Acta Tropica 140 (2014) 166–172

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Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga Karin Silva Caumo a,1 , Karina Mariante Monteiro b , Thiely Rodrigues Ott b , Vinicius José Maschio a , Glauber Wagner c , Henrique Bunselmeyer Ferreira b,∗ , Marilise Brittes Rott a a Laboratório de Parasitologia, Instituto de Ciências Básicas da Saúde, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal do Rio Grande do Sul, CEP: 90050170 Porto Alegre, RS, Brazil b Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, CEP: 91501-970 Porto Alegre, RS, Brazil c Laboratório de Doenc¸as Infecciosas e Parasitárias, Universidade do Oeste de Santa Catarina, CEP: 89600-000 Joac¸aba, SC, Brazil

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Article history: Received 25 April 2014 Received in revised form 31 July 2014 Accepted 8 August 2014 Available online 18 August 2014 Keywords: Acanthamoeba Trophozoite 2-DE LC–MS/MS Global protein analysis Pathogen–host interaction

a b s t r a c t Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC–MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3–10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC–MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Free-living amoebae (FLA) belonging to the genus Acanthamoeba are ubiquitously distributed in nature, and are adapted to live in a wide variety of natural and human-created environments (Schuster and Visvesvara, 2004; Caumo et al., 2009; Magliano et al., 2009; Carlesso et al., 2010; Winck et al., 2011; Siddiqui and Khan, 2012a). Acanthamoeba spp. have gained increasing attention from the scientific community over the years, due to their versatile roles in

∗ Corresponding author. Tel.: +55 51 3308 7768; fax: +55 51 3308 7309. E-mail addresses: [email protected] (K.S. Caumo), [email protected] (K.M. Monteiro), [email protected] (T.R. Ott), [email protected] (V.J. Maschio), [email protected] (G. Wagner), [email protected] (H.B. Ferreira), [email protected] (M.B. Rott). 1 Current address: Laboratório de Parasitologia Clínica, Centro de Ciências da Saúde, Departamento de Análises Clínicas, Universidade Federal de Santa Catarina, CEP: 88040-900 Florianópolis, SC, Brazil. http://dx.doi.org/10.1016/j.actatropica.2014.08.009 0001-706X/© 2014 Elsevier B.V. All rights reserved.

the ecosystem. The active trophozoite stage, which exhibits vegetative growth and is the infective form for mammalian hosts, feeds on bacteria, algae, and yeast. It is also a reservoir for pathogenic microorganisms that are resistant to phagocytosis by the amoebae, which may help to disperse important human pathogens such as Legionella pneumophila and Pseudomonas aeruginosa in the environment (Visvesvara et al., 2007; Siddiqui and Khan, 2012b). Several of the approximately 24 identified species of the genus Acanthamoeba have been linked to human disease, including Acanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba culbertsoni, Acanthamoeba healyi, and Acanthamoeba byersi (Visvesvara et al., 2007; Corsaro and Venditti, 2010; Visvesvara, 2010; Qvarnstrom et al., 2013). The growing importance of Acanthamoeba spp. in medical care and research during the last decade is due to their potential to infect human hosts, causing severe diseases, such as granulomatous amoebic encephalitis (GAE), a chronic brain infection that occurs more frequently in immunosuppressed individuals;

