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

34 pages, 2 tables, 1 figure.-- This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) licenseFoodborne parasitoses compared with bacterial and viral-caused diseases seem to be neglected, and their unrecognition is a serious issue. Parasitic diseases transmitted by food are currently becoming more common. Constantly changing eating habits, new culinary trends, and easier access to food make foodborne parasites’ transmission effortless, and the increase in the diagnosis of foodborne parasitic diseases in noted worldwide. This work presents the applications of numerous proteomic methods into the studies on foodborne parasites and their possible use in targeted diagnostics. Potential directions for the future are also providedThis work was funded by the “Development Program of the University of Warmia and Mazury in Olsztyn,” co-financed by the European Union under the European Social Fund from the Operational Program Knowledge Education Development. Robert Stryiński is a recipient of a scholarship from the program “Interdisciplinary Doctoral Studies in Biology and Biotechnology” (project number POWR.03.05.00-00-Z310/17), which is funded by the European Social Fund. This work was additionally co-funded by the GAIN-Xunta de Galicia (project number IN607D 2017/01) and the Spanish AEI/EU-FEDER PID2019-103845RB-C21 project. Mónica Carrera is supported by the Ramón y Cajal contract (Ministry of Science and Innovation of Spain)Peer reviewe

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

© 2020 by the authors.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.This work was funded by the GAIN-Xunta de Galicia, project number IN607D 2017/01. Mónica Carrera is supported by the Ramón y Cajal contract (Ministry of Science and Innovation of Spain).Peer reviewe

Shotgun Proteomics for L3 and L4 Anisakis simplex Development Stages

17 pages, 1 table, 5 figuresAnisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are present in many fish species and cephalopods all over the globe, might be consumed and develop occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and economic concern. In recent years, proteomic methods have gained greater acceptance among scientists involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)-based shotgun proteomics to define differences in proteomic composition between L3 and L4 development stages of A. simplex s.sThis work was supported by the “Development Program of the University of Warmia and Mazury in Olsztyn,” cofinanced by the European Union under the European Social Fund from the Operational Program Knowledge Education Development. R. S. is a recipient of a scholarship from the program Interdisciplinary Doctoral Studies in Biology and Biotechnology (POWR.03.05.00-00-Z310/17), which is funded by the European Social Fund. Part of this work was supported by “International scholarship exchange of doctoral students and academic staff (PROM)” financed by the polish National Agency for Academic Exchange (NAWA). The project was implemented by University of Warmia and Mazury in Olsztyn within the agreement with NAWA no. PPI/PRO/2019/1/00024/U/00001. The work was done by R. S. during the internship in Marine Research Institute in Vigo, Spain, and for that this work was also supported by the GAIN-Xunta de Galicia Project, Spain (IN607D 2017/01)N

The transcriptome of human intestinal epithelial cell line (CACO-2) exposed to the exosomes and whole larvae of Anisakis simplex s.s.

Póster.-- 15th International Congress of Parasitology (ICOPA 2022), August 21-26, CopenhagenAnisakis simplex is a parasitic nematode of marine organisms. Humans may be an accidental host for this species. The finding that parasitic nematodes can release exosomes was the breakthrough discovery. The secretion of exosomes as signaling molecules by parasitic nematodesHhas been poorly studied. This prompted us to characterize the transcriptome of host cells exposed to A. simplex exosomes as well as whole larvaeThis work was funded by National Science Centre of Poland, grant no. 2019/33/N/NZ6/01353. R. S. is also a recipient of a scholarship from the UE, grant no. POWR.03.05.00-00-Z310/17N

Transcriptomic differences in L3 and L4 stage larvae of Anisakis simplex s.s. from two different geographic populations - mRNA and long non-coding RNA expression patterns

Póster.-- 15th International Congress of Parasitology (ICOPA 2022), August 21-26, CopenhagenAnisakis simplex is a parasitic nematode of marine mammals. Humans can become infected by eating sea food contaminated with parasitic larvae. The ability of parasites to adapt to theenvironment or host is influenced by gene flow between populations and internal genetic drift. Therefore, estimating transcriptomic differences between populations is key to understanding the microevolutionary process in these taxaThis work was supported by National Science Centre of Poland, grant no. 2018/31/B/NZ9/01683N

Comparative proteomics analysis of Anisakis simplex s. s. – evaluation of the response of invasive larvae to ivermectin

Peer reviewe

Comparative proteomics analysis of Anisakis simplex s.s. - evaluation of the response of invasive larvae to ivermectin

Póster.-- 15th International Congress of Parasitology (ICOPA 2022), August 21-26, CopenhagenIvermectin (IVM) - an antiparasitic agent - has a beneficial effect on Anisakis simplex s. s. infection and is used for the treatment and prevention of anisakiasis in humans. However, the molecular mechanism of the action of IVM on A. simplex s. s. remains unknownThis work was funded by the GAIN-Xunta de Galicia, project number IN607D 2017/01. M. C. was supported by the Ramón y Cajal contract (Ministry of Science and Innovation of Spain)N

