Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi

Abstract

BackgroundBaylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines.MethodsIn this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera.ResultsBsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice.ConclusionThis study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.

Identification of a glutathione S-transferase gene of Physarum polycephalum as a biomarker for nanosized TiO2 exposure under dark conditions

“…An NCBI Blast search indicated that PpGST possessed a 'GST_N_Sigma_like domain' containing binding sites for glutathione (GSH) and a 'GST_C_like' domain containing a substrate-binding site. It has been reported that cytosolic GSTs contain thioredoxin-like domains with the topology bababba (Xie et al 2015). This secondary structure topology was also located in the PpGST identified in this study.…”

“…The secondary structure of the PpGST consisted of a bababba motif in the N terminus (ranging from residues 5 to 84) and a nearly complete a helix motif (ranging from residues 91 to 204) in the C-terminus. These domain characteristics are highly similar to typical GST features (Xie et al 2015), suggesting that a responsive GST gene from P. polycephalum for nTiO 2 exposure was obtained.…”

“…These domain characteristics are highly similar to typical GST features (Xie et al . ), suggesting that a responsive GST gene from P. polycephalum for nTiO 2 exposure was obtained.…”

“…It has been reported that cytosolic GSTs contain thioredoxin‐like domains with the topology βαβαββα (Xie et al . ). This secondary structure topology was also located in the PpGST identified in this study.…”

Parasites of the Giant Panda: A Risk Factor in the Conservation of a Species

“…For instance, an antibody detection enzyme-linked immunosorbent assay (ELISA) employing a B. schroederi glutathione S-transferase antigen was established for the detection anti-B. schroederi serum antibody (IgG) in experimentally infected mice, with a sensitivity of 79.1% and a specificity of 82.0% (Xie et al, 2015a). However, such an assay has not yet been assessed for the diagnosis of baylisascariasis or B. schroederi infection in giant pandas.…”