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amoebic keratitis (AK), a sight-threatening infection of the cornea that is related to contact lens misuse; and disseminated infections (Marciano-Cabral and Cabral, 2003; Visvesvara et al., 2007). Acanthamoeba spp. trophozoites also have been used extensively as model systems to study eukaryotic cell biology, because of their relatively large size, rapid growth in culture, and active motility (Horowitz and Hammer, 1990; Maciver and Hussey, 2002; Klopocka et al., 2009; Chrisman et al., 2010; Brzeska et al., 2012; Siddiqui and Khan, 2012a). The well-developed cytoskeleton of these organisms makes them especially good models for understanding actin cytoskeleton-based motility, and other molecular aspects of cell motility (Khan, 2006; Siddiqui and Khan, 2012c). Proteomic studies have been described for many protozoa, including Entamoeba histolytica, Giardia lamblia and Leishmania donovani (Tolstrup et al., 2007; Biller et al., 2009; Ali et al., 2012; Jerlstrom-Hultqvist et al., 2012; Pawar et al., 2012a; Faso et al., 2013). These analyses have revealed a diversity of proteins expressed by different parasitic species, helping to elucidate the molecular mechanisms of interaction with host species, and to identify potential biomarkers for diagnosis and targets for the development of new drugs or vaccines. For Acanthamoeba spp., the few proteomic studies have so far been limited to the investigation of protein expression during encystment (Bouyer et al., 2009; Leitsch et al., 2010). Although important for a better understanding of the biology of invasive forms, the repertoire of proteins expressed by Acanthamoeba spp. trophozoites has not yet been investigated. Analysis of the repertoire of proteins expressed by Acanthamoeba spp. trophozoites is expected to contribute to the elucidation of the mechanisms of virulence, and to the identification of diagnostic antigens and target proteins for therapy. Proteomic approaches based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) are powerful tools to capture the dynamics of global proteomic changes, with the simultaneous resolution and identification of large numbers of cellular proteins. These methods are particularly advantageous for the identification of stress-induced proteins along with their post-translational modifications, and to correlate altered protein abundance/modifications with physiological function(s) (Beranova-Giorgianni, 2003; Brewis and Brennan, 2010). In this study, we established the conditions for the 2-DE analysis of A. polyphaga trophozoites and identified most of the resolved proteins in order to provide a reference proteomic map. A gel-free LC–MS/MS analysis was also performed. Overall, 202 nonredundant proteins were identified, with 184 proteins identified from the 370 spots mapped in the 2-DE gel, along with 94 proteins identified by gel-free LC–MS/MS. Identified proteins were assigned to several functional classes: metabolism-related, cytoskeleton, post-translation modification, protein turnover and chaperones, surface-localized proteins, and proteins related to defense mechanisms. The importance of the identified repertoire of trophozoite proteins for the biology of A. polyphaga is discussed.

2. Material and methods 2.1. A. polyphaga strain, cultivation, and cell protein extracts A. polyphaga trophozoites of the T4 genotype were obtained from the American Type Culture Collection (ATCC 30872). This environmental isolate had its pathogenicity previously demonstrated (Veríssimo et al., 2013). Trophozoites were maintained in axenic cultures in peptone-yeast-glucose (PYG) medium, as previously described (Schuster, 2002), and samples for the proteomic analysis were directly taken from these cultures.

