Molecular & Biochemical Parasitology 239 (2020) 111311

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Molecular & Biochemical Parasitology

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Proteomic analysis of Ascocotyle longa (Trematoda: Heterophyidae) T metacercariae Karina M. Rebelloa,c,*, Juliana N. Borgesb, André Teixeirac, Jonas Peralesc, Cláudia P. Santosb,** a Laboratório de Estudos Integrados em Protozoologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil b Laboratório de Avaliação e Promoção da Saúde Ambiental, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil c Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

ARTICLE INFO ABSTRACT

Keywords: Ascocotyle longa is parasitic trematode with wide distribution throughout America, Europe, Africa, and Middle Mugil liza East. Despite the fact that this fish-borne pathogen has been considered an agent of human heterophyiasis in Fish-born pathogen Brazil, the molecules involved in the host-parasite interaction remain unknown. The present study reports the Proteins proteome profile ofA. longa metacercariae collected from the fishMugil liza from Brazil. This infective stage for Parasite humans, mammals and birds was analyzed using nLC-MS/MS approach. We identified a large repertoire of Helminth proteins, which are mainly involved in energy metabolism and cell structure. Peptidases and immunogenic Heterophyiasis proteins were also identified, which might play roles in host-parasite interface. Our data provided unprecedented insights into the biology of A. longa and represent a first step to understand the natural host-parasite interaction. Moreover, as the first proteome characterized in this trematode, it will provide an important resource for future studies.

1. Introduction find their way into their final hosts [14]. A. longa has been character­ ized by morphologic, ultrastructural, genetic, and ecological studies but Ascocotyle longa Ransom, 1920 is an intestinal fluke of fish-eating potential targets that could induce modulations on their hosts or al­ birds and mammals with a wide distribution throughout North and lergenic reaction in humans still need to be identified. South America, Europe, Africa and Middle East [1–5]. This parasite has The aim of the current study was to identify proteins in crude ex­ emerged as causative agent of fish-borne trematodiases [6–8]. tracts from A. longa metacercariae. Our detailed analyses provide Humans are infected by eating raw or undercooked freshwater fish deeper insights into parasite biology and our knowledge of protein parasitized by A. longa metacercariae. Upon ingestion, the excysted composition of the human infective stage of this trematode. Proteins metacercariae develop into adults in the small intestine [1]. This tre­ involved in host-pathogen interactions, immunogenic proteins and matode life cycle requires a gastropod mollusk as first intermediate molecules implicated in parasite´s metabolism were identified. host, mugilid fish as second intermediate host and piscivorous birds and mammals, including man as the definitive hosts 4[ ,6–10]. Simões and 2. Materials and methods colleagues described the snail Heleobia australis as first intermediate host and confirmed that mullet Mugil liza Valenciennes, 1836 plays the 2.1. Ethics statement role of a second intermediate host of A. longa in Rio de Janeiro, Brazil [11]. Mugil liza is widely distributed along the Atlantic coast of South This study was authorized by the Brazilian Institute of Environment America [12] and is a fishing resource of great social, economic, en­ and Renewable Natural Resources (IBAMA, license no. 15898-1). vironmental and cultural importance in the South and Southeastern Brazil [13]. In Rio de Janeiro A. longa was found parasitizing 100 % of 2.2. Fish sample and encysted metacercariae isolation the M. liza samples collected from Rodrigo de Freitas Lagoon [6] Poulin and Mauren discussed that such endoparasites with complex Six samples of fish M. liza were collected by local fishermen from life cycles require strategies to keep their different hosts alive and try to Rodrigo de Freitas Lagoon, Rio de Janeiro, Brazil (22°57′02″ S,

⁎ Corresponding author at: Laboratório de Estudos Integrados em Protozoologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. ⁎⁎ Corresponding author at: Laboratório de Avaliação e Promoção da Saúde Ambiental, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. E-mail addresses: [email protected] (K.M. Rebello), [email protected] (C.P. Santos). https://doi.org/10.1016/j.molbiopara.2020.111311 Received 2 June 2020; Received in revised form 20 July 2020; Accepted 21 July 2020 Available online 01 August 2020 0166-6851/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

