Saleh et al. Vet Res (2018) 49:37 https://doi.org/10.1186/s13567-018-0535-9

RESEARCH ARTICLE Open Access Quantitative shotgun proteomics distinguishes wound‑healing biomarker signatures in common carp skin mucus in response to Ichthyophthirius multifliis Mona Saleh1*, Gokhlesh Kumar1, Abdel‑Azeem Abdel‑Baki2,3, Mohamed A. Dkhil2,4, Mansour El‑Matbouli1 and Saleh Al‑Quraishy2

Abstract Ichthyophthirius multifliis is a ciliated protozoan parasite recognized as one of the most pathogenic diseases of wild and cultured freshwater fsh. Fish skin mucus plays a signifcant role against invading pathogens. However, the pro‑ tein-based modulation against infection with I. multifliis, of host fsh at this barrier is unknown. Thus, we investigated the skin mucus proteome of common carp using a shotgun proteomic approach at days 1 and 9 after I. multifliis exposure. We identifed 25 diferentially expressed in infected carp skin mucus. Upregulated proteins were mainly involved in metabolism, whereas downregulated proteins were mainly structural. This is the frst proteomic analysis of infected common carp skin mucus, and it provides novel information about proteome alteration caused by I. multifliis. Furthermore, we identifed novel proteins with yet unknown function in common carp following penetrat‑ ing injuries such as olfactomedin 4, , dermatopontin, papilin and I cytoskeletal 18. This analysis, therefore, represents a key for the search for potential biomarkers, which can help in a better understanding and monitoring of interactions between carp and I. multifliis. This proteomic study not only provides information on the -level pathways involved in fsh-ciliate interactions but also could represent a complementary system for studying tissue repair.

Introduction the modulation of the skin mucus proteome has been Intensive fsh farming raises the incidence of injuries investigated in response to infection, wounds, stress, or and diseases. Skin mucus acts as a mechanical, physical, after administration of diferent dietary supplements chemical, biological, and immunological barrier against [3–7]. any external stressor [1]. Skin mucus has become a novel and tissue repair are highly complex area of research and a true indicator of the immune status and indispensable processes to ensure the survival and of fsh. Te current advances in proteomics studies have health of an organism. Te regenerative competence been used for the characterization, identifcation, and difers considerably across organs and organisms and quantifcation of proteins [2]. Te skin mucus proteome requires the harmonized interaction of diferent cell has recently been studied in several fsh species. Tese types, signaling systems including cytokines, growth studies have revealed novel molecules involved in protec- factors, cellular matrix molecules, and diferent classes tion and immunity of this mucosal surface. Furthermore, of proteases, as well as their inhibitors [8]. However, the information on cellular and molecular mechanisms involved in these processes is still limited. *Correspondence: [email protected] Certain lower vertebrates such as teleost fsh have 1 Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria greater regenerative capacity than mammals. Tere- Full list of author information is available at the end of the article fore, they are used as a model for tissue regeneration in

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humans. Cordero et al. [1] investigated the modifca- glass slides, mucus was collected from the fsh skin while tion of the skin mucus proteome after inducing chronic avoiding blood contamination and excluding the ventral wounds in gilthead seabream (Sparus aurata) and found body surface close to the anal pore to prevent fecal con- that structural proteins, which are involved in tissue tamination. Collected mucus was transferred into 1.5-mL repair, were downregulated. However, little information microcentrifuge tubes, directly placed on ice, and then is available on the proteomic modifcation of fsh skin stored in a −80 °C freezer until further analysis. mucus in response to tissue damage caused by invading pathogens. Protein extraction Gonzalez et al. [9] reported a recruitment of neutro- Fish mucus was solubilized using 400 µL pre-cooled phils along with an initial upregulation, followed by the denaturing lysis bufer (7 M urea, 2 M thiourea, 4% downregulation of the proinfammatory cytokines (IL1β) CHAPS, and 1% DTT) containing mammalian protease and the chemokine receptor (CXCR1) in Ichthyoph- inhibitor cocktail (Sigma Aldrich, Vienna, Austria). Fish thirius multifliis-infected common carp skin, whereas mucus suspensions were disrupted by sonication. Te Jørgensen [10] directly visualized the recruitment of neu- lysates were then incubated overnight at 4 °C. Subse- trophils to infected areas in the skin. Tis is comparable quently, the lysates were vortexed, and then centrifuged to the expression pattern induced by mechanical at 18 000 × g for 30 min at 4 °C and the supernatants injuries and attributed to I. multifliis-induced injuries were collected. Te total protein concentration of each (penetrating wounds) at infection sites [9]. lysate was determined colorimetrically with a NanoDrop We hypothesized that a proteomic evaluation of skin 2000c (Termo Fisher Scientifc, USA) spectropho- mucus from non-exposed fsh against those exposed to tometer using a Pierce 660 nm Protein Assay (Pierce, I. multifliis would facilitate in identifying specifc mucus Termo Fisher Scientifc) according to the manufactur- components that are involved in carp (Cyprinus Carpio) er’s instructions. immune response to tissue damage caused by the para- site. Te aim of this study was to investigate the modu- Protein separation lation of the skin mucus after infection with I. multifliis Te protein samples (40 µg per lane) in biological and using quantitative proteomics to provide insights into the technical triplicate were subjected to electrophoresis in post-transcriptional and post-translational regulation of 10% polyacrylamide separating gels. After electrophore- skin mucus proteins. sis, the gels were stained with silver staining.