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Three identical and independent cultures (biological replicates) with approximately 1 × 108 trophozoites in the exponential growth phase were used for protein extraction. Cells were harvested at 2000 × g for 10 min and washed twice in phosphate-buffered saline (PBS) buffer (pH 7.2), prior to resuspension in 1 ml of 25 mM Tris–HCl, pH 7.2. Cell suspensions were then lysed by sonication (25 Hz in a VC601 Sonics and Materials Inc. sonicator) in an ice bath for five 30-s cycles with a 1-min interval between pulses. Lysates were centrifuged (18,000 × g, 15 min, 4 ◦ C) to separate soluble and insoluble protein fractions. Soluble proteins were quantified using a QubitTM quantitation fluorometer and Quant-itTM reagents (Invitrogen, USA). 2.2. Two-dimensional gel electrophoresis and gel image analysis Protein samples (2 mg) were precipitated overnight at −20 ◦ C with two volumes of ice-cold 20% (w/v) trichloroacetic acid/acetone. Protein precipitates were recovered by centrifugation (10 min at 18,000 × g) and washed five times with ice-cold acetone. The pellet was air-dried and solubilized in 350 ␮l isoelectric focusing (IEF) buffer containing 7 M urea, 2 M thio-urea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol (DTT), and 0.2% (v/v) ampholytes, pH 3–10 (Bio-Rad, Hercules, USA). The 17-cm immobilized pH gradient (IPG) strips (pH 3–10) were passively rehydrated with the cell extract sample in IEF buffer for 16 h, and IEF was performed in a Protean IEF cell system (Bio-Rad) with up to 50,000 VH at a maximum voltage of 10,000 V. Strips were equilibrated for 15 min in equilibration buffer (6 M urea, 30% glycerol, 2% SDS, and 0.375 M Tris, pH 8.8) containing 1% DTT for 15 min, and alkylated in equilibration buffer containing 4% iodoacetamide for an additional 15 min. In the second dimension, IPG strips were run vertically on SDS-PAGE 12% gels using PROTEAN® II xi 2D Cell (Bio-Rad). For each protein sample, three independent gels were run (technical replicates). Gels were stained with 0.1% Coomassie Brilliant Blue G (Acros, Geel, Belgium), scanned with a computer-assisted G-800 densitometer (Bio-Rad) and analyzed with the PDQuest Basic-8.0 software (Bio-Rad), followed by additional visual analysis. To determine experimental pI and MW coordinates for each single spot, 2-DE gels were calibrated using a select set of reliable identification landmarks distributed throughout the entire gel. 2.3. Sample preparation for mass spectrometry Protein spots were manually excised from Coomassie-stained 2-DE gels and in-gel digested with trypsin. Gel plugs were treated with three washes of 180 ␮l of 50% acetonitrile and 50 mM ammonium bicarbonate for 15 min each, followed by one wash with 180 ␮l of acetonitrile. After the washing procedures, gel plugs were dried by vacuum centrifugation and digested for 18–24 h at 37 ◦ C using 12 ␮l of 10 mg/ml modified porcine trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega), diluted to 25 mM in NH4 HCO3 . After tryptic digestion, peptides were extracted in two washes with 50 ␮l of 50% acetonitrile and trifluoroacetic acid (TFA) for 1 h. Extracted peptides were dried and resuspended in 10 ␮l of 0.1% TFA. For gel-free LC-ESI-Q-TOF MS/MS (LC–MS/MS) experiments, protein extracts were prepared from three identical and independent cultures (biological replicates). Protein samples were diluted in denaturing buffer (25 mM NH4 HCO3 /8 M urea, pH 8.9), reduced by adding DTT (0.02 ␮g/␮g protein), and carboxyamidomethylated with iodoacetamide (0.1 ␮g/␮g protein). Samples were further diluted with 25 mM NH4 HCO3 to a final urea concentration of 1 M and trypsin was added at a ratio of 0.01 ␮g/␮g protein. After digestion for 4 h at 37 ◦ C, an additional aliquot of enzyme was added, and samples were further incubated for 16–20 h at 37 ◦ C. The resulting

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peptides were desalted using OASIS® HLB Cartridge (Waters, USA) and eluted in 300 ␮l of 70% ACN/0.1% TFA. 2.4. Mass spectrometry analyses Peptides from digested protein spots and protein extracts were analyzed by on-line liquid chromatography/mass spectrometry (LC–MS/MS) using a Waters nanoACQUITY UPLC system coupled to a Waters Micromass Q-TOF Micro or Q-TOF Ultima API mass spectrometer (Waters MS Technologies, UK). The peptides were eluted from the reverse-phase column to the mass spectrometer at a flow rate of 200 nl/min with a 10–50% water/ACN 0.1% formic acid linear gradient over 10 min for peptides obtained from protein spots, and 45 min for peptides from protein extracts. Analyses were performed using the data-dependent acquisition (DDA) mode. For each MS spectrum, the three most intense multiple charged ions above the threshold (30 counts/s) were automatically selected for MS/MS fragmentation. The collision energies for peptide fragmentation were set using the charge state recognition files for +2, +3, and +4 peptide ions provided by MassLynx (Waters). MS/MS raw data were processed using Protein-Lynx Global Server 2.0 software (Waters), and peak lists were exported in the micromass (.pkl) format. For experiments with protein extracts, at least two independent LC–MS/MS runs were performed.