43°11′09″ W) and transported to the laboratory for dissection. The 2.7. Computational analysis and protein identification heart, spleen, liver, body musculature and intestine were collected and separated for artificial digestion. Two out of six fish samples were used Data sets were analyzed using Mascot search engine version 2.4.1 at each biological sample (n = 3). for protein identification: mass tolerance 10 ppm, fragment tolerance 0.60 Da, set as trypsin and allowance up to two missed clea­ vages, carbamidomethylation of cysteine as a fixed modification and 2.3. In vitro excystation and identification of A. longa metacercariae methionine oxidation and acetylation as variable modifications. Protein identification was conducted against a database containing sequences Briefly, fish organs and musculature of two mullets of each biolo­ from UniProtKB database (trematode – 362,357 entries). Brackish and gical sample were digested with pepsin solution at 37 °C for 2 h, as freshwater fish sequences (234,400 entries) were downloaded from described previously [15]. Fresh excysted metacercariae were washed UniprotKB and included to common mass spectra contaminants data­ with PBS in an attempt to remove host contaminants and were identi­ base. Search against this fish sequence database was also run to identify fied under a light microscope based on the presence of a single row of any host protein. 16 oral spines and gonotyl bipartite [11]. Then, samples were counted After the search, the data were statistically validated using Scaffold using a stereomicroscope and stored at −80 °C until further use. 4.4.7 software (Proteome Software) [18]. Protein and peptides were considered identified when their Peptide Prophet-calculated prob­ 2.4. Preparation of protein extract abilities were greater than 95 %. Proteins without proteotypic peptide identification were grouped to satisfy the principle of maximum par­ Protein extraction was performed by a combination of maceration of simony [19]. Proteins were classified according to UniProt-Gene­ On the metacercariae (approximately 500 parasites per tube) for 5 min in tology annotation available [20]. microcentrifuge tubes containing an abrasive resin (Sample Grinding Kit, GE Healthcare) and 100 μL of 40 μM Tris base, 1 % TX-100 and 1X 3. Results and discussion Complete Inhibitor Cocktail (Roche, Mannheim, Germany) followed by 10 consecutive freeze-thaw cycles in liquid nitrogen. The Each biological sample was analyzed in technical triplicate by re­ homogenate was centrifuged at 16,000×g for 10 min at 4 °C and the versed-phase nanochromatography coupled to nanoelectrospray high obtained supernatant was collected. resolution mass spectrometry for identification. We identified 849 proteins based on 4074 peptides, from these 736 were assigned to trematodes and 113 to fish species. Table S1 lists all the identifications 2.5. Protein digestion and sample preparation from all technical replicates of the three biological replicates together. Only proteins identified in all replicates were considered. They are Protein samples were precipitated overnight with cold ethanol/ separated by organism and organized in a nonincreasing order ac­ acetone [1(protein extract):4 (ethanol):4 (acetone) v/v] at – 20 °C. The cording to their number of peptide sequences. In the case of A. longa, precipitated proteins were sedimented at 16,000 ×g for 30 min, wa­ 614 proteins were assigned as having a predicted function, and 122 shed three times with ethanol/acetone/water (4:4:2 v/v) and solubi­ were assigned as uncharacterized proteins. lized in 20 μL of 0.4 M ammonium bicarbonate/ 8 M urea. Protein Our analysis of the A. longa metacercariae dataset showed a pre­ extracts were quantified using the BCA method employing bovine dominance of bioenergetics-related , ribosomal and structural/ serum albumin as standard [16]. Samples (50 μg) were than reduced motor proteins. Chaperones, , , calcium using 10 mM dithiothreitol (DTT) for 3 h at 37 °C and alkylated using binding proteins, , kinases, , proton pumps, nu­ 25 mM iodoacetamide (IAA) at room temperature for 15 min in the clear and immunogenic proteins as well as proteins involved in signal dark. Then, the concentration of urea was reduced to 1 M with water transduction and membrane trafficking were also identified (S1 Table). and the samples were digested using trypsin (Promega) at final ratio of The proteins were also classified into subcellular location, according to 1: 50 (w/w) overnight at 37 °C. Hydrolysis reactions were stopped with GO cellular component data, that were available for 80 % of the total formic acid at 1 % final concentration (v/v). The resulting peptide proteins (Fig. 1). mixtures were desalted using homemade tip columns packed with Poros R2 resin (Applied Biosystems, USA). Peptide samples were vacuum 3.1. Metabolic process and energy production molecules dried before resuspension in 1 % formic acid (v/v). Metacercariae of A. longa, as already described in other foodborne 2.6. Nano liquid chromatography-tandem mass spectrometry (nLC MS/ parasitic trematodes, are nonfeeding and are dependent on their own MS) glycogen for survival inside the host. Proteins of central carbon meta­ bolism that transforms carbon through glycolytic pathway and Krebs Peptide mixtures were analyzed by nLC ultra-high-pressure HPLC cycle into energy were identified, indicating that metacercaria is able to system coupled online to Q Exactive Plus mass spectrometer (both from obtain energy from both aerobic and anaerobic metabolism. Enzymes of Thermo Fisher Scientific) with a nanoelectrospray ion source. Peptide other catabolic pathways, such as pentose phosphate pathway, ga­ mixture (1 μg) was loaded onto a trap-column (100 μm i.d. × 20 mm lactose metabolism and beta oxidation of fatty acids were also identi­ long) packed with 200 Å (5 μm) Magic C18 (C18AQ; Michrom fied (Table 1). BioResources), followed by analytical separation on column (30 cm The glycolytic enzyme fructose 1,6-biphosphatase aldolase was long, 75 μm inner diameter). Peptides were eluted with gradient of 2 % identified and it is described as a vaccine candidate antigen against to 40 % of 0.1 % (v/v) formic acid in acetonitrile over 162 min, which several parasitic helminths, such as Onchocerca volvulus [21], Clonorchis was increased to 80 % in 4 min, followed by a washing step at this sinensis [22] and Trichinella spiralis [23]. It has also been described as concentration for 2 min and re-equilibration with 0.1 % (v/v) formic potential novel allergen in the parasite fish Anisakis simplex [24]. acid in water. Peptides eluted were directly electrosprayed into the Phosphofructokinase (PFK) that is most important regulatory en­ mass spectrometer and analyzed in positive ion mode. zyme of glycolytic pathway [25] was also identified in A. longa. Since, MS data was acquired using a data-dependent top-12 method dy­ several parasites utilize glycolysis as the main source of energy for their namically choosing the most abundant precursor ions in each survey for survival, this metabolic enzyme is an important drug target candidate higher energy collisional dissociation (HCD) fragmentation according [26]. Previous studies showed that PFK of schistosomes were selective to a previously described condition [17]. inhibited by antischistosomal trivalent antimonials [27]. In the

