A novel secretory poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

Abstract

Background: Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite-host interactions.

Methodology/principal findings: In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1:2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite.

Conclusions/significance: Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future.

Figure 1. Purification of native Ts-PCHTP with anion exchange chromatography followed by size exclusion chromatography.

(A) and Ni-NTA agarose purification (B). 1) markers; 2) soluble fraction after ultracentrifugation; 3) supernatant after 70% saturation with ammonium sulfate; 4) unbound fraction of anion exchange chromatography on DEAE-cellulose; 5) Ts-PCHTP after size exclusion chromatography on Superdex 75; 6) Ts-PCHTP after Ni-NTA chromatography. Proteins were visualized by Coomassie Blue staining.

Figure 2. Organization of ts-pchtp gene and protein chain.

A) Comparison between Ts-PCHTP cDNA and whole genome shotgun sequence, ABIR01001777 (start 65194–end 67089) from Trichinella spiralis showed 6 exons and 5 introns of the 1896 bp gene. B) Open reading frame of translated protein sequence contains signal peptide (exon 1), two homology poly-cysteine domains (exon 2–4 and exon 5–6) and histidine tail on C-term end (exon 6). The 2 potential glycosylation sites are shown. C) Comparison between poly-cystein domain 1 and 2. Data showed 38.5% identity at 174 amino acid. Conservative cysteine residues are in bold.

Figure 3. Comparison of the Ts-PCHTP pcd domains with Caenorhabditis elegans proteins and other nematode ESTs sequences.

Bordered groups pcd-1_T.sp and pcd-2_T.sp contains pcd domains of the Ts-PCHTP and related ESTs (Table S1); bordered group R01H10.4 contains ESTs pt39d02.y1 (Trichinella spiralis) and HTAB-aae58a04.b1 (Heterorhabditis bacteriophora); and bordered group T19C3.2 (YSV2_CAEEL) contains EST ph72c07.y1 (Ostertagia ostertagi) and hypothetical protein CBG15264 (C. briggsae). Homologous amino acids residues among groups are in grey boxes. Amino acids residues homologous for all proteins are shown with white letters.

Figure 4. Deglycosylation native Ts-PCHTP with N-Glycosidase F.

1) eluted protein fraction after Ni-NTA chromatography; 2) deglycosylation with N-Glycosidase F; 5) marker. Proteins were visualized by Coomassie Blue staining.

Figure 5. Total Reflection X-ray Fluorescence (TXRF) of the native Ts-PCHTP.

1 M of the native Ts-PCHTP bind about 1.5 M Fe; 2 M Zn and 2 M Ni.

Figure 6. Immunofluorescent localization of Ts-PCHTP in T. spiralis larvae.

(A) The intense staining of the encapsuled larva (L) after reaction with polyclonal serum. Nc - nurse cell, M - host muscle. (B) Labeling of the Ts-PCHTP in encapsuled larva (L) with anti-histidine antibody. Scale bar: A, B - 60 µm.

Figure 7. Immunogold electron microscopic localization of Ts-PCHTP in T. spiralis.

(A) Strong accumulation of gold particles in the cuticle (C), hypodermis (H) and muscle (M) layers, revealed by polyclonal serum. (B) Control sample. Scale bar: A, B - 3 µm.