experiments (three 2-DEs for each of the three replicate samples of A. polyphaga trophozoites). The 2-DE protein spot profiles were highly reproducible (∼90% matching between replicates), both in terms of the total number of protein spots, and in terms of their relative positions and intensities. About 370 protein spots were resolved on Coomassie-stained 2-DE gels, corresponding to proteins with molecular weights ranging from 19 to 188 kDa. A. polyphaga trophozoite protein spots resolved by 2-DE (pH 3–10) were submitted to ESI-Q-TOF MS/MS analysis for protein identification and 2-DE mapping. MS identification was obtained for 184 of the 370 resolved protein spots (Supplementary Table S1). One hundred and twelve of the 142 unique proteins were identified from single spots, while 30 of the identified proteins were represented by two or more distinct spots in the gel, suggesting post-translational modifications. Most of the spots corresponding to the same protein showed the same (or very similar) apparent molecular masses, with variation in pI. Other proteins showed variations in MW, which is suggestive of post-translational modification by proteolytic cleavage. Spots with protein identification are indicated in Fig. 1, and those possibly corresponding to post-translational modified proteins are indicated in bold in Supplementary Table S1. 3.2. Gel-free ESI-Q-TOF MS/MS analysis of A. polyphaga trophozoite protein extract

2.5. Database searching and bioinformatics analyses For peptide identification, raw MS data files were processed using Mascot Distiller. The data were searched using MASCOT software 2.0 (http://www.matrixscience.com, Matrix Science) against a local database of protein sequences (30,259) constructed based on the A. castellanii Neff strain genome obtained from GenBank (http://www.ncbi.nlm.nih.gov/protein/), (12/04/2013) (Clarke et al., 2013). We added porcine trypsin and human keratin to the databases as contaminant controls. The Mascot search parameters consisted of a maximum of one missed cleavage site, fixed carbamidomethyl alkylation of cysteines, variable oxidation of methionine, and a 0.1 mass unit tolerance on parent and fragment ions. The significance threshold was set at p < 0.05, and only peptides with individual ion scores above this significance threshold were considered for protein identification. The MS/MS spectra of protein identifications based on a single peptide and on borderline scores were manually inspected for acceptance. In addition, a decoy database search was used to estimate false discovery rates for LC–MS/MS analyses, resulting in a mean probability of 1.77% in searches against A. castellanii genome decoy sequences. Gene ontology (GO) terms were applied to the identified proteins using Blast2GO (Götz et al., 2008), where Blast and annotations were performed with default parameters. Blast2GO was also used to generate the pie charts of GO terms from molecular functions, biological processes and cellular components. 3. Results 3.1. Two-dimensional electrophoresis proteomic mapping of A. polyphaga trophozoites In order to resolve the proteins of A. polyphaga, we performed 2-DE in a pH range of 3–10 on strips from protein extracts from amoebae trophozoites maintained in long-term in vitro culture in standard conditions. As technical controls, all protein preparations and the subsequent 2-DE were repeated three times, and images representative of 2-DE gels in pH range 3–10 were selected for constructing the 2-DE reference map of A. polyphaga trophozoite proteins (Fig. 1). PDQuestTM software was used for the image analysis of representative 2-DE gels obtained from nine independent