2 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

Fig. 1. Pie chart represents computed GO cellular components (subcellular location) for proteins found from A. longa metacercariae. anaerobic energy metabolism, phosphoenolpyruvate carboxykinase allergen in A. simplex, being expected to be a useful tool for diagnosis (PEPCK) that is an important enzyme for many parasitic helminths [28] [41,42]. was also identified inA. longa. Moreover, structural differences between Tropomyosin is another important IgE target in many helminth in­ parasite PEPCK and mammalian enzyme homologues make them at­ fections [43]. It is highly conserved across multiple invertebrate species tractive as drug targets [25]. and is involved in cases of IgE cross-reactivity between mites and Additionally, we identified mitochondrial acetate: succinate CoA- parasitic nematode, such as O. volvulus, A. simplex and Ascaris spp. in A. longa. This enzyme produces acetate as a metabolic [44–46]. Of note, IgE response to the tropomyosin mite allergen was end product in metabolic route and are present in various parasites, increased in Onchocerca-infected individuals compared to uninfected especially those who inhabit anaerobic sites [29]. Since acetate is the subjects [46]. main end-product of parasite energy metabolism but not in their host, acetate formation constitutes a possibly interesting drug target in 3.3. Chaperones parasites. The 3 oxoacyl (acyl carrier protein) reductase was also identified in Heat shock proteins (HSPs) are highly conserved group of molecules our metacercariae proteome (Table 1). It has been investigated as a involved in several processes in mammals and microorganisms [47]. druggable target due to its importance to parasite survival. This enzyme Their main function is to act as molecular chaperones [48] but some are is involved in the fatty acid biosynthesis, which is part of lipid meta­ also able to stimulate cells of the innate immune system directly [49] bolism of parasites [30,31]. Taken together, our data showed that and thus, is being considered as an important target in various helminth metacercarial stage of A. longa is biochemically active, similar to those parasite infections [50]. Various chaperone proteins were identified in reported in other trematodes [32–34]. metacercarial stage of A. longa (Table 1). HSP70 from T. spiralis and C. sinensis induced protective immunity 3.2. Structural and cytoskeleton proteins activating dendritic cells in vivo and in vitro, respectively [51–53]. In Schistosoma mansoni, HSP70 is involved in cercaria-schistosomulum Not surprisingly, several proteins identified in A. longa lysates were transformation as well as play a pivotal role in the invasion by schis­ cytoskeleton-associated proteins, such as actin, myosin, paramyosin, tosome cercariae [54]. HSP90 was described as essential for filarial tubulin, tropomyosin, spectrin, dynein, filamin, troponin, talin and parasite Brugia pahangi [55] and was detected as one of the major vinculin (Table 1). Paramyosin is a multifunctional protein involved in constituents in secretome of Schistosoma japonicum being implicated in muscle physiological contraction and immunoregulation of helminths host-parasite immunomodulation [56]. [35]. As an immunogenic protein, paramyosin has been investigated for The glucose regulated proteins (GRP) of 78 kDa and 94 kDa encoded the protective effect in various parasitic helminths [36–39], being a by HSP70 and HSP90, respectively were also detected in metacercaria. potential vaccine candidate. It was also identified in cyst wall of C. They are endoplasmic reticulum (ER) chaperones essential in main­ sinensis metacercaria and induced combined Th1/Th2 response in taining ER homeostasis, including proteins synthesis, modification and murine model [40]. Moreover, paramyosin is a protein with conserved exported proteins [57,58]. These molecules are involved in antigen IgE-binding epitopes across species that has also been reported as an recognition in infection diseases [59,60] and have been considered

3 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

Table 1 List of identified some proteins with A. longa metacercariae (see S1 Table for the full list).