Materials and methods In‑gel digestion Animals and collection of samples Protein bands were excised manually from silver-stained Specifc pathogen-free common carp (11 ± 1 cm) were one-dimensional gels. After washing and destaining, obtained from a certifed Austrian hatchery and accli- bands were reduced with dithiothreitol and alkylated matized for 2 weeks under controlled laboratory condi- with iodoacetamide [12]. In-gel digestion was performed tions at 16 °C. Te fsh were fed 1% body weight per day using trypsin (Trypsin Gold, Mass Spectrometry Grade, using a commercial pellet diet (Garant Aqua, Pöchlarn, Promega, Madison, WI, USA) with a fnal trypsin concen- Austria). Before infection, 36 fsh were distributed tration of 20 ng/µL in 50 mM aqueous ammonium bicar- between six aquaria, six fsh per aquarium. Tere were bonate and 5 mM CaCl­ 2. In-gel digestion was performed two groups: exposed and non-exposed control. To mimic for 8 h at 37 °C using trypsin at a fnal concentration of natural exposure [11], the fsh were exposed to I. multifl- 20 ng/µL in 50 mM aqueous ammonium bicarbonate and iis by cohabitation with naturally infected giant gourami 5 mM ­CaCl2 [13]. Subsequently, peptides were extracted (Osphronemus goramy) obtained from a pet store. Te with three changes of 30 µL of 5% trifuoroacetic (TFA) giant gourami did not show any other ectoparasite or acid in 50% aqueous acetonitrile supported by ultrasoni- signs of a secondary bacterial infection. Te gouramis cation for 10 min per change. Extracted peptides were were also certifed as free from Aphanomyces invadans, dried down in a vacuum concentrator (Eppendorf, Ham- the causative agent of the epizootic ulcerative syndrome burg, Germany), and then re-dissolved in 0.1% aqueous and the Epizootic Haematopoietic Necrosis Virus, how- TFA before liquid chromatography–mass spectrometry ever, it should be kept in mind that they could harbour (LC–MS) injection. another disease. At 1 and 9 days post-exposure (dpe), Liquid chromatography–tandom mass spectrometry (LC– common carp (n = 3) from each of the exposed and non-exposed control groups were anesthetized using MS/MS) analysis 100 mg/L of ethyl 3-aminobenzoate methanesulfonate Peptides were separated on a nano-HPLC Ultimate (MS-222; Sigma, Darmstadt, Germany). Using sterile 3000 RSLC system (Dionex, USA). Te samples were Saleh et al. Vet Res (2018) 49:37 Page 3 of 12

pre-concentrated and desalted using a 5-mm Acclaim principal component analysis in the form of loading and PepMap μ-Precolumn (300 µm inner diameter, 5 µm par- score plots to get the frst impression of the overall data ticle size, and 100 Å pore size; Dionex, USA). For sample structure and assess the variability between technical and loading and desalting, 2% ACN in ultra-pure H­ 2O with biological replicates. 0.05% TFA was used as a mobile phase with a fow rate of To determine diferentially expressed proteins in the 5 µL/min. Peptides were separated on a 25-cm Acclaim mucus samples, statistical evaluation was performed in PepMap C18 column (75 µm inner diameter, 2 µm parti- R programming language [15]. Te diferential expres- cle size, and 100 Å pore size) with a fow rate of 300 nL/ sion of proteins was evaluated using one-way analysis of min. Te gradient started with 4% B (80% ACN with 0.1% variance (ANOVA) for each protein. For multiple test- formic acid) and increased to 35% B in 120 min. A wash- ing, the method of Benjamini and Hochberg [16] was ing step with 90% B was then performed. Mobile phase A used to control the FDR. Te diferences were considered contained ultra-pure ­H2O with 0.1% formic acid. signifcant if adjusted p values were smaller than the sig- nifcance level of α = 0.01. For such proteins, the honest Quadrupole time of fight (QTOF) mass spectrometry signifcant diference method of Tukey was applied as for sequential window acquisition of all theoretical mass post hoc test to assess the signifcance of the pairwise spectra (SWATH) measurements comparisons. Protein expression was considered difer- For mass spectrometric analysis, the LC was coupled to a ential if the adjusted p value was below α and the absolute high-resolution quadrupole time of fight mass spectrom- fold change was at least three (fold change < −3 or > +3). eter (Triple TOF 5600+, Sciex, USA). Data-independent PANTHER classifcation system was used for the clas- Sequential Window Acquisition of all Teoretical spectra sifcation of biological processes, cellular components, and (SWATH runs) technology based on MS2 quantifcation molecular functions of diferentially expressed proteins. To was used for quantitative measurements [14]. Peptides determine the network of diferentially expressed proteins, were fragmented in 35 fxed fragmentation windows of amino acid sequences of identifed proteins were BLAST 20 Da in the range of 400–1100 Da with an accumulation searched using the string tool based on the study conducted time of 50 min in TOF–MS and 80 min in product ion on Danio rerio [17]. Te representation of the protein–pro- mode. Te nano-HPLC system was operated by Chrome- tein network was analyzed at the confdence score of 0.15 in leon 6.8 (Dionex, USA) and the MS by Analyst Software the database, experiment, text mining, co-expression, neigh- 1.6 (Sciex, USA). borhood, gene fusion, and co-occurrence database.

