Comparative Proteomics Analysis of Anisakis simplex s.s.—Evaluation of the Response of Invasive Larvae to Ivermectin

Abstract

Ivermectin (IVM), an antiparasitic drug, has a positive effect against Anisakis simplex s.s. infection and has been used for the treatment and prevention of anisakiasis in humans. However, the molecular mechanism of action of IVM on A. simplex s.s. remains unknown. Herein, tandem mass tag (TMT) labeling and extensive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the effect of IVM on the proteome of A. simplex s.s. in vitro. During the study, 3433 proteins, of which 1247 had at least two protein unique peptides, were identified. Comparative proteomics analysis revealed that 59 proteins were differentially regulated (DRPs) in IVM-treated larvae, of which 14 proteins were upregulated and 38 were downregulated after 12 h of culture, but after 24 h, 12 proteins were upregulated and 22 were downregulated. The transcription level of five randomly selected DRPs was determined by real-time PCR as a supplement to the proteomic data. The functional enrichment analysis showed that most of the DRPs were involved in oxidoreductase activity, immunogenicity, protein degradation, and other biological processes. This study has, for the first time, provided comprehensive proteomics data on A. simplex s.s. response to IVM and might deliver new insight into the molecular mechanism by which IVM acts on invasive larvae of A. simplex s.s.

Quantitative Proteomics Comparison of Total Expressed Proteomes of Anisakis simplex Sensu Stricto, A. pegreffii, and Their Hybrid Genotype

“…Global proteomics analysis in anisakids nematodes is a very promising discipline [ 32 , 47 ] from clinical to diagnostic tools. There are approximately 20,000 genes in nematodes, however it is estimated that there are hundreds of thousands of proteins isoforms, most of them modified at postranscriptional stage.…”

“…Statistical comparison of proteomes is an efficient selection method of biomarkers in what is termed in proteomics “discovery phase” [ 26 , 27 ] Methodological developments in proteomics are applied to research in different topics relating to parasites [ 28 , 29 , 30 , 31 ]; however, few are devoted to taxonomic discrimination at a species or population level. The taxonomy of Anisakis is very stable and a wide range of intraspecific or genetic differences have been demonstrated [ 10 ] for which proteomic analysis can be applied for diagnostic purposes [ 32 ]. Limited results have been known on expressed proteome differences regarding any species of this important parasitic nematode genus.…”

Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe

“…Recently, for the first time, the global proteome of the third and fourth stage larvae of A. simplex was analyzed using quantitative proteomics based on tandem mass tag (TMT) [ 31 ]. In addition, the response to the invasive larvae of A. simplex s.s. to ivermectin (anthelminthic drug) was also evaluated using TMT-based methodology [ 250 ].…”

A Complex Proteomic Response of the Parasitic Nematode Anisakis simplex s.s. to Escherichia coli Lipopolysaccharide

“…The actin was used as the endogenous reference gene (44). The reaction was performed as described before (Polak et al 2020). In brief, the total RNA of the larvae was isolated using a Total RNA Mini Kit (A & A Biotechnology, Gdynia, Poland) and cDNA was obtained using TranScriba Kit (A & A Biotechnology, Gdynia, Poland) according to the manufacturer's protocols.…”