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.

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

Helminths 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 bacteria.

Shotgun Proteomics for L3 and L4 Anisakis simplex Development Stages.

Anisakis 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.s.

Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release.

BACKGROUND: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince. RESULTS: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases. CONCLUSION: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry.

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

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes' biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.

Proteomic and Bioinformatic Investigations of Heat-Treated Anisakis simplex Third-Stage Larvae.

Anisakis simplex third-stage larvae are the main source of hidden allergens in marine fish products. Some Anisakis allergens are thermostable and, even highly processed, could cause hypersensitivity reactions. However, Anisakis proteome has not been studied under autoclaving conditions of 121 °C for 60 min, which is an important process in the food industry. The aim of the study was the identification and characterization of allergens, potential allergens, and other proteins of heat-treated A. simplex larvae. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify 470 proteins, including allergens-Ani s 1, Ani s 2, Ani s 3, Ani s 4, Ani s 5-and 13 potential allergens that were mainly homologs of Anisakis spp., Ascaris spp., and Acari allergens. Ani s 2, Ani s 3, Ani s 5, and three possible allergens were found among the top 25 most abundant proteins. The computational analysis allowed us to detect allergen epitopes, assign protein families, and domains as well as to annotate the localization of proteins. The predicted 3D models of proteins revealed similarities between potential allergens and homologous allergens. Despite the partial degradation of heated A. simplex antigens, their immunoreactivity with anti-A. simplex IgG antibodies was confirmed using a Western blot. In conclusion, identified epitopes of allergenic peptides highlighted that the occurrence of Anisakis proteins in thermally processed fish products could be a potential allergic hazard. Further studies are necessary to confirm the IgE immunoreactivity and thermostability of identified proteins.

Effect of freezing on the metabolic status of L3 larvae of Anisakis simplex s. s.

The fish-borne parasite, Anisakis simplex s. s., triggers a disease called anisakiasis, that is associated with a gastrointestinal infection. The Anisakis is also associated with allergic response which may lead to anaphylactic shock. The A. simplex s. s. L3 larvae may be freeze tolerant despite when the nematodes will be cooled rapidly to -20 °C according to the sanitary authorities of the USA and the EU. The aim of this work was to study the metabolic status of A. simplex s. s. L3 larvae when frozen in terms of viability, expression of genes involved in the nematodes' survival of freezing, as well content of carbohydrates which play a cryoprotective role in thermal stress and are the main source of energy. The levels of trehalose were significantly higher after slow freezing treatment (p < .0001), than the fast freezing (p < .002). The lower temperatures induce changes, especially in trehalose synthesis gene expression, genes responsible for oxidative metabolism, and chaperone proteins, but we cannot state clearly whether these changes occur during freezing, or because they are already prevalent during cold acclimation. The induction of mentioned genes seems to be a common trait of both cold- and dehydration tolerance.

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

Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the 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. SIGNIFICANCE: A. simplex is a fish-borne parasite responsible for the human anisakiosis and allergic reactions around the world. The work describes for the first-time the comparison of the proteome of two A. simplex stages (L3 and L4). The strategy is based on four steps: (i) protein extraction, (ii) ultra-fast trypsin digestion under High-Intensity Focused Ultrasound (HIFU), (iii) TMT-isobaric mass tag labeling followed by high-pH reversed-phase fractionation and (iv) peptide analysis using a LTQ-Orbitrap Elite mass spectrometer. The workflow allows to select the most modulated proteins as proteomic signature of those specific development stages (L3 and L4) of A. simplex. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in future eradicate the anisakiosis disease.

Respiratory analysis as a tool to detect physiological changes in Anisakis larvae subjected to stress.

Human infection due to eating fish parasitized by live Anisakis larvae in the third stage is considered an important health problem, and the application of treatments to ensure their mortality in the fish products is crucial to prevent the risk of infection. Mobility is used to assess viability, but mobile larvae may not always be infective and immobile larvae may be erroneously considered as non-viable. The objective was to establish whether the analysis of respiratory activity by means of the oxygen consumption rate (OCR) of Anisakis could be used to identify subtle differences between larvae that were still considered viable in terms of their mobility but had been subjected to thermal and/or chemical stress. The metabolic modulators FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone] and sodium azide were used and the basal, maximum, spare and residual respiration rates calculated. Results showed that maximum respiratory capacity of larvae subjected to freezing significantly decreased immediately after thawing, but after some acclimatization, they recovered their capacity fully. However, when these larvae were stored at 4.6 °C, their mitochondria became dysfunctional faster than those of untreated larvae. OCR also showed that mitochondria of larvae were affected by incubation at 37 °C in NaCl or gastric juice. To conclude, OCR of Anisakis in the presence of metabolic modulators can help to identify subtle changes that occur in the larva. These measurements could be used to characterize larvae subjected to various stresses so that a broader picture of Anisakis pathogenic potential can be gained.

