Identification and profiling of Trichinella spiralis circulating antigens and proteins in sera of mice with trichinellosis

Abstract

Trichinellosis is a zoonotic disease caused by the ingestion of the Trichinella nematode. With a worldwide incidence of approximately 10,000 cases per year, Trichinella spiralis is responsible for most human infections. There are no specific signs or symptoms of this parasitic infection. Muscle biopsy is the gold diagnostic standard for trichinellosis, but the technique is invasive and unable to detect the early stage of infection. Although immunodiagnostics are also available, antibody detection usually occurs after 3 weeks and prolonged up to 19 years after the acute phase. Therefore, additional diagnostic biomarkers must be identified to improve trichinellosis diagnosis. This study aimed to measure concentration changes in mouse serum proteins prior to T. spiralis infection and 2, 4 and 8 weeks after infection, and to identify T. spiralis circulating proteins and antigens using mass spectrometry-based proteomics. Mouse muscle-related proteins including inter-alpha-trypsin inhibitor heavy chain H2, a protein involved in the response to muscle tissue damage, were up-regulated in mouse sera during the T. spiralis larvae invasion. Additionally, 33 circulatory parasite proteins were identified in infected mouse sera. Notably, T. spiralis long-chain fatty acid transport protein 1 could be detected in the early stage of infection and peroxidasin-like protein was identified 2, 4 and 8 weeks after infection. Seventeen T. spiralis circulating antigens were detected in mouse immune complexes, with PX domain protein being found 2, 4 and 8 weeks after infection. Because peroxidasin-like protein and PX domain protein were detected at all post-infection time points, sequence alignments of these proteins were performed, which showed they are conserved among Trichinella spp. and have less similarity to the human and murine sequences. Integrative analysis of T. spiralis biomarkers throughout the course of infection may reveal additional diagnostic targets to improve early diagnosis of trichinellosis.

Fig 1. SDS-PAGE of T. spiralis uninfected and infected mouse sera.

Lanes (from left to right): marker, uninfected, 2, 4 and 8 weeks post infection. The 11 horizontal sections show the regions excised for MS analyses.

Fig 2

Volcano plots comparing the expression of mouse proteins (blue dots) in mouse sera infected with T. spiralis 2 (a), 4 (b) and 8 weeks (c) post infection with those in uninfected sera. Statistically significant differential expression of proteins (red dots) is defined as a minimum 2-fold change relative to the uninfected condition (level of magnitude, vertical lines) and p < 0.05 (level of statistical significance, horizontal line).

Fig 3. Protein–protein interactions of up-regulated mouse serum proteins 8 weeks post T. spiralis infection.

Proteins identified by mass spectrometry were analyzed for their interactions using the STRING database. Red nodes represent proteins in the blood coagulation pathway which was predicted as the significant altered network after T. spiralis infection. The abbreviations for each protein are F2: Prothrombin, F2r: Thrombin receptor, Thbd: Thrombomodulin, Serpinc1: Serine/cysteine peptidase inhibitor clade c member 1, Serpind1: Serine/cysteine peptidase inhibitor clade d member 1, Gp1ba: Platelet glycoprotein.

Fig 4. SDS-PAGE of T. spiralis circulatory antigens from immune complex in T. spiralis uninfected and infected mouse sera.

The immune complexes in T. spiralis uninfected and infected mouse serum were enriched using protein A/G magnetic bead. and further separated by 12% gel electrophoresis. Lanes (from left to right): marker, uninfected, 2, 4 and 8 weeks post infection. The 11 horizontal sections show the regions excised for MS analyses.