Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella

Abstract

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results suggested that EtPDIL might be involved in sporulation in external environments and in host cell adhesion, invasion and development of E. tenella.

Figure 1. Full cDNA and deduced amino acid sequence of EtPDIL.

Underlined, start and stop codons; double underlined, CAAT box in the 5′-UTR; gray, non-classical thioredoxin-like motif (SXXC); wavy underlined, N-myristoylation site.

Figure 2. Multiple alignment analysis of EtPDIL of Eimera tenella with PDIL from other apicomplexan parasites.

Shown are sequences from Plasmodium cynomolgi (XP_004221713), Plasmodium vivax (XP_001614725), Eimeria mitis (CDJ34317.1). Deduced protein sequences were used in the Clustal W sequence alignment program. Asterisks, identical amino acids.

Figure 3. Quantitative real-time RT-PCR of EtPDIL expression in E. tenella developmental stages.

UO, unsporulated oocysts; SO, sporulated oocysts; Spz, sporozoites; Mrz, merozoites. Bars not sharing the same letters were significantly different (P<0.05).

Figure 4. Expression of recombinant EtPDIL in Escherichia coli by SDS-PAGE.

Lane 1, protein marker; lane 2, IPTG-induced recombinant EtPDIL protein at 0 h; lane 3, IPTG-induced recombinant EtPDIL protein at 6 h; lane 4, purified recombinant EtPDIL.

Figure 5. Western blots.

(A) Purified recombinant EtPDIL (diaminobenzidine as substrate). Rabbit sera against sporulated oocysts of E. tenella or anti-GST monoclonal antibody was used as primary antibody. Lane 1, protein marker. Lane 2, anti-GST monoclonal. Lane 3, antisporulated-oocysts serum. Lane 4, naive rabbit serum. (B) Protein lysates from four different life cycle stages of E. tenella. Rabbit sera against rEtPDIL or mouse monoclonal anti-α-tubulin was used as primary antibody. Spz, sporozoites; SO, sporulated oocysts; UO, unsporulated oocysts; Mrz, merozoites.

Figure 6. Localization of EtPDIL in different stages of E. tenella by indirect immuofluorescence using rEtPDIL antibody.

(A) Sporozoites (Spz) in PBS; (B) Spz in complete medium. Infected DF-1 cells were collected at indicated hours post infection (p.i.). (C) 2 h p.i., intracellular sporozoites (iSpz); (D) 12 h p.i., intracellular sporozoites (iSpz); (E) 24 h p.i., trophozoites (Tropho); (F) 48 h p.i., immature schizonts (iSc); (G) 60 h p.i., immature schizonts (iSc); (H) 68 h p.i., immature schizonts (iSc); (I) 72 h p.i., mature schizonts (mSc); (J) 85 h p.i., intracellular merozoites (iMrz); (K) Merozoites (Mrz) in PBS.

Figure 7. Inhibition of sporozoite invasion in vitro by antibody against rEtPDIL.

Anti- rEtPDIL, rabbit antiserum against recombinant EtPDIL protein; NA, naive rabbit serum. All assays were performed in triplicate. ** P<0.01 for differences between treatment with antibody against rEtPDIL and naïve rabbit serum at the same IgG concentration.