Efficiency of Target Larvicides Is Conditioned by ABC-Mediated Transport in the Zoonotic Nematode Anisakis pegreffii.

Anisakiasis is among the most significant emerging foodborne parasitoses contracted through consumption of thermally unprocessed seafood harboring infective Anisakis species larvae. The efficacy of the currently applied anthelminthic therapy in humans and in model organisms has not proven sufficient, so alternative solutions employing natural compounds combined with chemical inhibitors should be explored. By testing toxicity of the natural monoterpenes nerolidol and farnesol and the conventional anthelminthics abamectin and levamisole in the presence/absence of MK-571 and Valspodar, which inhibit the ABC transporter proteins multidrug resistance protein (MRP-like) and P-glycoprotein (P-gp), we determined the preliminary traits of Anisakis detoxifying mechanisms. We found that Anisakis P-gp and MRP-like transporters have a role in the efflux of the tested compounds, which could be useful in the design of novel anthelminthic strategies. As expected, transporter activation and efflux fluctuated over time; they were synchronously active very early postexposure, whereas the activity of one transporter dominated over the other in a time-dependent manner. MRP-like transporters dominated in the efflux of farnesol, and P-gp dominated in efflux of nerolidol, while both were active in effluxing levamisole. The highest toxicity was exerted by abamectin, a P-gp inhibitor per se, which also elicited the highest oxidative stress in treated Anisakis larvae. We suggest that ß-tubulin, observed for the first time as a core element in Anisakis cuticle, might represent an important target for the tested compounds.

Effect of ES products from Anisakis (Nematoda: Anisakidae) on experimentally induced colitis in adult zebrafish.

Inflammatory bowel disease (IBD) in developed countries is linked with elevated hygienic standards. One of the several factors involved in this question may be reduced exposure to the immunomodulatory effects of parasitic helminths. Several investigations on treatment of mice and humans with helminth-derived substances have supported this notion, but underlying mechanisms remain unclear. This study therefore dissects to what extent a series of immune-related genes are modulated in zebrafish with experimentally induced colitis following exposure to excretory-secretory (ES) products isolated from larval Anisakis, a widely distributed fish nematode. Adult zebrafish intrarectally exposed to the colitis-inducing agent TNBS developed severe colitis leading to 80% severe morbidity, but if co-injected (ip) with Anisakis ES products, the morbidity rate was 50% at the end of the experiment (48 hours post-exposure). Gene expression studies of TNBS-treated zebrafish showed clear upregulation of a range of genes encoding inflammatory cytokines and effector molecules and some induction of genes related to the adaptive response. A distinct innate-driven immune response was seen in both TNBS and TNBS + ES groups, but expression values were significantly depressed for several important pro-inflammatory genes in the TNBS + ES group, indicating protective mechanisms of Anisakis ES compounds on intestinal immunopathology in zebrafish.

Determination of Anti-Anisakis Simplex Antibodies and Relationship with αß and γδ Lymphocyte Subpopulations in Patients with Crohn's Disease.

BACKGROUND: The etiology of Crohn's disease (CD) is still unknown although new theories are based on defects in innate immunity. We have previously shown a decrease in γδ T cells in CD patients. Previous studies have shown a high prevalence of anti-A. simplex immunoglobulins in CD patients. The diminution of γδ T cells in the peripheral blood and intestinal mucosa of CD patients may create a state of immunosuppression that would facilitate A. simplex infection. AIMS: To study the antibody responses to Anisakis antigens in Crohn's disease patients and its relationship with αß and γδ T cell subsets. METHODS: We recruited 81 CD patients and 81 healthy controls. αß and γδ T cell subsets and anti-A. simplex antibodies were measured. RESULTS: Levels of anti-A. simplex IgG and IgM were significantly increased in CD patients. Almost 20% of CD patients were positive for IgG and IgM anti-A. simplex versus only 3.7 and 2.5%, respectively, in normal subjects. However, lower specific IgA levels were observed in the group of CD patients versus healthy subjects. We found an association between CD3 + CD8 + Î³Î´ subset and IgM anti-A. simplex levels. In ileal cases and stricturing behavior of CD, we observed the highest levels of specific antibodies with the exception of anti-A. simplex IgA. CONCLUSIONS: The relationship of specific antibodies with a γδ T cell deficiency makes these cell candidates to play a role in the immune response against Anisakis. In addition, anti-Anisakis antibodies could be considered as markers of risk of progression in CD.

