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ARTICLE IN PRESSG ModelCTROP 3437 1–7

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Contents lists available at ScienceDirect

Acta Tropica

jo ur nal home p age: www.elsev ier .com/ locate /ac ta t ropica

roteomic profiling of the infective trophozoite stage ofcanthamoeba polyphaga

arin Silva Caumoa,1, Karina Mariante Monteirob, Thiely Rodrigues Ottb,inicius José Maschioa, Glauber Wagnerc, Henrique Bunselmeyer Ferreirab,∗,arilise Brittes Rotta

Laboratório de Parasitologia, Instituto de Ciências Básicas e da Saúde, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federalo Rio Grande do Sul, CEP: 90050170 Porto Alegre, RS, BrazilLaboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, CEP: 91501-970 Porto Alegre, RS,razilLaboratório de Doenc as Infecciosas e Parasitárias, Universidade do Oeste de Santa Catarina, CEP: 89600-000 Joac aba, SC, Brazil

r t i c l e i n f o

rticle history:eceived 25 April 2014eceived in revised form 31 July 2014ccepted 8 August 2014vailable online xxx

eywords:canthamoebarophozoite-DE

a b s t r a c t

Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capableof 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 pathogenicmechanisms that have not yet been elucidated at the molecular level. We performed a comprehensiveanalysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MSand gel-free LC–MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphagaproteomic map in the pH range 3–10 was produced, with protein identification for 184 of 370 resolvedspots, 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

C–MS/MSlobal protein analysisathogen–host interaction

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 anAcanthamoeba species, and established foundations for prospective, comparative and functional studiesof proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and otherpathogenic amoebae.

© 2014 Published by Elsevier B.V.

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. Introduction

Free-living amoebae (FLA) belonging to the genus Acanthamoebare ubiquitously distributed in nature, and are adapted to live in aide variety of natural and human-created environments (Schuster

nd Visvesvara, 2004; Caumo et al., 2009; Magliano et al., 2009;

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

arlesso et al., 2010; Winck et al., 2011; Siddiqui and Khan, 2012a).canthamoeba spp. have gained increasing attention from the sci-ntific 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: k.caumo@ufsc.br (K.S. Caumo), karina@cbiot.ufrgs.br

K.M. Monteiro), thiely.ott@gmail.com (T.R. Ott), vinimaschio@yahoo.com.brV.J. Maschio), glauberwagner@gmail.com (G. Wagner), henrique@cbiot.ufrgs.brH.B. Ferreira), marilise.rott@ufrgs.br (M.B. Rott).

1 Current address: Laboratório de Parasitologia Clínica, Centro de Ciências daaúde, Departamento de Análises Clínicas, Universidade Federal de Santa Catarina,EP: 88040-900 Florianópolis, SC, Brazil.

ttp://dx.doi.org/10.1016/j.actatropica.2014.08.009001-706X/© 2014 Published by Elsevier B.V.

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the ecosystem. The active trophozoite stage, which exhibits vege-tative growth and is the infective form for mammalian hosts, feedson bacteria, algae, and yeast. It is also a reservoir for pathogenicmicroorganisms that are resistant to phagocytosis by the amoebae,which may help to disperse important human pathogens such asLegionella pneumophila and Pseudomonas aeruginosa in the environ-ment (Visvesvara et al., 2007; Siddiqui and Khan, 2012b).

Several of the approximately 24 identified species of the genusAcanthamoeba have been linked to human disease, includingAcanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoebaastronyxis, Acanthamoeba hatchetti, Acanthamoeba culbertsoni,Acanthamoeba healyi, and Acanthamoeba byersi (Visvesvara et al.,2007; Corsaro and Venditti, 2010; Visvesvara, 2010; Qvarnstromet al., 2013). The growing importance of Acanthamoeba spp. in med-

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

ical care and research during the last decade is due to their potentialto infect human hosts, causing severe diseases, such as granu-lomatous amoebic encephalitis (GAE), a chronic brain infectionthat occurs more frequently in immunosuppressed individuals;

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moebic keratitis (AK), a sight-threatening infection of the corneahat is related to contact lens misuse; and disseminated infectionsMarciano-Cabral and Cabral, 2003; Visvesvara et al., 2007).

Acanthamoeba spp. trophozoites also have been used exten-ively as model systems to study eukaryotic cell biology, becausef their relatively large size, rapid growth in culture, and activeotility (Horowitz and Hammer, 1990; Maciver and Hussey, 2002;

lopocka et al., 2009; Chrisman et al., 2010; Brzeska et al., 2012;iddiqui and Khan, 2012a). The well-developed cytoskeleton ofhese organisms makes them especially good models for under-tanding actin cytoskeleton-based motility, and other molecularspects of cell motility (Khan, 2006; Siddiqui and Khan, 2012c).

Proteomic studies have been described for many protozoa,ncluding Entamoeba histolytica, Giardia lamblia and Leishmaniaonovani (Tolstrup et al., 2007; Biller et al., 2009; Ali et al.,012; Jerlstrom-Hultqvist et al., 2012; Pawar et al., 2012a; Fasot al., 2013). These analyses have revealed a diversity of pro-eins expressed by different parasitic species, helping to elucidatehe molecular mechanisms of interaction with host species, ando identify potential biomarkers for diagnosis and targets for theevelopment of new drugs or vaccines. For Acanthamoeba spp.,he few proteomic studies have so far been limited to the inves-igation of protein expression during encystment (Bouyer et al.,009; Leitsch et al., 2010). Although important for a better under-tanding of the biology of invasive forms, the repertoire of proteinsxpressed by Acanthamoeba spp. trophozoites has not yet beennvestigated.