The gel-free analysis of the A. polyphaga trophozoite soluble protein extract by LC–MS/MS resulted in 99 protein identifications, 94 of which were unique (Supplementary Table S2). Each sample (biological replicate) was independently analyzed by MS twice to ensure data reproducibility. An approximately 96% correspondence in identified proteins was observed between replicate MS runs. Overall, the 2-DE/ESI-Q-TOF MS/MS and gel-free LC–MS/MS complementary proteomic approaches allowed the identification of 202 non-redundant A. polyphaga trophozoite proteins. Eighty-six proteins were identified by both experimental approaches, showing that they generated essentially complementary data sets. 3.3. Functional analysis of the identified proteins The functional annotation of the proteins identified in protein extracts from A. polyphaga trophozoites was based on GO terms. In this analysis, ≥1 GO terms were assigned for all proteins identified by 2-DE/ESI-Q-TOF MS/MS (184 proteins) and gel-free LC–MS/MS (99 proteins). The assigned GO terms for molecular function, biological process, and cellular components for these two sets of proteins are listed in Supplementary Tables S1 and S2, respectively, and are summarized in Fig. 2. Regarding biological process, the identified in the two sets were grouped into 14 GO categories. Metabolic process (GO:0008152), cellular process (GO:0009987), response to stimulus (GO:0050896), biological regulation (GO:0065007), and cellular component organization or biogenesis (GO:0071840) were the best represented processes, followed by developmental process (GO:0032502), multicellular organismal process (GO:0032501), reproduction (GO:0000003), signaling (GO:0023052), growth (GO:0040007), localization (GO:0051179), multi-organism process (GO:0051704), death (GO:0016265) and viral reproduction (GO:0050792). Catalytic activity (GO:0003824) and binding (GO:0005488) were the predominant molecular-function GO categories in both sets of proteins. Other well-represented categories of molecular function were structural molecule activity (GO:0005198), enzyme activity (GO:0030234), transporter activity regulator (GO:0005215), antioxidant activity (GO:0016209), enzyme regulator activity (GO:0030234), electron carrier (GO:0009055),

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Fig. 1. Representative 2-DE reference map of A. polyphaga trophozoite proteins. The proteins were separated on a linear pH range of 3–10, using IEF in the first dimension and 12% SDS-PAGE in the second dimension. Proteins were stained with Coomassie Brilliant Blue G. Molecular mass markers are shown on the left, and the acid-to-alkaline gradient is from left to right. Spots containing A. polyphaga proteins identified by LC–MS/MS are indicated by numbers that refer to spot numbers listed in Supporting information Table S1.

nucleic acid binding transcription factor activity (GO:), and receptor activity (GO:0004872). Finally, the proteins were classified into five groups of cell components. The predominant GO categories were cells (GO:0005623) and organelles (GO:0043226), followed by macromolecular complexes (GO:0032991), extracellular region (GO:0005576), and membrane-enclosed lumens (GO:0031974). 4. Discussion Acanthamoeba spp. have two stages in their life cycle, a dormant, free-living cyst stage, with minimal metabolic activity, and the infective trophozoite stage (Lorenzo-Morales et al., 2013). Trophozoites of Acanthamoeba spp. infect a variety of mammalian hosts and can cause infections in humans, as a result of complex interactions between the pathogen-host, environment, and even endosymbionts. Much of the damage caused by trophozoites in human corneal or brain infections involves several different pathogenic mechanisms that have not so far been elucidated at the molecular level. The elucidation of these mechanisms depends on the identification of proteins involved in the pathogen-host interplay, which will necessitate comprehensive proteomic studies. The genome of A. polyphaga has not yet been completely sequenced, but the genome sequence of the closely related species A. castellanii has been recently reported (Clarke et al., 2013). The genetic similarity among Acanthamoeba species, along with advances in mass-spectrometry techniques and protein identification software, has allowed efficient identification of A. polyphaga proteins in the present study, in which we combined complementary experimental strategies to analyze the proteome

of the trophozoite stage of A. polyphaga ATCC 30872. It was previously demonstrated (Veríssimo et al., 2013) that this isolate, while maintained in culture, as used here, is mildly pathogenic for rats, but, upon passage in the mammalian host, it undergoes activation of pathogenic traits and becomes more virulent. Therefore, the set of proteins identified in this study is representative of the proteome of the infective trophozoite stage of an A. polyphaga isolate prior to virulence activation. This is the largest proteome dataset of an Acanthamoeba species obtained to date, and the performed MS/MS analyses also generated quantitative estimates of the identified proteins (data not shown). Therefore, our proteomic dataset will be a useful reference in future comparative studies between samples of the same isolate after passage in the mammalian host, between cyst and trophozoite stages, or between virulent and avirulent strains. Bouyer et al. (2009) performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms of A. castellanii. Four of the 11 proteins that they identified (actophorin, elongation factor 2, heat shock protein, and enolase) were also found in our proteomic analysis of A. polyphaga trophozoites. More recently, a proteomic analysis of cysts of E. histolytica by Ali et al. (2012) resulted in the identification of 417 non-redundant proteins; this larger number of identified proteins was possible through the use of a more-sensitive mass spectrometer (Orbitrap) than those used in this study. While our 2-DE-ESI-Q-TOF MS/MS analysis generated a reference proteomic map with 142 identified proteins, corresponding to 184 spots identified of the 370 resolved spots detected, the reference 2DE proteomic map available for Leishmania (Viannia) braziliensis contains 101 identified spots, representing 75 protein entries