Data processing, quantifcation, and statistical evaluation Results Acquired raw data were assessed using ProteinPilot Protein identifcation software version 5.0 (Sciex, USA) for re-calibration and We identifed 1233 proteins in the skin mucus of common database searches using NCBI entries of Cyprinus (tax- carp. Twenty-fve structural and metabolism proteins onomy id: 7961). Mass tolerance in MS mode was set were diferentially expressed in infected carp skin mucus. with 0.05 and 0.1 Da in MSMS mode for the rapid re- Nineteen proteins were mainly structural and extracellu- calibration search, and 0.0011 Da in MS and 0.01 Da in lar matrix proteins, whereas six proteins have a distinctive MSMS mode for the fnal search. Te following sample role in metabolism (Tables 1 and 2). Tese proteins were parameters were used trypsin digestion, cysteine alkyla- associated with e.g. cellular, metabolic, developmental and tion set to iodoacetamide, and the processing parameter immune processes, as well as biological regulation, locali- was set to a rapid ID search efort. False discovery rate zation, response to a stimulus, and multicellular organ- analysis (FDR) was evaluated using the integrated tools ismal process. Further, they were implicated in binding, in ProteinPilot and was set to < 1% at the protein level. catalytic, receptor, signal transducer, structural, molecu- Te results of Information Dependent Acquisition (IDA) lar, and transporter activities. Te expression of top skin identifcation were used to create the SWATH ion library mucus proteins is shown in Figure 1. Te infected samples with the MS/MS (ALL) with SWATH Acquisition Micro- either show lower quantitative values of alpha, App 2.0 in PeakView 2.2 (both Sciex, USA). Peptides were lumican, dermatopontin, and papilin or higher quanti- chosen based on an FDR rate < 1%, excluding shared and tative values of keratin type I, myosin, and UDP-glucose modifed peptides. Up to six peptides per protein and 6-dehydrogenase than the control samples. up to six transitions per peptide were used. MarkerView 1.2.1 (Sciex) was used for the calculation of peak areas Structural and proteins of SWATH samples after retention time alignment and We identifed 11 members belonging to 6 collagen-alpha normalization using total area sums. Te resulting pro- family proteins (type I alpha 1a, type I alpha 2, type VI tein lists were then used for the visualization of data after alpha 1, type VI alpha 2, type VI alpha 3, and type XIV Saleh et al. Vet Res (2018) 49:37 Page 4 of 12 forming - forming mechanical injuries, pore mechanical injuries, activities lates its structure, and enhances its structure, lates and neutrophils macrophages recruitment actin flaments Protect cells from mechanical and non cells from Protect ‑ Binds collagen fbrils and regu Actin regulatory protein, stabilises Actin regulatory protein, Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Matrix component organisation Function 11.4* − 9.1* 2.6 − 10.9* − 5.2* − 5.3* − 31.2* − 8.3* − 88.2* − 4.5* − 9.9* − 35.7* − 37.1* 9 dpe 2.0 − 8.7* − 1.2 − 4.9* − 5.1* − 3.7* − 27.3* − 6.1* − 99.9* − 6.0* − 11.3* − 50.2* − 18.7* 1 dpe ], XP_016414194.1, 96% rhinocerous identity ], XP_018932781.1, carpio [ Cyprinus 96% identity ], [ Sinocyclocheilus rhinocerous XP_016385859.1, 90% identity like X3 [ Salmo isoform sala r], XP_014019344.1, 62% identity – lumican - like [ Sinocyclocheilus – – – collagen alpha - 2(VI) chain - like – – – – – collagen alpha - 2(I) chain like collagen alpha - 3(VI) chain - BLASTp results BLASTp 6 2 2 2 2 2 2 5 4 3 5 6 6 quantifed peptides Number of quantifed ] nus carpio ] carpio ] carpio [ Cyprinus ] carpio [ Cyprinus ] nus carpio ] carpio ] nus carpio ] nus carpio ] carpio keratin, type I cytoskeletal- 18 [ Cypri ] cypCar_00015496 carpio [ Cyprinus src substrate cortactin substrate src - likeCyprinus [ ] carpio dermatopontin - likeCyprinus [ collagen alpha - 1(XIV) chain - like ] cypCar_00016174 carpio [ Cyprinus collagen alpha - 1(XIV) chain - like collagen alpha - 1(VI)- chain - like [ Cypri cypCar_00043888, partial [ Cyprinus collagen alpha - 2(VI)- chain - like [ Cypri - collagen alpha - 1(I) chain like [ Cypri cypCar_00045321, partial [ Cyprinus ] cypCar_00022254 carpio [ Cyprinus Protein Diferentially expressed structural and extracellular matrix proteins of common carp of common proteins matrix structural and extracellular expressed Diferentially P_018921152.1 KTF82439.1 XP_018958339.1 XP_018950001.1 XP_018933497.1 KTF76685.1 XP_018962933.1 XP_018967802.1 KTF78707.1 XP_018967439.1 XP_018968199.1 KTF73577.1 KTG36050.1 Structural and extracellular matrix proteins 1 Table number NCBI accession Saleh et al. Vet Res (2018) 49:37 Page 5 of 12 ‑ ‑ forming - forming forming activities- forming activation tion, pore rearrangement cytes cytes mechanical injuries, pore mechanical injuries, activities Negative feedback efect on NF - κB efect feedback Negative Microtubule organization and stabiliza ‑ Microtubule organization metalloproteinase inhibitor, tissue inhibitor, metalloproteinase Transendothelial migration of leuko migration Transendothelial Transendothelial migration of leuko migration Transendothelial Protect cells from mechanical and non cells from Protect Function 1.3 3.5* − 11.4* 6* 2.5 − 3.5* 9 dpe 3.3* − 1 − 15* − 1.4 4.4* 1.2 1 dpe - subunit beta - like [ Sinocyclocheilus anshuiensis ], XP_016330044.1, 91% identity clocheilus anshuiensis ] [Danio rerio], XP_009289654.1, 48% [Danio rerio], identity ] rhinocerous identity – PREDICTED: neoverrucotoxin PREDICTED: papilin - like [ Sinocy XP_016354784.1, 92% identity myosin heavy chain, clone 203 myosin - 11 like [ Sinocyclocheilus myosin Sequence ID: XP_016405427.1, 90% – BLASTp results BLASTp 4 2 5 6 6 3 quantifed peptides Number of quantifed − 3 or > + 3. < 0.001 and fold change 0.001 and fold < ] carpio [ Cyprinus ] carpio ] carpio [ Cyprinus olfactomedin - 4 like X2 isoform cypCar_00043727, partial [ Cyprinus ] cypCar_00036737 carpio [ Cyprinus ] carpio [ Cyprinus LOC109057295 ] carpio X1 [ Cyprinus MYH16 isoform keratin, type I cytoskeletal 18 - like Protein continued XP_018924262.1 KTF72113.1 KTF75732.1 XP_018930066.1 XP_018980214.1 XP_018948461.1 1 Table to according signifcant (italic) statistically post-exposure values (dpe). * Denotes carp1 and 9 days at common exposed infected multifliis in Ichthyophthirius analyzed statistically was control) vs change (infected Fold p value with FDR-adjusted both ANOVA NCBI accession number NCBI accession Saleh et al. Vet Res (2018) 49:37 Page 6 of 12