Analysis of the repertoire of proteins expressed by Acan-hamoeba spp. trophozoites is expected to contribute to thelucidation of the mechanisms of virulence, and to the identi-cation of diagnostic antigens and target proteins for therapy.roteomic approaches based on two-dimensional gel electrophore-is (2-DE) and mass spectrometry (MS) are powerful tools toapture the dynamics of global proteomic changes, with theimultaneous resolution and identification of large numbersf cellular proteins. These methods are particularly advanta-eous for the identification of stress-induced proteins along withheir post-translational modifications, and to correlate alteredrotein abundance/modifications with physiological function(s)Beranova-Giorgianni, 2003; Brewis and Brennan, 2010).

In this study, we established the conditions for the 2-DEnalysis of A. polyphaga trophozoites and identified most of theesolved proteins in order to provide a reference proteomic map. Ael-free LC–MS/MS analysis was also performed. Overall, 202 non-edundant proteins were identified, with 184 proteins identifiedrom the 370 spots mapped in the 2-DE gel, along with 94 proteinsdentified by gel-free LC–MS/MS. Identified proteins were assignedo several functional classes: metabolism-related, cytoskeleton,ost-translation modification, protein turnover and chaperones,urface-localized proteins, and proteins related to defense mech-nisms. The importance of the identified repertoire of trophozoiteroteins for the biology of A. polyphaga is discussed.

. Material and methods

.1. A. polyphaga strain, cultivation, and cell protein extracts

A. polyphaga trophozoites of the T4 genotype were obtainedrom the American Type Culture Collection (ATCC 30872). This envi-onmental isolate had its pathogenicity previously demonstrated

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

Veríssimo et al., 2013). Trophozoites were maintained in axenicultures in peptone-yeast-glucose (PYG) medium, as previouslyescribed (Schuster, 2002), and samples for the proteomic analysisere directly taken from these cultures.

PRESSca xxx (2014) xxx–xxx

Three identical and independent cultures (biological replicates)with approximately 1 × 108 trophozoites in the exponential growthphase were used for protein extraction. Cells were harvested at2000 × g for 10 min and washed twice in phosphate-buffered saline(PBS) buffer (pH 7.2), prior to resuspension in 1 ml of 25 mMTris–HCl, pH 7.2. Cell suspensions were then lysed by sonication(25 Hz in a VC601 Sonics and Materials Inc. sonicator) in an ice bathfor five 30-s cycles with a 1-min interval between pulses. Lysateswere centrifuged (18,000 × g, 15 min, 4 ◦C) to separate soluble andinsoluble protein fractions. Soluble proteins were quantified usinga QubitTM quantitation fluorometer and Quant-itTM reagents (Invi-trogen, 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) trichloroaceticacid/acetone. Protein precipitates were recovered by centrifugation(10 min at 18,000 × g) and washed five times with ice-cold ace-tone. The pellet was air-dried and solubilized in 350 �l isoelectricfocusing (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 pHgradient (IPG) strips (pH 3–10) were passively rehydrated with thecell extract sample in IEF buffer for 16 h, and IEF was performedin a Protean IEF cell system (Bio-Rad) with up to 50,000 VH ata maximum voltage of 10,000 V. Strips were equilibrated for15 min in equilibration buffer (6 M urea, 30% glycerol, 2% SDS, and0.375 M Tris, pH 8.8) containing 1% DTT for 15 min, and alkylated inequilibration buffer containing 4% iodoacetamide for an additional15 min. In the second dimension, IPG strips were run vertically onSDS-PAGE 12% gels using PROTEAN® II xi 2D Cell (Bio-Rad). Foreach protein sample, three independent gels were run (technicalreplicates). Gels were stained with 0.1% Coomassie Brilliant Blue G(Acros, Geel, Belgium), scanned with a computer-assisted G-800densitometer (Bio-Rad) and analyzed with the PDQuest Basic-8.0software (Bio-Rad), followed by additional visual analysis. Todetermine experimental pI and MW coordinates for each singlespot, 2-DE gels were calibrated using a select set of reliableidentification landmarks distributed throughout the entire gel.

2.3. Sample preparation for mass spectrometry

Protein spots were manually excised from Coomassie-stained2-DE gels and in-gel digested with trypsin. Gel plugs were treatedwith three washes of 180 �l of 50% acetonitrile and 50 mM ammo-nium bicarbonate for 15 min each, followed by one wash with180 �l of acetonitrile. After the washing procedures, gel plugswere dried by vacuum centrifugation and digested for 18–24 h at37 ◦C using 12 �l of 10 mg/ml modified porcine trypsin (TrypsinGold, Mass Spectrometry Grade, Promega), diluted to 25 mM inNH4HCO3. After tryptic digestion, peptides were extracted in twowashes with 50 �l of 50% acetonitrile and trifluoroacetic acid (TFA)for 1 h. Extracted peptides were dried and resuspended in 10 �l of0.1% TFA.