Description Species Uniprot accession Unique peptides Total Peptides

ENERGY METABOLISM Alpha-1,4 glucan phosphorylase Opisthorchis viverrini A0A075ABK6 16 27 Schistosoma mansoni G4VSJ5 Opisthorchis viverrini A0A075ABK6 L-lactate dehydrogenase Clonorchis sinensis Q1M156 4 15 Opisthorchis viverrini, A0A075A688 Schistosoma haematobium A0A094ZC89 Phosphoglycerate kinase Opisthorchis viverrini A0A075A297 5 14 Fasciola hepatica Q45UT3 Glyceraldehyde-3-phosphate dehydrogenase Clonorchis sinensis H2KPE2 9 16 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Clonorchis sinensis, G7YQG7 7 18 Schistosoma haematobium A0A094ZF21 Methylmalonyl-CoA mutase Clonorchis sinensis G7YST9 1 9 Fructose-bisphosphate aldolase Clonorchis sinensis G7YD93 15 17 G7YM44 Schistosoma mansoni P53442 Propionyl-CoA carboxylase beta chain Clonorchis sinensis G7YFK3 3 8 Glycerol-3-phosphate dehydrogenase Clonorchis sinensis G7YMD6 11 12 Opisthorchis viverrini A0A074Z180 Schistosoma mansoni Q5QEI6 Succinate–CoA [ADP/GDP-forming] subunit alpha, mitochondrial Opisthorchis viverrini, A0A075A326 8 8 Clonorchis sinensis G7YUT2 Fumarate hydratase class I Clonorchis sinensis H2KP23 2 9 Aamy domain-containing protein Opisthorchis viverrini A0A074ZWZ6 5 6 Phosphoglycerate mutase Clonorchis sinensis B2KYE8 6 6 ATP-dependent 6-phosphofructokinase Clonorchis sinensis H2KSW1 2 8 Opisthorchis viverrini A0A074ZTV8 Glycogen [starch] synthase Schistosoma mansoni G4VGQ5 1 8 Triosephosphate Opisthorchis viverrini A0A074Z863 3 5 2-oxoglutarate dehydrogenase E1 component Clonorchis sinensis H2KTC5 1 9 AcetylCoA_hyd_C domain-containing protein Opisthorchis viverrini A0A074ZTG3 3 5 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial, Opisthorchis viverrini A0A075AJ94 5 11 Schistosoma japonicum Q5MB10 Enolase Schistosoma haematobium A0A095AJN4 2 6 Pyruvate kinase Opisthorchis viverrini A0A074Z5Q7 3 5 Malate dehydrogenase Clonorchis sinensis, G7YWM7 8 11 Schistosoma mansoni G4V7Z7 Mitochondrial acetate:succinate CoA-transferase Fasciola hepatica C6EUD4 2 4 Propionyl-CoA carboxylase alpha chain, mitochondrial Schistosoma haematobium A0A094ZUC3 1 5 Glycogen debranching enzyme Clonorchis sinensis G7YQV3 2 6 Cytosolic malate dehydrogenase Clonorchis sinensis Q5DM86 4 7 Isocitrate dehydrogenase [NAD] subunit, mitochondrial Clonorchis sinensis H2KQU1 4 7 H2KTA2 Schistosoma japonicum C1LFY3 Phosphoenolpyruvate carboxykinase [GTP] Schistosoma haematobium A0A095BVS7 1 2 Transketolase Clonorchis sinensis G7Y2J3 5 6 Schistosoma mansoni G4V9A6 6-phosphogluconate dehydrogenase, decarboxylating Opisthorchis viverrini A0A074ZNY6 1 3 Long-chain acyl-CoA synthetase Clonorchis sinensis G7YK44 1 2 Aconitate hydratase, mitochondrial Opisthorchis viverrini A0A074ZDF6 2 3 Acetyl-CoA acyltransferase Clonorchis sinensis G7YAA0 1 1 1-alkyl-2-acetylglycerophosphocholine esterase Clonorchis sinensis H2KSQ3 1 1 Malic enzyme Opisthorchis viverrini A0A074Z9L0 3 5 Clonorchis sinensis G7YC97 Long-chain-fatty-acid–CoA ligase Schistosoma mansoni C4QPH5 1 2 Aldehyde dehydrogenase (NAD+) Clonorchis sinensis H2KSW5 2 2 Pyruvate carboxylase Schistosoma mansoni G4V7Q4 1 2 UTP–glucose-1-phosphate uridylyltransferase Opisthorchis viverrini A0A075A5U2 2 3 Enoyl-CoA hydratase/long-chain 3-hydroxyacyl-CoA dehydrogenase Clonorchis sinensis G7YW38 3 3 Transaldolase Clonorchis sinensis G7YEJ9 1 2 Peroxisomal 3 2-trans-enoyl-CoA isomerase Clonorchis sinensis G7YW60 1 1 Lysosomal alpha-glucosidase Clonorchis sinensis H2KPA9 3 3 Glycerol-3-phosphate acyltransferase 3 Clonorchis sinensis G7YJB8 1 1 UDP-glucose 4-epimerase Clonorchis sinensis H2KQ61 1 1 Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Schistosoma haematobium A0A095C8C0 2 2 Glucose-6-phosphate isomerase Clonorchis sinensis H2KV24 1 2 Schistosoma mansoni G4VPH5 3-oxoacyl-[acyl-carrier-protein] reductase Clonorchis sinensis G7YFI1 1 1 Enoyl-CoA hydratase Clonorchis sinensis G7YGA2 2 2 Glycogenin-related Schistosoma mansoni G4V5G7 1 2 Phosphoacetylglucosamine mutase Clonorchis sinensis H2KS69 1 1 Pyruvate dehydrogenase E1 component subunit alpha Opisthorchis viverrini A0A075AAV0 1 1 Glycerol kinase Clonorchis sinensis H2KSG0 1 1 Phosphotransferase Opisthorchis viverrini A0A075AI13 1 1 (continued on next page)