Table 2 Diferentially expressed metabolism proteins of common carp NCBI Accession number Protein Number of quantifed BLASTp 1 dpe 9 dpe Function peptides results

Metabolism proteins XP_018962399.1 UDP-glucose 6-dehydroge‑ 6 – 1.6 4.4* Catalyzes the conversion of nase [Cyprinus carpio] glucose 6-phosphate to 6-phosphogluconate XP_018964044.1 clustered mitochondria 2 – 1.5 3.5* Regulate mitochondrial protein homolog [Cypri- metabolism nus carpio] XP_018973586.1 arachidonate 5-lipoxyge‑ 4 – 4* 10.1* Transforms essential fatty nase-like [Cyprinus carpio] − − acid (EFA) substrates into leukotrienes XP_018937956.1 arachidonate 5-lipoxyge‑ 4 – 3* 6* Transforms essential fatty nase-like [Cyprinus carpio] − − acid (EFA) substrates into leukotrienes XP_018936787.1 PDZ and LIM domain 4 – 1.0 3.0* Bind to the NF-κB subunit protein 1-like [Cyprinus − p65 and inhibits its tran‑ carpio] scriptional activity XP_018979180.1 relA-associated inhibitor- 4 – 2.1 2.8 Bind to the NF-κB subunit like [Cyprinus carpio] − p65 and inhibits its tran‑ scriptional activity