For gel-free LC-ESI-Q-TOF MS/MS (LC–MS/MS) experiments,protein extracts were prepared from three identical and indepen-dent cultures (biological replicates). Protein samples were dilutedin denaturing buffer (25 mM NH4HCO3/8 M urea, pH 8.9), reducedby adding DTT (0.02 �g/�g protein), and carboxyamidomethylatedwith iodoacetamide (0.1 �g/�g protein). Samples were further

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

diluted with 25 mM NH4HCO3 to a final urea concentration of 1 Mand trypsin was added at a ratio of 0.01 �g/�g protein. After diges-tion for 4 h at 37 ◦C, an additional aliquot of enzyme was added, andsamples were further incubated for 16–20 h at 37 ◦C. The resulting

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eptides were desalted using OASIS® HLB Cartridge (Waters, USA)nd eluted in 300 �l of 70% ACN/0.1% TFA.

.4. Mass spectrometry analyses

Peptides from digested protein spots and protein extracts werenalyzed by on-line liquid chromatography/mass spectrometryLC–MS/MS) using a Waters nanoACQUITY UPLC system coupledo a Waters Micromass Q-TOF Micro or Q-TOF Ultima API masspectrometer (Waters MS Technologies, UK). The peptides wereluted from the reverse-phase column to the mass spectrometert a flow rate of 200 nl/min with a 10–50% water/ACN 0.1% formiccid linear gradient over 10 min for peptides obtained from proteinpots, and 45 min for peptides from protein extracts. Analyses wereerformed using the data-dependent acquisition (DDA) mode. Forach MS spectrum, the three most intense multiple charged ionsbove the threshold (30 counts/s) were automatically selected forS/MS fragmentation. The collision energies for peptide fragmen-

ation were set using the charge state recognition files for +2, +3,nd +4 peptide ions provided by MassLynx (Waters). MS/MS rawata were processed using Protein-Lynx Global Server 2.0 softwareWaters), and peak lists were exported in the micromass (.pkl)ormat. For experiments with protein extracts, at least two inde-endent LC–MS/MS runs were performed.

.5. Database searching and bioinformatics analyses

For peptide identification, raw MS data files were processedsing Mascot Distiller. The data were searched using MAS-OT software 2.0 (http://www.matrixscience.com, Matrix Science)gainst a local database of protein sequences (30,259) constructedased on the A. castellanii Neff strain genome obtained fromenBank (http://www.ncbi.nlm.nih.gov/protein/), (12/04/2013)

Clarke et al., 2013). We added porcine trypsin and human ker-tin to the databases as contaminant controls. The Mascot searcharameters consisted of a maximum of one missed cleavage site,xed carbamidomethyl alkylation of cysteines, variable oxidationf methionine, and a 0.1 mass unit tolerance on parent and frag-ent ions. The significance threshold was set at p < 0.05, and only

eptides with individual ion scores above this significance thresh-ld were considered for protein identification. The MS/MS spectraf protein identifications based on a single peptide and on border-ine scores were manually inspected for acceptance. In addition, aecoy database search was used to estimate false discovery ratesor LC–MS/MS analyses, resulting in a mean probability of 1.77% inearches against A. castellanii genome decoy sequences.

Gene ontology (GO) terms were applied to the identifiedroteins using Blast2GO (Gotz et al., 2008), where Blast and anno-ations were performed with default parameters. Blast2GO waslso used to generate the pie charts of GO terms from molecularunctions, biological processes and cellular components.

. Results

.1. Two-dimensional electrophoresis proteomic mapping of A.olyphaga trophozoites

In order to resolve the proteins of A. polyphaga, we performed-DE in a pH range of 3–10 on strips from protein extracts frommoebae trophozoites maintained in long-term in vitro culturen standard conditions. As technical controls, all protein prepa-

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

ations and the subsequent 2-DE were repeated three times, andmages representative of 2-DE gels in pH range 3–10 were selectedor constructing the 2-DE reference map of A. polyphaga tropho-oite proteins (Fig. 1). PDQuestTM software was used for the image

PRESSca xxx (2014) xxx–xxx 3

analysis of representative 2-DE gels obtained from nine indepen-dent experiments (three 2-DEs for each of the three replicatesamples of A. polyphaga trophozoites). The 2-DE protein spot pro-files were highly reproducible (∼90% matching between replicates),both in terms of the total number of protein spots, and in termsof their relative positions and intensities. About 370 protein spotswere resolved on Coomassie-stained 2-DE gels, corresponding toproteins with molecular weights ranging from 19 to 188 kDa.

A. polyphaga trophozoite protein spots resolved by 2-DE (pH3–10) were submitted to ESI-Q-TOF MS/MS analysis for proteinidentification and 2-DE mapping. MS identification was obtainedfor 184 of the 370 resolved protein spots (Supplementary Table S1).One hundred and twelve of the 142 unique proteins were iden-tified from single spots, while 30 of the identified proteins wererepresented by two or more distinct spots in the gel, suggestingpost-translational modifications. Most of the spots correspond-ing to the same protein showed the same (or very similar)apparent molecular masses, with variation in pI. Other proteinsshowed variations in MW, which is suggestive of post-translationalmodification by proteolytic cleavage. Spots with protein identifica-tion are indicated in Fig. 1, and those possibly corresponding topost-translational modified proteins are indicated in bold in Sup-plementary Table S1.

3.2. Gel-free ESI-Q-TOF MS/MS analysis of A. polyphagatrophozoite protein extract

The gel-free analysis of the A. polyphaga trophozoite soluble pro-tein 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 toensure data reproducibility. An approximately 96% correspondencein identified proteins was observed between replicate MS runs.

Overall, the 2-DE/ESI-Q-TOF MS/MS and gel-free LC–MS/MScomplementary proteomic approaches allowed the identificationof 202 non-redundant A. polyphaga trophozoite proteins. Eighty-sixproteins were identified by both experimental approaches, show-ing that they generated essentially complementary data sets.