4 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

Table 1 (continued)

Description Species Uniprot accession Unique peptides Total Peptides

Beta-galactosidase Opisthorchis viverrini A0A074YY46 1 1 Lipoyl-binding domain-containing protein Opisthorchis viverrini A0A074Z881 3 4 STRUCTURAL AND MOTOR PROTEINS Actin Opisthorchis viverrini A0A075A1W9 2 57 Schistosoma japonicum C1LDB7 Clonorchis sinensis H2KRT9 Alpha-parvin Clonorchis sinensis H2KT73 1 1 Alpha-actinin sarcomeric Clonorchis sinensis H2KTW4 18 22 Actin-related protein 2 Opisthorchis viverrini A0A074ZPT1 4 4 Actin-like protein 3 Schistosoma japonicum H2KVF0 2 3 Adenylyl cyclase-associated protein Opisthorchis viverrini A0A075AA54 1 1 Arp2/3 complex34 kDa subunit Opisthorchis viverrini A0A074ZA47 1 2 F-actin-capping protein subunit alpha Clonorchis sinensis G7Y925 1 1 F-actin-capping protein subunit beta Clonorchis sinensis G7YWN0 1 1 Alpha-centractin (Centractin) (Centrosome-associated actin homolog) (Actin-RPV) (ARP1) Schistosoma japonicum C7TQQ1 3 4 Actinin alpha Clonorchis sinensis G7Y532 1 1 Band 4.1-like protein 5 Clonorchis sinensis H2KS79 1 1 Collagen type XV alpha Clonorchis sinensis G7YPD8 1 1 Dynein light chain Schistosoma haematobium A0A095B4L9 17 27 Opisthorchis viverrini, A0A075A9J2 Clonorchis sinensis G7Y6P6 H2KT57 G7YW13 Schistosoma haematobium H2KT57 Dynein heavy chain 1 cytosolic Clonorchis sinensis G7Y725 18 26 Dynein heavy chain 10 axonemal Clonorchis sinensis G7Y5V4 1 1 Dynein light chain roadblock-type Clonorchis sinensis G7YML0 1 1 Filamin Clonorchis sinensis G7Y5W0 30 43 Filamin-C Clonorchis sinensis G7Y7C5 2 10 Fimbrin Schistosoma mansoni Q26574 1 4 Laminin alpha 1/2 Clonorchis sinensis G7YIN3 1 1 Lamin Dm0 Clonorchis sinensis H2KUU0 12 15 Microtubule-associated protein 1 Clonorchis sinensis H2KQ37 1 1 Microtubule-associated protein RP/EB family Clonorchis sinensis G7Y5Z7 1 1 Myosin heavy chain Clonorchis sinensis H2KRJ7 12 128 Schistosoma mansoni Q02456 Clonorchis sinensis G7YNH2 3 16 Myosin_tail_1 domain-containing protein Opisthorchis viverrini A0A075AJR2 3 35 Myosin regulatory light chain invertebrate Clonorchis sinensis H2KT77 2 2 Myosin essential light chain striated adductor muscle Clonorchis sinensis G7YQQ2 1 1 Myosin-2 essential light chain Clonorchis sinensis G7YS07 3 3 Myosin regulatory light chain 2, smooth muscle minor isoform (G1) Schistosoma japonicum C1L626 5 16 Schistosoma haematobium A0A095APS3 Fasciola hepatica D0VAX0 Paramyosin Schistosoma haematobium A5A6F8 10 34 Schistosoma japonicum B1PS36 Paragonimus westermani Q1 × 6H7 Plastin-2 Clonorchis sinensis G7YNU0 7 33 Putative vinculin Schistosoma mansoni G4VDU1 1 1 Putative gelsolin Schistosoma mansoni G4VIJ2 1 1 Severin Clonorchis sinensis H2KQN2 6 6 G7YBE5 Smoothelin Clonorchis sinensis G7YCD7 1 1 Spectrin repeat-containing domain protein Opisthorchis viverrini A0A075AI56 6 24 Spectrin alpha chain Clonorchis sinensis G7Y6J8 3 69 Schistosoma haematobium A0A094ZS46 Spectrin beta chain Opisthorchis viverrini A0A075A6I1 37 50 Schistosoma mansoni G4VDE6 Talin Clonorchis sinensis H2KNJ2 1 4 Titin, putative Schistosoma mansoni G4 M018 4 13 Clonorchis sinensis G7YG49 Transgelin Clonorchis sinensis H2KV59 7 10 Schistosoma japonicum C1LMV1 Tubulin alpha chain Opisthorchis viverrini A0A074Z4W4 7 34 A0A074Z808 A0A074ZQC9 Clonorchis sinensis G7YF88 H2KTH3 Tubulin beta chain Schistosoma mansoni G4VTA4 3 22 Opisthorchis viverrini A0A074ZWB8 Tubulin polymerization-promoting protein Clonorchis sinensis G7YGH2 2 3 Tubulin polymerization-promoting member 3 Clonorchis sinensis H2KPC6 1 1 Tropomyosin Schistosoma haematobium Q26503 1 14 Clonorchis sinensis Q23758 Troponin T Clonorchis sinensis H2KQE7 7 14 (continued on next page)