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

18 pages, 8 figures, 1 table.-- This is an open access article under the CC BY licenseHelminths are masters at manipulating host's immune response. Especially, parasitic nematodes have evolved strategies that allow them to evade, suppress, or modulate host's immune response to persist and spread in the host's organism. While the immunomodulatory effects of nematodes on their hosts are studied with a great commitment, very little is known about nematodes' own immune system, immune response to their pathogens, and interactions between parasites and bacteria in the host's organism. To illustrate the response of the parasitic nematode Anisakis simplex s.s. during simulated interaction with Escherichia coli, different concentrations of lipopolysaccharide (LPS) were used, and the proteomic analysis with isobaric mass tags for relative and absolute quantification (tandem mass tag–based LC–MS/MS) was performed. In addition, gene expression and biochemical analyses of selected markers of oxidative stress were determined. The results revealed 1148 proteins in a group of which 115 were identified as differentially regulated proteins, for example, peroxiredoxin, thioredoxin, and macrophage migration inhibitory factor. Gene Ontology annotation and Reactome pathway analysis indicated that metabolic pathways related to catalytic activity, oxidation–reduction processes, antioxidant activity, response to stress, and innate immune system were the most common, in which differentially regulated proteins were involved. Further biochemical analyses let us confirm that the LPS induced the oxidative stress response, which plays a key role in the innate immunity of parasitic nematodes. Our findings, to our knowledge, indicate for the first time, the complexity of the interaction of parasitic nematode, A. simplex s.s. with bacterial LPS, which mimics the coexistence of helminth and gut bacteria in the host. The simulation of this crosstalk led us to conclude that the obtained results could be hugely valuable in the integrated systems biology approach to describe a relationship between parasite, host, and its commensal bacteriaThis work was funded by the GAIN-Xunta de Galicia, project number IN607D 2017/01 and the Spanish AEI/EU-FEDER PID2019-103845RB-C21 project. Part of this work was supported by “International scholarship exchange of doctoral students and academic staff (PROM)” financed by the Polish National Agency for Academic Exchange (NAWA)Peer reviewe

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

29 pages, 9 figures, 2 tables.-- This is an open access article distributed under the Creative Commons Attribution LicenseAnisakis 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 anisakiasisThis work was supported by National Science Centre of Poland, grant no. 2018/31/B/NZ9/01683. Additionally, this work was supported by the “Development Program of the University of Warmia and Mazury in Olsztyn”, co-financed by the European Union under the European Social Fund from the Operational Program “Knowledge Education Development”. Robert Stryinski is a recipient of a scholarship from the program “Interdisciplinary Doctoral Studies in Biology and Biotechnology” (POWR.03.05.00-00-Z310/17), which is funded by the European Social Fund. The LC-MS/MS analysis part of this work was funded by the GAIN-Xunta de Galicia, project number IN607D 2017/01 and the Spanish AEI/EU-FEDER PID2019-103845RB-C21 projectPeer reviewe

Proteome profiling of L3 and L4 Anisakis simplex development stages by TMT-based quantitative proteomics

6th International Congress on Analytical Proteomics, Caparica, 8th-11th July 2019Anisakis simplex is a parasitic nematode, which is a threat to the health of people all over the world [1]. The consumption of raw or inadequately prepared fish containing A. simplex larvae, due to their ability to penetrate the mucous membrane of the gastrointestinal trac t, as well as severe human allergic reactions, can cause illness called anisakiosis [2, 3]. In this work, using TMT- based (tandem mass tags) quantitative proteomics the global proteome of L3 and L4 development stage of A. simplex was analyzed. The experiment was divided into four stages: (a) extraction of the L3 and L4 larvae proteins, (b) trypsin digestion assisted with high intensity focused ultrasound (HIFU), (c) TMT-isobaric mass tag labeling, and (d) global proteome analysis (LC-MS/MS) of L3 and L4 A. simplex development stages using a LTQ-Orbitrap Elite mass spectrometer [4, 5, 6]. In this study, we create a reference proteome dataset for each of the two development stages of A. simplex, L3 and L4. Total of 2443 different proteins were identified. Analysis of the most modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in the future eradicate anisakiosis. Demonstrated (TMT) -based quantitative proteomics approach could be a universal tool to analyze global proteomic dynamics in parasitic nematode stagesThis work was supported by the Ramón Areces Foundation (XVII National grant), and as part of the project "Development Program of the University of Warmia and Mazury in Olsztyn" no. POWR.03.05.00-00-Z310/ 17Peer reviewe