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

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

Proteome profiling of the parasitic nematode Anisakis simplex s. s.

99 pagesAnisakis simplex is a parasitic nematode that causes anisakiasis in humans. Consumption of fish containing live A. simplex larvae may pose a health risk because of their ability to penetrate the gastrointestinal mucosa, as may foods contaminated with parasite allergens, which can cause severe allergic reactions in humans. The increasing popularity of eating raw fish and the rapid increase in the population of this nematode, as well as the expansion of its range, are leading to an increase in the number of cases of anisakiasis. So far, the biology of this parasite does not seem to be fully understood. Therefore, it seems useful to thoroughly study the proteome of this nematode, as it has not been fully characterized so far, which may shed light on the molecular mechanisms of the parasite's growth and development, as well as host-parasite interactions. The main objective of this study was to identify and characterize the proteome of the parasitic nematode A. simplex at global and tissue levels. As a result of the first experiment, the global proteomes of the two developmental stages, L3 and L4, of the A. simplex nematode were described. The most accurate and comprehensive proteome of this species was identified and characterized for the first time using ultra highperformance liquid chromatography coupled with high resolution tandem mass spectrometry (LC-MS/MS) and isobaric tandem mass tagging (TMT) of peptides for relative quantification of proteins. The second experiment sought to identify nematode proteins in the tissues responsible for host contact, i.e., intestine and cuticle. By using a similar proteomic workflow and complex analysis of proteomic data, comprehensive sets of proteins characteristic of the nematode tissues studied were identified. The results on the global proteome and the tissue proteome of A. simplex may serve as a basis for the development of new drugs against anisakiasis and, in the future, alleviate the symptoms of this disease or eliminate it in humans. Moreover, using quantitative TMT-based proteomics, the presented methodology can serve as a universal tool for the analysis of global proteome dynamics in the stages and tissues of parasitic nematodesN

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

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

Infective larvae of Anisakis simplex (Nematoda) accumulate trehalose and glycogen in response to starvation and temperature stress

Anisakis simplex L3 larvae infect fish and other seafood species such as squid or octopi; therefore, humans consuming raw or undercooked fish may become accidental hosts for this parasite. These larvae are induced to enter hypometabolism by cold temperatures. It is assumed that sugars (in particular trehalose and glycogen) are instrumental for survival under environmental stress conditions. To elucidate the mechanisms of environmental stress response in A. simplex, we observed the effects of starvation and temperature on trehalose and glycogen content, the activity of enzymes metabolizing those sugars, and the relative expression of genes of trehalose and glycogen metabolic pathways. The L3 of A. simplex synthesize trehalose both in low (0°C) and high temperatures (45°C). The highest content of glycogen was observed at 45°C at 36 h of incubation. On the second day of incubation, tissue content of trehalose depended on the activity of the enzymes: TPS was more active at 45°C, and TPP was more active at 0°C. The changes in TPP activity were consistent with the transcript level changes of the TPP gene, and the trehalose level, while glycogen synthesis correlates with the expression of glycogen synthase gene at 45°C; this suggests that the synthesis of trehalose is more essential. These results show that trehalose plays a key role in providing energy during the thermotolerance and starvation processes through the molecular and biochemical regulation of trehalose and glycogen metabolism

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

Osthole Inhibits Expression of Genes Associated with Toll-like Receptor 2 Signaling Pathway in an Organotypic 3D Skin Model of Human Epidermis with Atopic Dermatitis

The Toll-like receptor (TLR) family signature has been linked to the etiopathology of atopic dermatitis (AD), a chronic inflammatory skin disease associated with skin barrier dysfunction and immune system imbalance. We aimed to investigate whether osthole (a plant-derived compound) can inhibit the genetic profile of key genes associated with TLR2 signaling (TIRAP, MyD88, IRAK1, TRAF6, IκBα, NFκB) after stimulation with LPS or histamine in a 3D in vitro model of AD. Overexpression of the aforementioned genes may directly increase the secretion of proinflammatory cytokines (CKs) and chemokines (ChKs), which may exacerbate the symptoms of AD. Relative gene expressions were quantified by qPCR and secretion of CKs and ChKs was evaluated by ELISA assay. LPS and histamine increased the relative expression of genes related to the TLR2 pathway, and osthole successfully reduced it. In summary, our results show that osthole inhibits the expression of genes associated with the TLR signaling pathway in a skin model of AD. Moreover, the secretion of CKs and ChKs after treatment of AD with osthole in a 3D skin model in vitro suggests the potential of osthole as a novel compound for the treatment of AD

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

Peer reviewe

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