5 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

Table 1 (continued)

Description Species Uniprot accession Unique peptides Total Peptides

Troponin I 4 Clonorchis sinensis H2KQ84 8 10 Schistosoma haematobium A0A094ZZF6 Tropomodulin Clonorchis sinensis G7YE54 1 1 Calcium binding protein containing EF hand domains Opisthorchis viverrini A0A074ZI95 1 2 CHAPERONES Chaperonin GroEL Clonorchis sinensis G7YUU9 3 12 GrpE protein homolog Clonorchis sinensis H2KQH8 1 1 DnaJ domain protein Opisthorchis viverrini A0A074ZVL8 4 6 DnaJ homolog subfamily B member 4 Clonorchis sinensis G7Y3V9 3 5 GrpE protein homolog Clonorchis sinensis H2KQH8 1 1 Heat shock 70 kDa protein 5 Clonorchis sinensis H2KR48 2 15 Heat shock 70 kDa protein 1/8 Clonorchis sinensis H2KSA9 3 19 Hsp90 protein Opisthorchis viverrini A0A074Z7V0 23 30 Molecular chaperone DnaK Clonorchis sinensis G7YNS8 8 15 78 kDa glucose-regulated protein Schistosoma haematobium A0A095CGL8 1 10 T-complex protein 1 subunit alpha Schistosoma mansoni Q94757 1 7 T-complex protein 1 subunit delta Clonorchis sinensis H2KNK7 11 12 Schistosoma japonicum C1L5N9 T-complex protein 1 subunit gamma Opisthorchis viverrini A0A074ZMD9 4 9 T-complex protein 1 subunit eta Schistosoma japonicum C1LIN3 2 5 Schistosoma haematobium A0A094ZHR7 T-complex protein 1 subunit theta Schistosoma haematobium A0A095CBQ7 1 2 Putative chaperonin containing t-complex protein 1, zeta subunit, tcpz Schistosoma mansoni G4V5L4 2 6 Opisthorchis viverrini A0A1S8WVZ7 Phosphoenolpyruvate carboxykinase (GTP) Clonorchis sinensis H2KQ97 3 16 Opisthorchis viverrini A0A074ZKX7 Fasciola sp. A0A0H5AS27 IMMUNOGENIC PROTEINS 20 kDa calcium-binding protein (Antigen SM20) Schistosoma japonicum Q86ET3 2 5 Major egg antigen Clonorchis sinensis H2KTV6 4 4 G7YE15 Tegument antigen Clonorchis sinensis G7YAJ7 4 4 PEPTIDASES AND INHIBITORS Opisthorchis viverrini A0A074Z517 1 1 Aspartyl aminopeptidase Clonorchis sinensis G7YJ41 2 2 CAAX prenyl protease Clonorchis sinensis H2KUU2 3 3 Calpain Clonorchis sinensis G7YHP4 1 5 Calpain- B Clonorchis sinensis G7YU18 1 6 Cathepsin D Clonorchis sinensis G7YV90 1 1 Mitochondrial-processing peptidase subunit beta (M16 family) Schistosoma mansoni G4LYP2 4 6 Clonorchis sinensis G7Y663 Kyphoscoliosis peptidase Clonorchis sinensis H2KTC4 5 5 H2KPM4 Leukotriene-A4 Clonorchis sinensis G7Y9I2 2 2 Lon protease homolog, mitochondrial Clonorchis sinensis G7YNX1 3 5 Peptidase A2 domain-containing protein Opisthorchis viverrini A0A074ZZM6 4 7 26S protease regulatory subunit 4 Clonorchis sinensis G7Y889 4 8 26S protease regulatory subunit 7 Clonorchis sinensis H2KTA5 5 7 Proteasome complex Clonorchis sinensis G7YQA6 10 10 G7YA15 B5G4Y1 Proteasome subunit alpha type Clonorchis sinensis E4W3S2 6 7 H2KUP1 Schistosoma mansoni G4VDG1 Proteasome subunit beta Opisthorchis viverrini A0A075AAC7 1 1 HTRA2, mitochondrial Schistosoma haematobium A0A094ZJV9 1 1 Signal peptidase complex subunit 2 Clonorchis sinensis B5G4Y6 2 2 Signal peptidase complex subunit 3 Clonorchis sinensis A0A074ZX75 1 1 Tissue factor pathway inhibitor 2 Clonorchis sinensis H2KNJ4 3 3 Ubiquitin carboxyl-terminal hydrolase Clonorchis sinensis H2KP94 3 3 G7YH74 Xaa-Pro Clonorchis sinensis G7YUN1 1 1 Kinesin-like protein Clonorchis sinensis G7YBN7 1 1 SJCHGC02536 protein Schistosoma japonicum Q5DDG6 2 4 MATH domain protein Opisthorchis viverrini A0A074Z2K9 1 1 ANTIOXIDANTS Cytochrome C Oxidase Schistosoma japonicum C1LPG9 2 2 Dihydrolipoyl dehydrogenase Schistosoma haematobium A0A094ZIZ2 2 2 Glutathione S-transferase Clonorchis sinensis G7Y784 1 1 Glutathione peroxidase Clonorchis sinensis A0SWV9 1 1 Protein disulfide-isomerase A6 Clonorchis sinensis G7YPJ9 1 1 Thioredoxin reductase (NADPH) Clonorchis sinensis G7YV41 1 1 Superoxide dismutase [Cu-Zn] Clonorchis sinensis Q6B7T2 1 1

