Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis

Abstract

Anisakis simplex s. s. is a parasitic nematode of marine mammals and causative agent of anisakiasis in humans. The cuticle and intestine of the larvae are the tissues most responsible for direct and indirect contact, respectively, of the parasite with the host. At the L4 larval stage, tissues, such as the cuticle and intestine, are fully developed and functional, in contrast to the L3 stage. As such, this work provides for the first time the tissue-specific proteome of A. simplex s. s. larvae in the L4 stage. Statistical analysis (FC ≥ 2; p-value ≤ 0.01) showed that 107 proteins were differentially regulated (DRPs) between the cuticle and the rest of the larval body. In the comparison between the intestine and the rest of the larval body at the L4 stage, 123 proteins were identified as DRPs. Comparison of the individual tissues examined revealed a total of 272 DRPs, with 133 proteins more abundant in the cuticle and 139 proteins more abundant in the intestine. Detailed functional analysis of the identified proteins was performed using bioinformatics tools. Glycolysis and the tricarboxylic acid cycle were the most enriched metabolic pathways by cuticular and intestinal proteins, respectively, in the L4 stage of A. simplex s. s. The presence of two proteins, folliculin (FLCN) and oxoglutarate dehydrogenase (OGDH), was confirmed by Western blot, and their tertiary structure was predicted and compared with other species. In addition, host–pathogen interactions were identified, and potential new allergens were predicted. The result of this manuscript shows the largest number of protein identifications to our knowledge using proteomics tools for different tissues of L4 larvae of A. simplex s. s. The identified tissue-specific proteins could serve as targets for new drugs against anisakiasis.

Metabolomic analysis reveals a differential adaptation process of the larval stages of Anisakis simplex to the host environment

“…Combining a genetically defensible model organism such as C. elegans with the functional evaluation of metabolomics and/or lipidomics holds great promise for expanding our knowledge of metabolism and metabolic regulation ( Salzer and Witting, 2021 ). While there are numerous published studies on the genomes ( Mattiucci et al, 2016 ; Coghlan et al, 2019 ; Łopieńska-Biernat et al, 2019 ), transcriptomes ( Cavallero et al, 2018 ; Kim et al, 2018 ; Llorens et al, 2018 ; Łopieńska-Biernat et al, 2018 ; Cavallero et al, 2020 ), and proteomes ( Stryiński et al, 2019 ; Stryiński et al, 2022 ; Polak et al, 2020 ; Mierzejewski et al, 2021 ; Kochanowski et al, 2022 ) of A. simplex , less attention has been paid to its metabolome. Metabolic changes between L3 and L4 larval stages of A. simplex have been studied previously, however in a narrow range, e.g., of one metabolic pathway ( Łopieńska-Biernat et al, 2008 ; Łopieńska-Biernat et al, 2018 ; Łopieńska-Biernat et al, 2019 ), and almost nothing is known about the complement of an intermediate or end product of metabolism in Anisakis nematodes, especially in the stages found in humans (L3 and L4).…”

Re‐evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1–3

“…This was followed by mapping of allergens in the Anisakis proteome using the nano LC‐ESI‐MS/MS technique (Fæste et al., 2014 ), quantification of A. simplex proteins in food matrix by LC‐MS/MS (Fæste et al., 2016 ), fast monitoring of parasites in fish products using parallel reaction monitoring (PRM) MS (Carrera et al., 2016 ) and quantification of proteomes of A. simplex s.s., A. pegreffii and their hybrids using iTRAQ labelling and subsequent nano LC‐ESI‐MS/MS (Arcos et al., 2020 ). Shotgun sequence identification of L3 and L4 proteins based on isobaric mass labelling (Tandem Mass Tag, TMT) (Stryiński et al., 2019 , 2022 ) was then undertaken, followed by the construction of a spectral library of Anisakis spp. using MALDI‐TOF MS supported by LC‐ESI‐MS/MS (Marzano et al., 2020 ).…”