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Proteomic Analysis of Ascocotyle Longa (Trematoda: Heterophyidae) T Metacercariae Karina M

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Proteomic Analysis of Ascocotyle Longa (Trematoda: Heterophyidae) T Metacercariae Karina M Molecular & Biochemical Parasitology 239 (2020) 111311 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology journal homepage: www.elsevier.com/locate/molbiopara 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, enzyme 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 Protease 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 enzymes, ribosomal and structural/ serum albumin as standard [16]. Samples (50 μg) were than reduced motor proteins. Chaperones, proteases, oxidoreductases, calcium using 10 mM dithiothreitol (DTT) for 3 h at 37 °C and alkylated using binding proteins, isomerases, kinases, transferases, 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
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