6 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311 potential drug targets in parasitic infections [61,62]. mitochondrial homeostasis as well as cell survivor [84]. Another mi­ We also identified members of TCP-1 chaperonin family, which tochondrial serine peptidase identified was Lon peptidase homolog includes seven different subunits and GroEL, 60 kDa heat shock protein (LON), which is an ATP-dependent multifunctional enzyme highly (HSP60). The cytosolic HSP60/TCP-1 complex is essential for proper conserved through all phylogenetic kingdoms. It plays a pivotal role in folding of many proteins and cell cycle [63]. Unexpectedly, we iden­ selective degradation of misfolded and damaged polypeptides and tified peptidase A2 domain-containing protein that is a peptidase-cha­ maintains mitochondrial genome integrity [85]. Downregulation of perone protein. This unique model molecule is a chaperone of TCP-1 LON leads to apoptosis and disruption of mitochondrial structure, chaperonin family, which displays both remodel protein and aspartic causing cell death [86]. peptidase functions on a single polypeptide. Similar structure has been Different proteasome forms, containing different subunits were de­ described for heat-shock protein DegP from Escherichia coli [64]. tected in the metacercarial extract. The proteasome is an intra cellular multi-catalytic peptidase system with many subunits that plays a key 3.4. Immunogenic proteins role in the degradation of many cytoplasmic and nuclear protein [87]. The catalytic core of the proteasome is a threonine peptidase, and the Only two immunogenic proteins were found in A. longa meta­ proteins modified by proteasome proteolysis seems to be involved in cercaria proteome, the tegument antigen and major egg antigen essential cell processes in parasites, including trematodes [88]. (Table 1). Tegument antigen is a protein of 20.8 kDa that contains C- Different members of papain-like cysteine peptidase superfamily terminal dynein light chain-like (DLC-like) domain and two EF-hand were identified in A. longa lysates. Our data revealed the presence of motifs in N-terminal [65]. The presence of EF-hand structure gives it a two calpain homologs, which belongs to peptidase family C2 (calpain similar structure to EF-hand family of allergens. Therefore, this protein family, clan CA). They are intracellular calcium-dependent cysteine is also called 20.8 Tegument-Allergen-Like (TAL). In S. mansoni, is a peptidases involved in many important cellular functions in several dominant IgE target and in C. sinensis stimulates Immunoglobulin A organisms such as apoptosis, vesicular trafficking, cell differentiation (IgA) but not IgG response [66,67]. and signal transduction [89,90]. They have been found and char­ Major egg antigen belongs to the small heat shock protein (sHsp20) acterized in S. mansoni and S. japonicum, and were identified as po­ family. sHsps are group of ATP-independent molecules that has been tential vaccine candidates [91,92]. We also identified kyphoscoliosis described in pathogens [68]. Their main function is to act as molecular peptidase and ubiquitinyl (families C9 and C12) in A. longa chaperones, but some are also able to stimulate cells of the innate im­ metacercariae. Kyphoscoliosis peptidase was described in cercariae and mune system during helminth infection [69–71]. schistosomula of S. japonicum and appears to be involved in host in­ We identified a glycosylphosphatidylinositol (GPI)-anchored surface vasion [77]. Ubiquitinyl hydrolases are intracellular peptidases that glycoprotein within the A. longa metacercaria proteome; however, its remove ubiquitin from ubiquitinylated proteins and peptides expressed role remains unclear. GPI-anchored proteins are commonly described in throughout S. mansoni life cycle and seem to be important for early adult flukes tegument and play an essential role in the in the host-pa­ schistosomula development of trematode [93]. Some studies reported thogen interaction [72–74]. Immunization with S. mansoni GPI-an­ the association of cysteine peptidase activity and metacercarial en­ chored glycoproteins induced partial protection in mice against cer­ cystment [94]. carial challenge and reduced the pathology associated with trematode Regarding the metallopeptidases, representatives of four families infection [75]. could be identified in A. longa metacercariae. Peptidases of M1-family contain membrane bound or cytosolic zinc that act at the 3.5. Proteases and inhibitors free N-terminus end of polypeptides. Most members of the family are alanyl , but also includes leukotriene A4 hydrolase Analysis of the proteome of A. longa metacercariae identified pep­ [95]. We identified both in the metacercariae extract. M1 family tidases of five catalytic classes (aspartic, cysteine, serine, threonine, and members are found among all kingdoms, except viruses and are es­ metallopeptidases) as well subunits of the 26S proteasome regulatory sential to Plasmodium falciparum growth and development and are complex and signal peptidase complex subunits (Table 1). considered an attractive target for antimalarial agents [96]. Our pipeline identified two aspartic peptidases from families A1 and Mitochondrial processing peptidase beta-subunit is a zinc me­ A2, respectively. In trematodes, cathepsin D (family A1) has been talloendopeptidases, member of M16 family [95] also identified in in­ shown to be mainly involved in helminths nutrition, due to its essential fective stages. This enzyme was detected in proteomic analyses of the role in hemoglobin digestion [76]. Cathepsin D has been detected in tegument surface of S. mansoni schistosomula, and it is considered a cercaria and schistosomula in S. japonicum, as well as in metacercariae potential therapeutic target for schistosomiasis [97]. M18 aspartyl of C. sinensis [77]. However, the exact role of this enzyme in those early aminopeptidase (AAP) detected here in A. longa metacercariae is the stages is still unknown. Peptidase A2 domain -containing protein be­ first record of this zinc dependent enzyme in trematodes. Although longs to retropepsin family (A2) and was detected in crude meta­ widely distributed in bacteria and eukaryotes, M18 family peptidases cercarial extract. T. spirialis and Brugia malayi genomes encode retro­ have been detailed characterized only in Saccharomyces cerevisiae [98], pepsin-like aspartic peptidase [78,79] and its role is an as-yet humans [99]. P. falciparum [100] and Toxoplasma gondii [101]. undiscovered. Xaa-Pro dipeptidase, also called to proline dipeptidase belongs to Our results revealed the presence of members of some serine pep­ M24B peptidase family [95] has been identified inA. longa lysates. This tidases families. Two enzymes of S26 family (signal peptidase complex peptidase was found amongst the Fasciola hepatica extracellular vesicles subunits 2 and 3) that are an integral membrane protein that have been surface proteins [102] and in content of exosome-like vesicles derived identified in prokaryotes and eukaryotes80 [ ]. These enzymes are from S. mansoni [103]. serine peptidases that cleave the signal peptides from secretory proteins Peptidase family M48 includes zinc- that are integral targeted to the lumen of the endoplasmic reticulum [81]. Signal pep­ membrane proteins associated with the endoplasmic reticulum and tidases identified in Taenia solium genome seem to be responsible for golgi complex [95]. CAAX prenyl peptidase detected in crude meta­ processing precursor proteins to mature forms [82,83]. A mitochondrial cercarial extract belongs to the subfamily M48A and appears to be in­ serine peptidase HTRA2 (high temperature requirement A serine pep­ volved in host-invasion of S. japonicum cercariae [77]. tidase 2) was also detected in A. longa metacercaria proteome. This The tissue factor pathway inhibitor- 2 (TFPI-2) was the only pepti­ proteolytic enzyme is an ATP- independent characterized by a trypsin- dase inhibitor identified in metacercaria lysates. It is a member of the like peptidase domain with one or two C-terminal PDZ domain that Kunitz-type serine peptidase that possesses both anti-inflammatory and combined peptidase/chaperone function. It is crucial regulator of anticoagulatory effects [104] and generated partial protection against