Fold change (infected vs control) was statistically analyzed in Ichthyophthirius multifliis exposed infected common carp at 1 and 9 days post-exposure (dpe). * Denotes values (italic) statistically signifcant according to both ANOVA with FDR-adjusted p value < 0.001 and fold change < 3 or > 3. − + alpha 1). In addition, we identifed the following pro- were involved in the protein–protein interaction net- teins: Src substrate cortactin-like, dermatopontin-like, work. Details of protein abbreviation, node colour, edge papilin-like, neoverrucotoxin, olfactomedin 4, lumican, interaction, network stats and functional enrichment are myosin (n = 2), and keratin (n = 2). Most of the structural described in Additional fle 1. Te data showed that colla- proteins were downregulated (Table 1). Te extracellular gen type I alpha was the central node of protein–protein matrix proteins such as olfactomedin 4, neoverrucotoxin, interaction analysis. Tese diferentially up- and down- myosin, and keratin were upregulated. Te extracellu- regulated proteins showed two KEGG pathways: extra- lar matrix components that are hallmarks of the wound cellular matrix receptor interaction and focal adhesion. matrix [18] were identifed with diferential quantitative Pfam and InterPro database evaluation (as a part of the values in infected and non-infected carp. Tese compo- string) revealed enrichment in collagen triple helix repeat nents include collagen alpha, neoverrucotoxin (contains and von Willebrand factor, type A domain-containing fbronectin type III domain), olfactomedin 4, and lumi- proteins. Similarly, on the same dataset, the PANTHER can. Collagen alpha members were extremely down- analysis found an increase in the following biological regulated in infected carp. Tese observations link the processes: cellular, metabolic, biological regulation, and downregulation of the collagen alpha to penetrating response to a stimulus. wounds caused by I. multifliis, and lead to the hypothesis that defciency in collagen alpha may refect an impaired Discussion healing state during parasite attachment to carp skin. In vertebrates, the mucosal immune system has crucial functions against infections. It prevents the uptake of Metabolism proteins microorganisms and foreign substances and avoids the We identifed the following metabolism proteins (Table 2): development of destructive immune responses against UDP-glucose 6-dehydrogenase, clustered mitochondria invasive pathogens. Te protective role of fsh skin mucus protein homolog (CLUH), PDZ/LIM domain protein 1-like, is of immense economic signifcance, as infectious dis- RelA-associated inhibitor (RAI)-like, and arachidonate eases limit intensive aquaculture globally. 5-lipoxygenase (ALOX5)-like (n = 2). Te expression of these In this study, the expression of 11 collagen-alpha mem- proteins was upregulated at 9 dpe, except for ALOX5-like. bers was extremely downregulated likely because of the increased production of parasite and carp proteases. It Protein–protein interaction network has been suggested that fsh parasites release proteolytic As shown in Figure 2, eight proteins (six collagen alpha enzymes to degrade collagen and other structural mol- family proteins, lumican, and dermatopontin proteins) ecules to assist in disruption of external epithelia as an Saleh et al. Vet Res (2018) 49:37 Page 7 of 12

Figure 1 Expression plots of top candidate carp skin mucus proteins. Plots show proteins up and down-regulation of carp skin mucus at 0 (control), 1, and 9 days post-exposure. invasion strategy [19]. I. multifliis proteolytic repertoire extracellular matrix and tissue remodeling to limit pro- (degradome) includes 254 protease homologs, approxi- teolysis and subtle regulation of immune processes [23]. mately 3.1% of the proteome [20]. In another study, cortisol and lice have been reported to are extracellular matrix proteins that play a equally downregulate a large number of motor proteins structural role in the body of humans and animals. Col- that have considerable roles in wound contraction and lagen degradation and downregulation are believed to healing [21]. Te downregulation of collagens and other limit the peripheral damage of healthy tissues, frst by structural proteins was in parallel with the induction of releasing metalloproteinases to cleave collagen fbrils and metalloproteinases that degrade extracellular matrix [21]. then taking up the resulting fragments for wound heal- Cortisol and, to a lesser extent, lice enhanced the colla- ing. Te responses because of skin damage signify a com- gen-degrading matrix metalloproteinases. Te down- plex cascade of events that involves several overlapping expression of carp collagen proteins after exposure to I. stages including hemostasis, infammation, proliferation, multifliis suggests a process of massive degradation of and maturation. Te enzymes that destroy components collagen aimed at tissue remodeling and wound healing of the extracellular matrix are involved in both infam- of carp skin in a similar response pattern as for Atlantic mation and tissue repair and can be considered a bridge salmon during the salmon louse infection experiments between these phases [21]. It has been reported that conducted by Krasnov et al. [21]. several matrix metalloproteinases are upregulated in Te expression of carp Src substrate cortactin-like Atlantic salmon skin during the early stages of infec- protein was downregulated (1.2-fold) at 1 dpe but then tion by salmon louse [22]. Tese enzymes have a wide increased (2.6-fold) at 9 dpe resulting in a considerable range of functions including the massive degradation of relative upregulation (3.3-fold). Cortactin regulates actin Saleh et al. Vet Res (2018) 49:37 Page 8 of 12