3.3. Functional analysis of the identified proteins

The functional annotation of the proteins identified in proteinextracts from A. polyphaga trophozoites was based on GO terms. Inthis analysis, ≥1 GO terms were assigned for all proteins identifiedby 2-DE/ESI-Q-TOF MS/MS (184 proteins) and gel-free LC–MS/MS(99 proteins). The assigned GO terms for molecular function, biolog-ical process, and cellular components for these two sets of proteinsare listed in Supplementary Tables S1 and S2, respectively, and aresummarized in Fig. 2.

Regarding biological process, the identified in the twosets were grouped into 14 GO categories. Metabolic process(GO:0008152), cellular process (GO:0009987), response to stimu-lus (GO:0050896), biological regulation (GO:0065007), and cellularcomponent organization or biogenesis (GO:0071840) were thebest 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 pro-cess (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

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

sets of proteins. Other well-represented categories of molec-ular function were structural molecule activity (GO:0005198),enzyme regulator activity (GO:0030234), transporter activ-ity (GO:0005215), antioxidant activity (GO:0016209), enzyme

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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 dimensionand 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-alkalineg LC–Mi

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radient is from left to right. Spots containing A. polyphaga proteins identified bynformation Table S1.

egulator activity (GO:0030234), electron carrier (GO:0009055),ucleic acid binding transcription factor activity (GO:), and receptorctivity (GO:0004872).

Finally, the proteins were classified into five groups of cell com-onents. The predominant GO categories were cells (GO:0005623)nd organelles (GO:0043226), followed by macromolecular com-lexes (GO:0032991), extracellular region (GO:0005576), andembrane-enclosed lumens (GO:0031974).

. Discussion

Acanthamoeba spp. have two stages in their life cycle, a dor-ant, free-living cyst stage, with minimal metabolic activity, and

he infective trophozoite stage (Lorenzo-Morales et al., 2013).rophozoites of Acanthamoeba spp. infect a variety of mammalianosts and can cause infections in humans, as a result of com-lex interactions between the pathogen-host, environment, andven endosymbionts. Much of the damage caused by trophozoitesn human corneal or brain infections involves several differentathogenic mechanisms that have not so far been elucidated at theolecular level. The elucidation of these mechanisms depends on

he identification of proteins involved in the pathogen-host inter-lay, which will necessitate comprehensive proteomic studies.

The genome of A. polyphaga has not yet been completely

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

equenced, but the genome sequence of the closely relatedpecies A. castellanii has been recently reported (Clarke et al.,013). The genetic similarity among Acanthamoeba species, alongith advances in mass-spectrometry techniques and protein

S/MS are indicated by numbers that refer to spot numbers listed in Supporting

identification software, has allowed efficient identification of A.polyphaga proteins in the present study, in which we combinedcomplementary experimental strategies to analyze the proteomeof the trophozoite stage of A. polyphaga ATCC 30872. It was previ-ously 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 activationof pathogenic traits and becomes more virulent. Therefore, the setof proteins identified in this study is representative of the pro-teome of the infective trophozoite stage of an A. polyphaga isolateprior to virulence activation. This is the largest proteome datasetof an Acanthamoeba species obtained to date, and the performedMS/MS analyses also generated quantitative estimates of the iden-tified proteins (data not shown). Therefore, our proteomic datasetwill be a useful reference in future comparative studies betweensamples of the same isolate after passage in the mammalian host,between cyst and trophozoite stages, or between virulent and avir-ulent strains.

Bouyer et al. (2009) performed two-dimensional gel elec-trophoresis to compare protein expression in trophozoite and cystforms of A. castellanii. Four of the 11 proteins that they iden-tified (actophorin, elongation factor 2, heat shock protein, andenolase) were also found in our proteomic analysis of A. polyphagatrophozoites. More recently, a proteomic analysis of cysts of E.

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

histolytica by Ali et al. (2012) resulted in the identification of417 non-redundant proteins; this larger number of identified pro-teins was possible through the use of a more-sensitive massspectrometer (Orbitrap) than those used in this study. While our

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ig. 2. Functional analysis of proteins identified from A. polyphaga trophozoitennotated according to biological processes, molecular functions and cellular comps indicated in the sectors of the circle.

-DE-ESI-Q-TOF MS/MS analysis generated a reference proteomicap with 142 identified proteins, corresponding to 184 spots

dentified of the 370 resolved spots detected, the referenceDE proteomic map available for Leishmania (Viannia) brazilien-is contains 101 identified spots, representing 75 protein entries

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

Cuervo et al., 2007). The reference proteomic maps of Trichomonasaginalis for three pH ranges (3–10, 4–7, 6–11) contain, over-ll, 247 spots representing 164 different proteins (Huang et al.,009).

functional annotation of the proteins was based on Gene Ontology. Proteins were (level 2), using the Blast2GO tool. The distribution of the proteins in each category

Our 2-DE analyses provided evidence of post-translationalprocessing for several A. polyphaga trophozoite proteins, in theform of more than one spot assigned to the same protein.Post-translational modifications modulate the activity of mosteukaryotic proteins, and can determine their location, turnover,

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

and interactions with other proteins (Mann and Jensen, 2003). Pro-tein variants or isoforms may result from biologically importantpost-translational modifications, ranging from chemical modifica-tions to proteolytic cleavage (Ambatipudi et al., 2006). Information