7 K.M. Rebello, et al. Molecular & Biochemical Parasitology 239 (2020) 111311

S. mansoni infection in murine model [105,106]. Kunitz-type serine [4] L.A. Barros, L.A. Arruda, V.S. Gomes, D. Côrrea, R.M. Pinto, First natural infection peptidase inhibitor has also been described in L3 larvae of A. simplex by Ascocotyle (Phagicola) longa Ransom (Digenea, Heterophyidae) in an avian host, Ardea cocoi Linnaeus (Aves, Ciconiiformes, Ardeidae) in Brazil, Rev. Bras. Zool. 19 and A. pegreffii, both are causative agents of the fishborne gastro­ (2002) 151–155. intestinal infection named anisakiasis [107,108]. This inhibitor was [5] S.R. Martorelli, A. Lino, P. Marcotegui, M.M. Montes, P. Alda, C.J. Panei, demonstrated to be a major allergen and a useful tool for the diagnosis Morphological and molecular identification of the fish-borne metacercaria of Ascocotyle (Phagicola) longa Ransom, 1920 in Mugil liza from Argentina, Vet. of A. simplex allergy in humans [109,110]. Parasitol. 190 (2012) 599–603. [6] C.P. Santos, K.C. Lopes, V. da Silva Costa, E.G. dos Santos, Fish-borne tremato­ 3.6. Antioxidant proteins dosis: potential risk of infection by Ascocotyle (Phagicola) longa (Heterophyidae), Vet. Parasitol. 193 (2013) 302–306. [7] P.P. Chieffi, O.H. Leite, R.M.D. Souza-Dias, D.M.A.V. Torres, A.C.S. Mangini, The parasitic trematode must survive in the hostile environment of Human parasitism by Phagicola sp. (Trematoda, Heterophyidae) in Cananéia, São the host's to allow the continuation of its life cycle. As part of its stra­ Paulo State, Brazil, Rev. Inst. Med. Trop. São Paulo 32 (1990) 285–288. tegies, the parasite encodes antioxidant enzymes to protect itself [8] N.M. Hung, H. Madsen, B. Fried, Global status of fish-borne zoonotic trematodiasis in humans, Acta Parasitol. 58 (2013) 231–258. against reactive oxygen and nitrogen species generated by both en­ [9] E.M. Pereira, G. Muller, E. Secchi, J. Pereira Jr., A.L. 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