Figure 2 The protein–protein interaction network of carp skin mucus proteins. In this network, nodes are proteins, lines represent the predicted functional associations, and the number of lines represents the strength of predicted functional interactions between proteins. Eight proteins including six collagen alpha family proteins (col1a2, col1a1a, col6a1, col6a2, col6a3, col14a1), lumican (lum), and dermatopontin (DPT) were involved in the protein–protein interaction network. The fgure shows that collagen type I alpha is the central node of the protein–protein interaction network. assembly and cell migration by stimulating actin-related particularly during re-epithelialization by promoting protein Arp 2/3-mediated actin polymerization. High migration [27]. Te considerable downregu- cortactin expression is associated with cell motility, lation of the carp dermatopontin-like protein was likely invasion, and metastasis, and the elevated expression of to limit infammation and tissue damage caused by I. cortactin correlates with the poor prognosis in human multifliis. carcinomas [24]. Watt et al. [25] reported that protein Te expression of carp papilin protein was downregu- tyrosine phosphorylation occurs within seconds of injury lated (11-fold) after exposure to I. multifliis. Papilin to the surface of intact articular cartilage, as does the interacts with several extracellular matrix components activation of mitogen-activated protein kinases (MAPKs) and enzymes and is essential for embryonic develop- and IκB kinase (IKK). Te tyrosine-phosphorylated pro- ment of Drosophila melanogaster and Caenorhabditis tein cortactin was identifed together with adhesion elegans [28]. In vitro, papilin non-competitively inhibits kinase and paxillin as the substrates of Src family kinases. procollagen N proteinase, an ADAMTS metalloprotein- Injury to the surface of intact articular cartilage has been ase. Targeted disruption of papilin by RNAi experiments suggested to activate Src-like kinases, as well as MAPKs on Drosophila embryos led to a repeated decrease in and IKK involved in NF-κB regulation [25]. Te modula- survival rates, correlated to muscle defects [28]. Tus, it tion of the carp Src substrate cortactin-like protein was has been suggested that papilin infuences cell rearrange- likely to modulate NF-κB activation, aimed at reducing ments in muscle tissue. Te downregulation of the carp infammation and tissue damage caused by I. multifliis. papilin was likely because of tissue damage as a result of In this study, the expression of the carp dermatopon- the development and growth of I. multifliis. tin-like protein was considerably downregulated (10.9- Te expression of lumican protein was downregu- fold) at 9 dpe. Dermatopontin regulates the assembly lated (ninefold) in infected carp. Lumican has several of the extracellular matrix through the acceleration of matricellular roles as an extracellular matrix constituent collagen and fbronectin fbrillogenesis [26]. Recently, it and as a matrikine involving in cell proliferation, gene has been suggested that it is involved in wound healing, expression, and wound healing [22]. Lumican regulates Saleh et al. Vet Res (2018) 49:37 Page 9 of 12