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bout post-translational modifications of proteins in Acanthamoebapp. is sparse, but our findings indicate that these phenomenaay be frequent. The biological significance of post-translationalodification varies depending on the protein and the type(s) ofodification.The GO classification carried out to functionally annotate the

roteins identified here resulted in data comparable to those ofeference proteomes of other pathogens (Huang et al., 2009, 2012;awar et al., 2012b). Most of the proteins were assigned to thelasses of cellular processes and metabolism. These results aren agreement with previous microarray and EST analyses, whichevealed that genes related to energy production and conversion,arbohydrate transport and metabolism, cytoskeleton, transla-ion, ribosomal structure and biogenesis, and protein turnovernd chaperone categories are predominantly overexpressed in therophozoite stage in comparison to the cyst stage (Moon et al.,011). This predominance of proteins involved in cellular processesnd metabolism may be a consequence of the vegetative growthnd increased cellular activity exhibited by the A. polyphaga activerophozoite stage.

Some of the metabolism-related proteins identified in the A.olyphaga trophozoite proteome, especially those with the bio-ogical functions of energy and carbohydrate metabolism, suchs glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase,ransaldolase, citrate synthase and malate dehydrogenase, havelso been described as multifunctional. For example, GAPDH,nolase, and transaldolase from a variety of pathogenic orga-isms have the ability to bind plasminogen, which may inducelasmin-mediated proteolysis, degrading the extracellular matrixnd facilitating invasion of and migration in the host (Sotillo et al.,010; Wang et al., 2011). These proteins are interesting targets foruture studies, as they may play roles in pathogen–host interac-ions, including evasion of the immune response in the infectiousrocess by A. polyphaga.

The A. polyphaga proteins identified that belong to theytoskeleton-associated protein group contain structural proteinsactin, actin-like protein) and proteins that regulate the stabilityf the polymers made by these molecules (coronin, actophorin).hey provide a venue for future studies on this protozoan, as manyiological processes, such as cell motility and morphological trans-ormation, require remodeling of the cytoskeleton in response tontracellular and extracellular signals, and the ability to undergo

orphological changes may be related to virulence and patho-enesis in Acanthamoeba spp. Previous studies have shown thatorphological transformation occurs when A. culbertsoni attaches

o collagen and laminin, and actin rearrangement was found toe a requisite for invasion (Rocha-Azevedo et al., 2009). For thisransformation to occur in mammalian cells and parasitic proto-oa, the actin cytoskeleton must undergo rearrangement in order tostablish focal points of adhesion (Martin et al., 2002). In addition,ata suggest that the interaction between Acanthamoeba spp. andhe extracellular matrix is mediated by protein receptors that cannduce major cytoskeletal rearrangements. These rearrangementsave been shown to lead to conformational changes, and may be

ollowed by activation of signal transduction pathways that affectotility and protease secretion (Rocha-Azevedo et al., 2009, 2010).The group of proteins related to protein turnover and chaper-

nes, which includes HSP70, HSP90, HSP91, HSP82, HPS20, ATPaseith chaperone activity, proteasome, chaperone DnaK, peroxire-oxin, ubiquitin, and calreticulin, among others, was also wellepresented in the A. polyphaga trophozoite proteome. Severalf these proteins are potentially involved in pathogen survival

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

echanisms, and may be important for A. polyphaga pathogen-city. HSP70 was previously identified as an antigenic proteiny 2DE immunoblot experiments using infected rat serum with. polyphaga (unpublished results). Heat shock proteins, such as

PRESSca xxx (2014) xxx–xxx

HSP70, are considered to be inducible protective proteins that arecritical for pathogen survival, as well as immune-reactive proteinsthat are important in parasitic infection (Wang et al., 2009).

Peroxiredoxins (Prxs) belong to the peroxidase family, which isfound in different organisms including yeasts, protozoa and meta-zoans. Prxs possess antioxidant functions (protecting cells fromattack by reactive oxygen species) and have a role in receptorsignaling, protein phosphorylation, transcriptional regulation andphagocytosis (Dzik, 2006). Peroxiredoxin 2 was identified in thisstudy, which suggests that A. polyphaga trophozoites may produceperoxiredoxin as a protection against H2O2. Peroxiredoxin was alsoidentified in the proteomes of Naegleria fowleri and Toxoplasmagondii, and was characterized as an important antigenic protein,implicated in host cell invasion and in facilitating suppression ofthe immune response of hosts (Kim et al., 2009; Ma et al., 2009).

Although the A. polyphaga trophozoite protein extracts wereenriched in soluble proteins, several membrane proteins werealso identified. Membrane proteins are very difficult to solubi-lize by commonly used solubilization buffers, which often causestheir underrepresentation in 2-DE (Gorg et al., 2004). Some pro-teins or protein families were identified as exposed on the plasmamembrane of A. polyphaga. These proteins include GDP-mannosepyrophosphorylase, the serine hydroxymethyltransferase, a cal-reticulin, a coronin (an actinin-like protein), the Rab family GTPase,the C2 domain containing protein, TolA protein, and a LIM proteinassociated with lipid rafts in the plasma membrane. Studies haveidentified these proteins as surface molecules of E. histolytica, andsome of these molecules, including C2 proteins, calreticulin, LIMand Rab proteins have helped to elucidate mechanisms of viru-lence and have been identified as proteins differentially expressedin virulent E. histolytica and avirulent Entamoeba dispar (Davis et al.,2009; Wilson et al., 2012; Biller et al., 2014). The identificationof these membrane proteins opens possibilities for future studieson the pathogenesis, virulence factors, and drug interaction in A.polyphaga and other acanthamoebas.