Expression patterns and structural modelling of Hsp70 and Hsp90 in a fish-borne zoonotic nematode Anisakis pegreffii.

Heat shock proteins (HSPs) are essential molecular chaperones that are highly conserved across organisms. They have a pivotal function in responding to thermal stress and are responsible for many cellular functions. Here, we aimed to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts. We examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of Anisakis pegreffii. The open reading frame of Hsp70 of A. pegreffii was 1950 bp, and deduced amino acid sequence showed high homology with those of other nematodes. Heatmap analysis revealed sequence identity of Hsp70 and Hsp90 in 13 important parasitic species, human and yeast. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. ApHsp70 and ApHsp90 may play important roles in Anisakis in response to thermal stress and might be important molecules in the development of A. pegreffii, which has implications for its control.

ANISERP: a new serpin from the parasite Anisakis simplex.

BACKGROUND: Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). METHODS: The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. RESULTS: The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. CONCLUSIONS: Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).

Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family.

BACKGROUND: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. METHODOLOGY/PRINCIPAL FINDINGS: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. CONCLUSIONS/SIGNIFICANCE: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.

Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss).

Excretory/secretory (ES) products are molecules produced by parasitic nematodes, including larval Anisakis simplex, a parasite occurring in numerous marine fish hosts. The effects of these substances on host physiology have not been fully described. The present work elucidates the influence of ES substances on the fish immune system by measuring immune gene expression in spleen and liver of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with ES products isolated from A. simplex third stage larvae. The overall gene expression profile of exposed fish showed a generalized down-regulation of the immune genes tested, suggesting a role of ES proteins in immunomodulation. We also tested the enzymatic activity of the ES proteins and found that lipase, esterase/lipase, valine and cysteine arylamidases, naphthol-AS-BI-phosphohydrolase and α-galactosidase activities were present in the ES solution. This type of hydrolytic enzyme activity may play a role in nematode penetration of host tissue. In addition, based on the notion that A. simplex ES products may have an immune-depressive effect (by minimizing immune gene expression) it could also be suggested that worm enzymes directly target host immune molecules which would add to a decreased host immune response and increased worm survival.

Identification of a 24 kDa excretory secretory protein in Anisakis simplex.

A gene coding for a 24 kDa protein (22 U homologous; As22U) was isolated from the Anisakis simplex third-stage larvae cDNA library during expressed sequence tag analysis. As22U was 636 bp long, and was found to code for 212 amino acid residues with a calculated mass of 23.5 kDa and a PI of 9.06. The As22U deduced amino acid sequence harbored a signal peptide region and 16 highly conserved cysteine residues, and it was identified in both the total extracts and excretory secretory (ES) protein of A. simplex. Its molecular weight was measured at 24 kDa via western blot analysis. The expression levels of thymic stromal lymphopoietin, IL-25, and CXCL1 (Gro-α) genes were increased at 6h after recombinant As22U treatment in mouse intestinal epithelial cells. Additionally, thymus and activation-regulated chemokine gene levels were increased at 14 h after treatment. Although we do not currently have sufficient evidence to determine whether As22U plays a role as an allergen, this remains possible. Further in vivo studies may provide some insight as to the allergenic properties of As22U.

First record of anisakid juveniles (Nematoda) in the European seabass Dicentrarchus labrax (family: Moronidae), and their role as bio-indicators of heavy metal pollution.

This study assessed the anisakid nematode distribution pattern in the fish collected from coasts of Mediterranean Sea, Egypt, during the period September 2010-April 2011. Two hundred thirty out of 300 (76.7%) Dicentrarchus labrax (European seabass) marine fishes belonging to family Moronidae were dissected and found to be infected with larva three nematodes. The larvae had been studied by light and scanning electron microscopy. The present work represents the first record of the presence of the parasite in this fish in the Mediterranean Sea. The concentrations of some heavy metals (Pb, Zn, Fe, Cd, Cu, Mn, Ni) in parasites as well as in tissues of fish were measured. The presented results showed that the nematode parasites are able to accumulate heavy metals in their tissues and in some cases that they are able to accumulate large amounts of heavy metals in a higher amount than host tissues. This demonstrated their sustainability as bioindicators of environmental pollution by removing heavy metals and help in the survival of fish.