collagen fbrillogenesis, which is involved in the shap- infection by salmon louse [22]. Te upregulation of the ing and sustainment of clear corneas, promoting corneal myosin proteins was likely to support the migration of epithelial wound healing and the integrity of numerous leucocytes to the sites of infection as an active immune other connective tissues such as sclera, skin, and as a response of carp and to protect against tissue damage chemokine gradient maker [29]. In addition to regulating caused by I. multifliis. Te expression of carp MYH16 the collagen fbril architecture, lumican supports neutro- isoform X1 myosin protein was considerably upregulated phil recruitment and invasion after corneal damage and (fourfold) at 1 dpe and then decreased (2.5-fold) at 9 dpe infammation [30]. Lumican-defcient wounded mice after I. multifliis exposure, correlating with previously corneas showed delayed healing, reduced recruitment of reported leukocyte recruitment patterns [9, 10]. macrophages and neutrophils, and no induction of the In this study, the expression of neoverrucotoxin subunit proinfammatory cytokines, tumor necrosis factor-alpha beta protein was upregulated (3.5-fold) in the skin mucus (TNFα), and IL1β. Lumican is highly conserved between of infected carp at 9 dpe. that contain fbronectin zebrafsh and mammals, such as human and mouse, in type III and neoverrucotoxin subunit domains were pre- respect to gene structure, expression patterns, and pro- dicted to be involved in microtubule organization and tein function [31]. Tus, the downregulation of lumican stabilization [36]. Neoverrucotoxin in fsh venom has a was likely to reduce infammatory response and tissue mechanism of action where it has been shown to func- damage caused by I. multifliis and correlates with pre- tion by forming pores in cell membranes [37]. Te skin vious fndings on fsh mimicking tissue toxins in Gobiodon have a range of biological functions, injuries and leukocyte recruitment [9]. including parasite and predator prevention [38]. Te In this study, carp keratin I cytoskeletal 18 protein was upregulation of the neoverrucotoxin subunit beta protein upregulated (11.4-fold) after I. multifliis infection, while was likely to defend fsh against the parasite. However, its keratin I cytoskeletal 18-like was downregulated (3.5-fold) specifc role in carp immune response against I. multifl- at 9 dpe. Keratin has been identifed in the skin mucus of iis should be thoroughly explored. gilthead seabream [32], Atlantic cod [33], and European In this study, olfactomedin 4 was upregulated (3.3-fold) sea bass [1]. Keratin is a cytoskeletal protein whose pri- at 1 dpe but then was downregulated (1.3-fold) at 9 dpe. mary function is to protect cells from mechanical and Olfactomedin 4 was originally detected in the prepa- non-mechanical injuries. In addition, it has been shown rations of chemosensory dendritic cilia obtained from that the keratin of fsh mucus possesses antibacterial olfactory epithelium of the bullfrog, Litobathes catesbe- activity because of its pore-forming properties [34]. Kera- ianus [39]. In addition, it is upregulated in infammatory tin turnover is dependent on the ubiquitin–proteasome bowel diseases and Helicobacter pylori-infected patients. pathway, and its expression levels can be altered upon Olfactomedin 4 is the target gene of nuclear factor kappa injury. Recently, it has been reported that the expression B (NF-κB) pathway and has a negative feedback efect of keratin I and II was downregulated in cod mucus, fol- on NF-κB activation induced by H. pylori infection [40]. lowing a vibrio infection, and in gilthead seabream in In a transcriptomic profling study, several central sig- response to chronic wounds [1, 5]. Te carp keratin I natures of catfsh responses following columnaris infec- cytoskeletal 18 proteins were diferentially regulated after tion were identifed. A suppression of NF-κB signaling I. multifliis infection, suggesting they may play diferent and olfactomedin 4 expression was observed in catfsh sequential and/or site dependant roles in carp immune gills, following columnaris infection [41]. Olfactomedin 4 response aimed at protection against the parasite. expression was upregulated at 1 dpe and then reduced at Te expression of carp myosin proteins was consider- 2 dpe. Te expression pattern of olfactomedin 4 was sim- ably upregulated after I. multifliis infection. In fsh, dis- ilar to that observed in catfsh gills, following columnaris ease resistance has been suggested to be associated with infection as reported by Sun et al. [41], likely to reduce the expression of myosin heavy chain protein. Te prot- excessive tissue damage caused by carp infammatory eomic profling of zebrafsh fns was recently determined response induced by I. multifliis. after a hemorrhagic septicemia Rhabdovirus infection Diferentially regulated metabolism proteins were iden- using two-dimensional diferential gel electrophoresis tifed in carp skin mucus (Table 2). In this study, the carp [35]. Several diferentially expressed proteins identi- ALOX5 proteins were downregulated. Te lipoxygenases fed were related to the and involved in fn are lipid peroxidizing enzymes, which have been involved regeneration including myosin [35]. A gene expression in the biosynthesis of pro- and anti-infammatory media- study of Atlantic salmon (Salmo salar) showed diferen- tors [42]. It transforms EFA substrates into leukotrienes. tial expression of the transcripts of some proteins that Te overproduction of leukotrienes is a major cause of have a major role in the transendothelial migration of infammation in asthma, allergic rhinitis, and osteoar- leukocytes, including myosin during the early stages of thritis. In addition, they play a role in the pathogenesis Saleh et al. Vet Res (2018) 49:37 Page 10 of 12