Our proteomic analyses surveyed, for the first time, therepertoire of proteins expressed by A. polyphaga. We providedtrophozoite reference protein sets that serve as foundationsfor future prospective, comparative and functional studies of A.polyphaga proteins involved in molecular mechanisms that arecrucial for its development, survival and pathogenicity. This A.polyphaga proteome map will be a useful reference for immunolog-ical studies aiming toward the identification of antigenic proteins.Comparative proteomic analyses between samples of the organismcultured under different conditions, including stress and nutri-tional states, and between pathogenic and non-pathogenic strainswill allow the identification of differentially expressed proteinsrelated to pathogen survival, development and infectivity. Upon theidentification of important antigens and differentially expressedproteins, it will be possible to experimentally address the functionof these proteins and to define protein targets for the developmentof new drugs, immunodiagnostic methods and vaccines.

Acknowledgments

We acknowledge the Unidade de Química de Proteínas e Espec-trometria de Massas (Uniprote-MS) at the Centro de Biotecnologia(Universidade Federal do Rio Grande do Sul, Brazil) and the MassSpectrometry Laboratory at the Brazilian Biosciences National Lab-oratory (LNBio), CNPEM, Campinas, Brazil, for their support withthe mass spectrometry analyses (project MAS 11532). We are grate-

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

ful to Dr. Janet W. Reid for revision of the English text. This studywas supported by grants from the Conselho Nacional de Desen- Q3volvimento Científico e Tecnológico (CNPq) and the Coordenac ão Q4de Aperfeic oamento de Pessoal de Nível Superior (CAPES).

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ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.actatropica.014.08.009.

eferences

li, I.K., Haque, R., Siddique, A., Kabir, M., Sherman, N.E., Gray, S.A., Cangelosi, G.A.,Petri, W.A., 2012. Proteomic analysis of the cyst stage of Entamoeba histolytica.PLoS Negl. Trop. Dis. 6, e1643.

mbatipudi, K., Old, J., Guilhaus, M., Raftery, M., Hinds, L., Deane, E., 2006. Proteomicanalysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii).Comp. Biochem. Physiol. D Genomics Proteomics (The Netherlands), 283–291.

eranova-Giorgianni, S., 2003. Proteome analysis by two-dimensional gel elec-trophoresis and mass spectrometry: strengths and limitations. TrAC, TrendsAnal. Chem. 22, 273–281.

iller, L., Matthiesen, J., Kuhne, V., Lotter, H., Handal, G., Nozaki, T., Saito-Nakano,Y., Schumann, M., Roeder, T., Tannich, E., Krause, E., Bruchhaus, I., 2014. The cellsurface proteome of Entamoeba histolytica. Mol. Cell. Proteomics 13, 132–144.

iller, L., Schmidt, H., Krause, E., Gelhaus, C., Matthiesen, J., Handal, G., Lotter, H.,Janssen, O., Tannich, E., Bruchhaus, I., 2009. Comparison of two geneticallyrelated Entamoeba histolytica cell lines derived from the same isolate with dif-ferent pathogenic properties. Proteomics 9, 4107–4120.

ouyer, S., Rodier, M.H., Guillot, A., Héchard, Y., 2009. Acanthamoeba castellanii: pro-teins involved in actin dynamics, glycolysis, and proteolysis are regulated duringencystation. Exp. Parasitol. 123, 90–94.

rewis, I.A., Brennan, P., 2010. Proteomics technologies for the global identificationand quantification of proteins. In: Advances in Protein Chemistry and StructuralBiology 2010. Elsevier Inc., The Netherlands, pp. 1–44.

rzeska, H., Guag, J., Preston, G.M., Titus, M.A., Korn, E.D., 2012. Molecular basisof dynamic relocalization of Dictyostelium myosin IB. J. Biol. Chem. 287,14923–14936.

arlesso, A.M., Artuso, G.L., Caumo, K., Rott, M.B., 2010. Potentially pathogenic acan-thamoeba isolated from a hospital in Brazil. Curr. Microbiol. 60, 185–190.

aumo, K., Frasson, A.P., Pens, C.J., Panatieri, L.F., Frazzon, A.P., Rott, M.B., 2009. Poten-tially pathogenic Acanthamoeba in swimming pools: a survey in the southernBrazilian city of Porto Alegre. Ann. Trop. Med. Parasitol. 103, 477–485.

hrisman, C.J., Alvarez, M., Casadevall, A., 2010. Phagocytosis of Cryptococcus neofor-mans by, and nonlytic exocytosis from, Acanthamoeba castellanii. Appl. Environ.Microbiol. 76, 6056–6062.

larke, M., Lohan, A.J., Liu, B., Lagkouvardos, I., Roy, S., Zafar, N., Bertelli, C., Schilde,C., Kianianmomeni, A., Burglin, T.R., Frech, C., Turcotte, B., Kopec, K.O., Synnott,J.M., Choo, C., Paponov, I., Finkler, A., Soon Heng Tan, C., Hutchins, A.P., Wein-meier, T., Rattei, T., Chu, J.S., Gimenez, G., Irimia, M., Rigden, D.J., Fitzpatrick, D.A.,Lorenzo-Morales, J., Bateman, A., Chiu, C.H., Tang, P., Hegemann, P., Fromm, H.,Raoult, D., Greub, G., Miranda-Saavedra, D., Chen, N., Nash, P., Ginger, M.L., Horn,M., Schaap, P., Caler, L., Loftus, B., 2013. Genome of Acanthamoeba castellaniihighlights extensive lateral gene transfer and. Genome Biol. 14, R11.