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Fig. 2. Functional analysis of proteins identified from A. polyphaga trophozoites. The functional annotation of the proteins was based on Gene Ontology. Proteins were annotated according to biological processes, molecular functions and cellular components (level 2), using the Blast2GO tool. The distribution of the proteins in each category is indicated in the sectors of the circle.

(Cuervo et al., 2007). The reference proteomic maps of Trichomonas vaginalis for three pH ranges (3–10, 4–7, 6–11) contain, overall, 247 spots representing 164 different proteins (Huang et al., 2009). Our 2-DE analyses provided evidence of post-translational processing for several A. polyphaga trophozoite proteins, in the form of more than one spot assigned to the same protein. Post-translational modifications modulate the activity of most eukaryotic proteins, and can determine their location, turnover,

and interactions with other proteins (Mann and Jensen, 2003). Protein variants or isoforms may result from biologically important post-translational modifications, ranging from chemical modifications to proteolytic cleavage (Ambatipudi et al., 2006). Information about post-translational modifications of proteins in Acanthamoeba spp. is sparse, but our findings indicate that these phenomena may be frequent. The biological significance of post-translational modification varies depending on the protein and the type(s) of modification.

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The GO classification carried out to functionally annotate the proteins identified here resulted in data comparable to those of reference proteomes of other pathogens (Huang et al., 2009, 2012; Pawar et al., 2012b). Most of the proteins were assigned to the classes of cellular processes and metabolism. These results are in agreement with previous microarray and EST analyses, which revealed that genes related to energy production and conversion, carbohydrate transport and metabolism, cytoskeleton, translation, ribosomal structure and biogenesis, and protein turnover and chaperone categories are predominantly overexpressed in the trophozoite stage in comparison to the cyst stage (Moon et al., 2011). This predominance of proteins involved in cellular processes and metabolism may be a consequence of the vegetative growth and increased cellular activity exhibited by the A. polyphaga active trophozoite stage. Some of the metabolism-related proteins identified in the A. polyphaga trophozoite proteome, especially those with the biological functions of energy and carbohydrate metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, transaldolase, citrate synthase and malate dehydrogenase, have also been described as multifunctional. For example, GAPDH, enolase, and transaldolase from a variety of pathogenic organisms have the ability to bind plasminogen, which may induce plasmin-mediated proteolysis, degrading the extracellular matrix and facilitating invasion of and migration in the host (Sotillo et al., 2010; Wang et al., 2011). These proteins are interesting targets for future studies, as they may play roles in pathogen–host interactions, including evasion of the immune response in the infectious process by A. polyphaga. The A. polyphaga proteins identified that belong to the cytoskeleton-associated protein group contain structural proteins (actin, actin-like protein) and proteins that regulate the stability of the polymers made by these molecules (coronin, actophorin). They provide a venue for future studies on this protozoan, as many biological processes, such as cell motility and morphological transformation, require remodeling of the cytoskeleton in response to intracellular and extracellular signals, and the ability to undergo morphological changes may be related to virulence and pathogenesis in Acanthamoeba spp. Previous studies have shown that morphological transformation occurs when A. culbertsoni attaches to collagen and laminin, and actin rearrangement was found to be a requisite for invasion (Rocha-Azevedo et al., 2009). For this transformation to occur in mammalian cells and parasitic protozoa, the actin cytoskeleton must undergo rearrangement in order to establish focal points of adhesion (Martin et al., 2002). In addition, data suggest that the interaction between Acanthamoeba spp. and the extracellular matrix is mediated by protein receptors that can induce major cytoskeletal rearrangements. These rearrangements have been shown to lead to conformational changes, and may be followed by activation of signal transduction pathways that affect motility and protease secretion (Rocha-Azevedo et al., 2009, 2010). The group of proteins related to protein turnover and chaperones, which includes HSP70, HSP90, HSP91, HSP82, HPS20, ATPase with chaperone activity, proteasome, chaperone DnaK, peroxiredoxin, ubiquitin, and calreticulin, among others, was also well represented in the A. polyphaga trophozoite proteome. Several of these proteins are potentially involved in pathogen survival mechanisms, and may be important for A. polyphaga pathogenicity. HSP70 was previously identified as an antigenic protein by 2DE immunoblot experiments using infected rat serum with A. polyphaga (unpublished results). Heat shock proteins, such as HSP70, are considered to be inducible protective proteins that are critical for pathogen survival, as well as immune-reactive proteins that are important in parasitic infection (Wang et al., 2009). Peroxiredoxins (Prxs) belong to the peroxidase family, which is found in different organisms including yeasts, protozoa and