of cardiovascular [43], hyperproliferative [44], and neu- chemokines, and immunoreceptors [53]. Gonzalez et al. rological [45] diseases. Te fnding that zebrafsh (Danio [9] identifed two putative molecules of the NF-κB signal- rerio) expresses a functional ALOX5, together with the ing pathway, which show strong similarity (63–68%) with observation that most other human leukotriene-relevant the zebrafsh IκBα in addition to a predicted molecule genes have an ortholog in the zebrafsh genome, suggests that is similar to NF-κB. It was the frst time that mol- the biological relevance of leukotriene signaling in lower ecules from the NF-κB signaling pathway were described vertebrates [46]. Te downregulation of the ALOX5 pro- in I. multifliis-infected carp. Te upregulation of the RAI teins was likely to reduce carp infammatory response protein expression likely inhibits NF-κB and decreases because of the invasion and development of I. multifliis. cytokine production to reduce infammation and tissue UDP-Glucose-6-phosphate dehydrogenase was overex- damage induced by I. multifliis. pressed (4.4-fold) in the skin mucus of infected carp at PDZ/LIM domain 1-like protein was considerably 9 dpe. Tis protein catalyzes the conversion of glucose upregulated (3.0-fold) at 9 dpe. Te actin-associated pro- 6-phosphate to 6-phosphogluconate [47]. Te most cru- tein family members containing PDZ/LIM protein–pro- cial function of this enzyme is the biosynthesis of glycosa- tein interaction motifs are involved in the heart and fn minoglycans and other essential components of several development of zebrafsh [54]. In salmon, alpha-actinin- extracellular matrix molecules, including chondroitin associated LIM protein containing PDZ and LIM motifs sulfate, heparan sulfate, and hyaluronic acid. Te pro- builds multi-protein complexes linking actin in muscles duction of these molecules could afect several develop- and non-muscular tissues [55]. PDLIM1, another mem- mental processes. Te changes in Glucose-6-phosphate ber of LIM proteins, has been reported to negatively dehydrogenase (G6PD) enzyme activity have been stud- regulate NF-κB-mediated signaling in the cytoplasm. ied in liver and gill tissues of rainbow trout after exposure PDLIM1 sequestered the p65 subunit of NF-κB in the to organophosphate pesticide chlorpyrifos [48]. Acute cytoplasm and suppressed its nuclear translocation in exposure to chlorpyrifos exhibited apoptotic efects in an IκBα-independent manner [56]. Te upregulation the liver and gill tissues associated with a time-dependent of PDZ/LIM domain 1-like protein was likely because decrease in G6PD enzyme activity at all concentrations of the suppression of carp immune response to reduce tested [48]. Te upregulation of G6PD likely activates tis- infammation and tissue damage caused by I. multifliis sue repair against damage caused by I. multifliis. attachment and growth. However, the specifc role of this Te CLUH expression was upregulated during I. mul- protein in carp skin mucus needs to be determined. tifliis infection and considerably increased (3.5-fold) at In this study, we identifed proteins that were entirely 9 dpe. Furthermore, CLUH is a cytosolic RNA-binding novel in the context of the fsh host response to I. mul- protein specifc for a subset of mRNAs encoding mito- tifliis, such as olfactomedin 4, lumican, dermatopontin, chondrial proteins [49]. Tus, CLUH was considered as papilin and I cytoskeletal 18. Te modulation of these an excellent candidate for performing a post-transcrip- proteins in the fsh skin mucus suggests a role in immune tional regulatory function. In HeLa cells, CLUH was response aimed at protecting against tissue damage reported to bind several mRNAs involved in intermediate caused by this parasite. Proteases from parasitic proto- metabolism and oxidative phosphorylation [49]. CLUH zoa have been involved in host invasion and emergence, defciency was found to afect respiratory function and encystment and excystment, cytoadherence, stimulation mitochondrial DNA content, distribution, and ultras- and evasion of host immune responses, and catabolism tructure [50]. Te upregulation of CLUH protein expres- of host proteins for nutrients [57]. Te cysteine protease, sion apparently co-ordinates carp immune response to cathepsin L was diferentially expressed among all life cope with adverse efects caused by this ciliate. stages and was suggested to play important roles in host- In this study, the expression of RAI protein was modu- pathogen interactions [58, 59]. Indeed, the largest up- lated after I. multifliis infection. Te expression of this regulation of cathepsin L cysteine protease was observed protein was downregulated (1.2-fold) at 1 dpe, but then in the infective theront [59]. Hence, proteases from I. increased (2.8-fold) at 9 dpe resulting in a considerable multifliis have been suggested to suppress fsh immune relative upregulation (3.3-fold). RAI was reported to bind response, facilitate host invasion and support degrada- to the NF-κB subunit p65 and inhibit its transcriptional tion of host cells [59]. We suggest that the degradation activity. RAI inhibited the action of NF-κB in a transient of collagens and the modulation of the other structural luciferase gene expression assay and the endogenous proteins lead to the downregulation of the infammatory NF-κB activity induced by TNFα [51]. RAI can efectively response by targeting the NF-κB pathway and binding to block HIV-1 replication [52]. NF-κB is an inducible tran- the NF-κB subunit p65 to inhibit its transcriptional activ- scription factor in cells involved in immune and infam- ity aimed at reducing infammation and protecting fsh matory responses. It induces the expression of cytokines, against tissue damage caused by I. multifliis. However, Saleh et al. Vet Res (2018) 49:37 Page 11 of 12

specifc, functional studies are required to support this (Lepeophtheirus salmonis). Comp Biochem Physiol Part D Genom Proteom 4:159–167 suggestion and investigate the role of these diferentially 4. Easy RH, Ross NW (2010) Changes in Atlantic salmon Salmo salar mucus expressed proteins in tissue damage and wound healing components following short- and long-term handling stress. J Fish Biol to obtain a comprehensive understanding of the con- 77:1616–1631 5. Rajan B, Lokesh J, Kiron V, Brinchmann MF (2013) Diferentially expressed tributions of the fsh mucus layer in defense against I. proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural multifliis. Using quantitative proteomics, the obtained infection with Vibrio anguillarum. BMC Vet Res 9:103 results provide information on carp-I. multifliis inter- 6. 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MS, GK, AAAB and MAD analyzed the data. MS wrote the 12. Jiménez CR, Huang L, Qiu Y, Burlingame AL (2001) In-gel digestion of manuscript. GK, AAAB, MAD, MEM and SAQ revised the manuscript. All authors proteins for MALDI-MS fngerprint mapping. Curr Protoc Protein Sci 16:4 read and approved the fnal manuscript. 13. Shevchenko A, Wilm M, Vorm O, Man M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Acknowledgements Chem 68:850–858 The authors would like to thank Prof. Dr. Ebrahim Razzazi-Fazeli and the team 14. Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. of the VetCore Proteomics unit at University Veterinary Medicine, Vienna, Science 312:212–217 for the proteomic analyses. This study was funded by the national plan for 15. R Core Team (2015) R. a language and environment for statistical com‑ Science, technology and innovation (MAARIFAH), Project ID: 13-NAN2121-02, puting. 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