orsaro, D., Venditti, D., 2010. Phylogenetic evidence for a new genotype of Acan-thamoeba (Amoebozoa, Acanthamoebida). Parasitol. Res. 107, 233–238.

uervo, P., de Jesus, J.B., Junqueira, M., Mendonc a-Lima, L., González, L.J., Betancourt,L., Grimaldi, G., Domont, G.B., Fernandes, O., Cupolillo, E., 2007. Proteome analy-sis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresisand mass spectrometry. Mol. Biochem. Parasitol. 154, 6–21.

avis, P.H., Chen, M., Zhang, X., Clark, C.G., Townsend, R.R., Stanley, S.L., 2009. Pro-teomic comparison of Entamoeba histolytica and Entamoeba dispar and the roleof E. histolytica alcohol dehydrogenase 3 in virulence. PLoS Negl. Trop. Dis. 3,e415.

zik, J.M., 2006. Molecules released by helminth parasites involved in host coloniza-tion. Acta Biochim. Pol. (Poland), 33–64.

aso, C., Bischof, S., Hehl, A.B., 2013. The proteome landscape of Giardia lambliaencystation. PLoS One 8, e83207.

org, A., Weiss, W., Dunn, M.J., 2004. Current two-dimensional electrophoresis tech-nology for proteomics. Proteomics 4, 3665–3685.

otz, S., Garcia-Gomez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles,M., Talon, M., Dopazo, J., Conesa, A., 2008. High-throughput functional anno-tation and data mining with the Blast2GO suite. Nucleic Acids Res. (England),3420–3435.

orowitz, J.A., Hammer, J.A., 1990. A new Acanthamoeba myosin heavy chain. Cloningof the gene and immunological identification of the polypeptide. J. Biol. Chem.265, 20646–20652.

uang, K.Y., Chien, K.Y., Lin, Y.C., Hsu, W.M., Fong, I.K., Huang, P.J., Yueh, Y.M., Gan,R.R., Tang, P., 2009. A proteome reference map of Trichomonas vaginalis. Parasitol.Res. 104, 927–933.

uang, K.Y., Shin, J.W., Huang, P.J., Ku, F.M., Lin, W.C., Lin, R., Hsu, W.M., Tang, P., 2012.Functional profiling of the Tritrichomonas foetus transcriptome and proteome.Mol. Biochem. Parasitol. 187, 60–71.

Please cite this article in press as: Caumo, K.S., et al., Proteomic profiliActa Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.08.009

erlstrom-Hultqvist, J., Stadelmann, B., Birkestedt, S., Hellman, U., Svard, S.G., 2012.Plasmid vectors for proteomic analyses in Giardia: purification of virulence.Eukaryot. Cell 11, 864–873.

han, N.A., 2006. Acanthamoeba: biology and increasing importance in humanhealth. FEMS Microbiol. Rev. 30, 564–595.

PRESSca xxx (2014) xxx–xxx 7

Kim, J.H., Yang, A.H., Sohn, H.J., Kim, D., Song, K.J., Shin, H.J., 2009. Immunodomi-nant antigens in Naegleria fowleri excretory–secretory proteins were potentialpathogenic factors. Parasitol. Res. 105, 1675–1681.

Klopocka, W., Redowicz, M.J., Wasik, A., 2009. Regulation of cortical cytoskele-ton dynamics during migration of free-living amoebae. Postepy Biochem. 55,129–137.

Leitsch, D., Köhsler, M., Marchetti-Deschmann, M., Deutsch, A., Allmaier, G.,Duchêne, M., Walochnik, J., 2010. Major role for cysteine proteases during theearly phase of Acanthamoeba castellanii encystment. Eukaryot. Cell 9, 611–618.

Lorenzo-Morales, J., Martin-Navarro, C.M., Lopez-Arencibia, A., Arnalich-Montiel, F.,Pinero, J.E., Valladares, B., 2013. Acanthamoeba keratitis: an emerging diseasegathering importance worldwide? Trends Parasitol.

Ma, G.Y., Zhang, J.Z., Yin, G.R., Zhang, J.H., Meng, X.L., Zhao, F., 2009. Toxoplasmagondii: proteomic analysis of antigenicity of soluble tachyzoite antigen. Exp.Parasitol. (United States), 41–46.

Maciver, S.K., Hussey, P.J., 2002. The ADF/cofilin family: actin-remodeling proteins.Genome Biol. 3, reviews3007.

Magliano, A.C., da Silva, F.M., Teixeira, M.M., Alfieri, S.C., 2009. Genotyping,physiological features and proteolytic activities of a potentially pathogenicAcanthamoeba sp. isolated from tap water in Brazil. Exp. Parasitol. 123, 231–235.

Mann, M., Jensen, O.N., 2003. Proteomic analysis of post-translational modifications.Nat. Biotechnol. (United States), 255–261.

Marciano-Cabral, F., Cabral, G., 2003. Acanthamoeba spp. as agents of disease inhumans. Clin. Microbiol. Rev. 16, 273–307.

Martin, K.H., Slack, J.K., Boerner, S.A., Martin, C.C., Parsons, J.T., 2002. Integrin con-nections map: to infinity and beyond. Science (United States), 1652–1653.