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metazoans. Prxs possess antioxidant functions (protecting cells from attack by reactive oxygen species) and have a role in receptor signaling, protein phosphorylation, transcriptional regulation and phagocytosis (Dzik, 2006). Peroxiredoxin 2 was identified in this study, which suggests that A. polyphaga trophozoites may produce peroxiredoxin as a protection against H2 O2 . Peroxiredoxin was also identified in the proteomes of Naegleria fowleri and Toxoplasma gondii, and was characterized as an important antigenic protein, implicated in host cell invasion and in facilitating suppression of the immune response of hosts (Kim et al., 2009; Ma et al., 2009). Although the A. polyphaga trophozoite protein extracts were enriched in soluble proteins, several membrane proteins were also identified. Membrane proteins are very difficult to solubilize by commonly used solubilization buffers, which often causes their underrepresentation in 2-DE (Gorg et al., 2004). Some proteins or protein families were identified as exposed on the plasma membrane of A. polyphaga. These proteins include GDP-mannose pyrophosphorylase, the serine hydroxymethyltransferase, a calreticulin, a coronin (an actinin-like protein), the Rab family GTPase, the C2 domain containing protein, TolA protein, and a LIM protein associated with lipid rafts in the plasma membrane. Studies have identified these proteins as surface molecules of E. histolytica, and some of these molecules, including C2 proteins, calreticulin, LIM and Rab proteins have helped to elucidate mechanisms of virulence and have been identified as proteins differentially expressed in virulent E. histolytica and avirulent Entamoeba dispar (Davis et al., 2009; Wilson et al., 2012; Biller et al., 2014). The identification of these membrane proteins opens possibilities for future studies on the pathogenesis, virulence factors, and drug interaction in A. polyphaga and other Acanthamoeba species. Our proteomic analyses surveyed, for the first time, the repertoire of proteins expressed by A. polyphaga. We provided trophozoite reference protein sets that serve as foundations for future prospective, comparative and functional studies of A. polyphaga proteins involved in molecular mechanisms that are crucial for its development, survival and pathogenicity. This A. polyphaga proteome map will be a useful reference for immunological studies aiming toward the identification of antigenic proteins. Comparative proteomic analyses between samples of the organism cultured under different conditions, including stress and nutritional states, and between pathogenic and non-pathogenic strains will allow the identification of differentially expressed proteins related to pathogen survival, development and infectivity. Upon the identification of important antigens and differentially expressed proteins, it will be possible to experimentally address the function of these proteins and to define protein targets for the development of new drugs, immunodiagnostic methods and vaccines. Acknowledgments We acknowledge the Unidade de Química de Proteínas e Espectrometria de Massas (Uniprote-MS) at the Centro de Biotecnologia (Universidade Federal do Rio Grande do Sul, Brazil) and the Mass Spectrometry Laboratory at the Brazilian Biosciences National Laboratory (LNBio), CNPEM, Campinas, Brazil, for their support with the mass spectrometry analyses (project MAS 11532). We are grateful to Dr. Janet W. Reid for revision of the English text. This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.actatropica.2014.08.009.

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