Characterisation of a niche-specific excretory-secretory peroxiredoxin from the parasitic nematode Teladorsagia circumcincta

Abstract

Background: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae).

Results: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H₂O₂) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells.

Conclusions: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.

Keywords: Anti-oxidant; Excretory–secretory; Niche; Peroxidase; Peroxiredoxin.

Fig. 1

2D gels of excretory–secretory (ES) proteins of fourth-stage T. circumcincta larvae. ES proteins (23 µg of each) were separated by 2D gel electrophoresis and stained with SimplyBlue^TM SafeStain (Invitrogen). Protein spots upregulated in either T. circumcincta L4 mucosal-dwelling ES proteins or T. circumcincta L4 lumen-dwelling ES proteins are numbered and their protein identifications are shown in Table 1. The two lower panels show 3-dimensional images generated from the areas outlined in the dashed boxes on the gels with the relevant spots circled

Fig. 2

Stage specific expression of Tci-Prx1. a qPCR gene expression analysis of Tci-Prx1 mRNA in T. circumcincta stage 3 larvae (L3); sodium hypochlorite exsheathed T. circumcincta L3 (xL3 NaOCl); carbon dioxide exsheathed T. circumcincta L3 (xL3); fourth-stage T. circumcincta larvae (L4); and adult T. circumcincta worms (Adult). b qPCR gene expression analysis of Tci-Prx1 mRNA in T. circumcincta L4 lumen-dwelling (Lumen) worms and T. circumcincta L4 mucosal-dwelling (Mucosal) worms. Each value is the mean ± SEM, n = 3. There were no significant differences between lumen and mucosal expression levels (t-test, t = 0.8106, df = 2, P = 0.724). All data were normalized to Tci-β-tubulin and shown relative to either L3 expression (a) or Lumen expression (b)

Fig. 3

Phylogenetic analysis of Tci-Prx1 and related peroxiredoxins. Neighbour joining phylogenetic reconstruction of Tci-Prx1 (red) and selected AhpC-Prx1 subfamily (typical 2-cys) peroxiredoxins from helminths and vertebrate species (as indicated in figure). The tree is rooted using alkyl hydroperoxide reductase C (AhPC) from Escherichia coli. The reliability of the tree was determined using 1000 bootstrap replications, bootstrap values > 50% are shown. All sequences are available from NCBI using the accession numbers in the phylogenetic tree. Where protein isoforms exist, the longest isoform is included in the alignment. The scale-bar represents 0.06 substitutions per amino acid site. Peroxiredoxins with a signal peptide are indicated (*)

Fig. 4

Immune recognition of rTci-Prx1 using sera from T. circumcincta trickle-infected sheep. Purified recombinant Tci-Prx1 and other recombinant T. circumcincta ES proteins including Tci-MEP-1 and mut Tci-APY-1 (500 ng of each) were resolved by SDS-PAGE and proteins transferred to nitrocellulose membranes. Blots were probed with pooled sera from 7 helminth-naïve six-month-old sheep at either day 0 (prior to T. circumcincta trickle infection); or day 112 (post T. circumcincta trickle infection). Blots shown in a were probed with anti-goat/sheep IgG-HRP secondary antibodies. Blots shown in b were probed with anti-sheep IgA-HRP secondary antibodies. Specifically bound antibodies were visualised using DAB staining. Arrows indicate presence of either Tci-MEP-1 (57 kDa) or mut Tci-APY-1 (38 kDa)

Fig. 5

Biochemical analysis of wild-type and mutant Tci-Prx1. a SDS-PAGE analysis of purified wild-type (wt) and mutant (mut) Tci-Prx1 (5 µg of each protein/lane) under reducing and non-reducing conditions. Presence of monomer (1-mer, 23.3 kDa) and dimer (2-mer, 46.6 kDa) is indicated. b Peroxidase activity of wt and mut Tci-Prx1. Each reaction contained 40 µM of H₂O₂ and either wt Tci-Prx1 or mut Tci-Prx1, and diminishment of peroxide was monitored by FOX assay. Each value is the mean ± SEM, n = 5. Significant differences between wt Tci-Prx1 and mut Tci-Prx1 are shown (t-test, t = 6.768, df = 4, P < 0.001)

Fig. 6

In vitro stimulation of macrophages with recombinant Tci-Prx1. qPCR gene expression analysis of markers of M1 activation (IL-6, iNOS, TNF-α) and M2 activation (arginase and MRC-1) in RAW 264.7 cells following stimulation with IL4 (20 ng/ml), IFN-γ (100 ng/ml), wt Tci-Prx1 (20 µg/ml) and mut Tci-Prx1 (20 µg/ml). Unstimulated negative control cells (-ve) were run in parallel and incubated with culture media only. All data were normalized to β-actin and shown relative to media only (-ve control) expression levels