Moon, E.K., Xuan, Y.H., Chung, D.I., Hong, Y., Kong, H.H., 2011. Microarray analysis ofdifferentially expressed genes between cysts and trophozoites of Acanthamoebacastellanii. Korean J. Parasitol 49, 341–347.

Pawar, H., Sahasrabuddhe, N.A., Renuse, S., Keerthikumar, S., Sharma, J., Kumar,G.S., Venugopal, A., Sekhar, N.R., Kelkar, D.S., Nemade, H., Khobragade, S.N.,Muthusamy, B., Kandasamy, K., Harsha, H.C., Chaerkady, R., Patole, M.S., Pandey,A., 2012a. A proteogenomic approach to map the proteome of an unsequencedpathogen. Proteomics 12, 832–844.

Pawar, H., Sahasrabuddhe, N.A., Renuse, S., Keerthikumar, S., Sharma, J., Kumar,G.S., Venugopal, A., Sekhar, N.R., Kelkar, D.S., Nemade, H., Khobragade, S.N.,Muthusamy, B., Kandasamy, K., Harsha, H.C., Chaerkady, R., Patole, M.S., Pandey,A., 2012b. A proteogenomic approach to map the proteome of an unsequencedpathogen—Leishmania donovani. Proteomics 12, 832–844.

Qvarnstrom, Y., Nerad, T.A., Visvesvara, G.S., 2013. Characterization of a newpathogenic Acanthamoeba Species, A. byersi n. sp., isolated from a human withfatal amoebic encephalitis. J. Eukaryot. Microbiol. 60, 626–633.

Rocha-Azevedo, B., Jamerson, M., Cabral, G.A., Marciano-Cabral, F., 2010.Acanthamoeba culbertsoni: analysis of amoebic adhesion and invasion on extra-cellular matrix components collagen I and laminin-1. Exp. Parasitol. 126, 79–84.

Rocha-Azevedo, B.D., Jamerson, M., Cabral, G.A., Silva-Filho, F.C., Marciano-Cabral, F.,2009. Acanthamoeba interaction with extracellular matrix glycoproteins: biolog-ical and biochemical characterization and role in cytotoxicity and invasiveness.J. Eukaryot. Microbiol. 56, 270–278.

Schuster, F.L., 2002. Cultivation of pathogenic and opportunistic free-living amebas.Clin. Microbiol. Rev. 15, 342–354.

Schuster, F.L., Visvesvara, G.S., 2004. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int. J. Parasitol. 34, 1001–1027.

Siddiqui, R., Khan, N.A., 2012a. Biology and pathogenesis of Acanthamoeba. Parasit.Vectors 5, 6.

Siddiqui, R., Khan, N.A., 2012b. War of the microbial worlds: who is the beneficiaryin Acanthamoeba—bacterial interactions? Exp. Parasitol. 130, 311–313.

Siddiqui, R., Khan, N.A., 2012c. Acanthamoeba is an evolutionary ancestor ofmacrophages: a myth or reality? Exp. Parasitol. 130, 95–97.

Sotillo, J., Valero, M.L., Sánchez Del Pino, M.M., Fried, B., Esteban, J.G., Marcilla, A.,Toledo, R., 2010. Excretory/secretory proteome of the adult stage of Echinostomacaproni. Parasitol. Res. 107, 691–697.

Tolstrup, J., Krause, E., Tannich, E., Bruchhaus, I., 2007. Proteomic analysis of Enta-moeba histolytica. Parasitology 134, 289–298.

Veríssimo, C.M., Maschio, V.J., Correa, A.P., Brandelli, A., Rott, M.B., 2013. Infectionin a rat model reactivates attenuated virulence after long-term axenic cultureof Acanthamoeba spp. Mem. Inst. Oswaldo Cruz. 108 (7), 832–835.

Visvesvara, G.S., 2010. Amebic meningoencephalitides and keratitis: challenges indiagnosis and treatment. Curr. Opin. Infect. Dis. 23, 590–594.

Visvesvara, G.S., Moura, H., Schuster, F.L., 2007. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri,and Sappinia diploidea. FEMS Immunol. Med. Microbiol. 50, 1–26.

Wang, X., Chen, W., Hu, F., Deng, C., Zhou, C., Lv, X., Fan, Y., Men, J., Huang, Y., Sun,J., Hu, D., Chen, J., Yang, Y., Liang, C., Zheng, H., Hu, X., Xu, J., Wu, Z., Yu, X.,2011. Clonorchis sinensis enolase: identification and biochemical characteriza-tion of a glycolytic enzyme from excretory/secretory products. In: Molecularand Biochemical Parasitology. Crown 2011. Elsevier B.V, The Netherlands, pp.135–142.

Wang, Y., Cheng, Z., Lu, X., Tang, C., 2009. Echinococcus multilocularis: proteomicanalysis of the protoscoleces by two-dimensional electrophoresis and massspectrometry. Exp. Parasitol. 123, 162–167.

ng of the infective trophozoite stage of Acanthamoeba polyphaga.

Wilson, I.W., Weedall, G.D., Hall, N., 2012. Host-Parasite interactions in Entamoebahistolytica and Entamoeba dispar: what have we learned from their genomes?Parasite Immunol. 34, 90–99.

Winck, M.A., Caumo, K., Rott, M.B., 2011. Prevalence of acanthamoeba from tap waterin Rio Grande do Sul, Brazil. Curr. Microbiol. 63, 464–469.

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