pro-inflammatory cytokines (TNFα (a), IL-1 (b), IL-6 (c)) and co-stimulatory markers (CD40 (d), CD80 (e) and CD86 (f)) were evaluated on bovine PBMCs following 72 h of incubation with 0.1, 1 and 10 μg/ml concentrations of rHc-CBP-1. Data are reported as the mean (M) of two biological and three technical replicates. Statistically significant differences between media controls vs samples, inhibitor vs mix of inhibitor and rHc-CBP-1 (0.1, 1 and 10 µg/ml) and rHc-CBP-1 (0.1, 1 and 10 µg/ml) vs mix of inhibitor and rHc-CBP-1 (0.1, 1 and 10 µg/ml) are defined as follows: (*), ( +), (#) = p ≤ 0.05; (**), (+ +), (##) = p ≤ 0.01; (***), (+ + +), (###) = p ≤ 0.001. The arrows indicate the direction of change in transcript abundance normalized to media only