Cell Surface Proteome Analysis of Human-Hosted Trypanosoma cruzi

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Cell Surface Proteome Analysis of HumanHosted Trypanosoma cruzi Life Stages Rayner M. L. Queiroz, Sébastien CHARNEAU, Izabela M.D. Bastos, Jaime M. Santana, Marcelo Valle de Sousa, Peter Roepstorff, and Carlos André Ornelas Ricart J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr401120y • Publication Date (Web): 30 Jun 2014 Downloaded from http://pubs.acs.org on July 1, 2014

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Journal of Proteome Research

Cell Surface Proteome Analysis of HumanHosted Trypanosoma cruzi Life Stages Rayner M. L. Queiroz§‡, Sébastien Charneau§, Izabela M. D. Bastos §, Jaime M. Santana §, Marcelo V. de Sousa §, Peter Roepstorff‡ and Carlos A. O. Ricart§ § Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, Brazil ‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

KEYWORDS Trypanosoma cruzi, trypomastigote, amastigote, subproteome, plasma membrane, cell surface enzymes, cell surface trypsinization, biotinylation.

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ABSTRACT

Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins in the PM subproteomes, including enzymes that might be suitable candidates for drug intervention. The comparison of the cell surface proteome among the life forms revealed some potentially stage-specific enzymes, although the majority was shared by both stages. Bioinformatic analysis showed that the vast majority of the identified proteins are membrane-derived and/or possess predicted transmembrane domains. They are mainly involved in host cell infection, protein adhesion, cell signaling and the modulation of mammalian host immune response. Several virulence factors and proteins potentially capable of acting at a number of metabolic pathways of the host and also to regulate cell differentiation of the parasite itself were also found. INTRODUCTION Chagas' disease is a parasitic illness endemic mostly in Latin America, which is caused by the protozoan Trypanosoma cruzi. This parasite has a digenetic life-cycle with four major life stages: epimastigotes and metacyclic trypomastigotes in the insect vectors and trypomastigotes and amastigotes in the mammalian hosts 1, 2. So far there is no immunoprophylactic vaccine or effective chemotherapic treatment considering that the drugs commonly used to treat human


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infection, nifurtimox and benznidazol, have high toxicity and do not prevent the progression of the chronic form of Chagas' disease 3. Before cell invasion, T. cruzi infective life forms must gain access and form stable adhesions to the host cell surface. Some T. cruzi surface proteins are able to bind to components of extracellular matrix or to host cell surface receptors and others display hydrolytic activities against components of extracellular matrix 4-6. In fact, plasma membrane (PM) proteins are essential to safeguard cell integrity, to cell-cell communication, to membrane transport, as well as transmembrane signaling processes in response to extracellular stimuli where cytoplasmatic domains of transmembrane proteins may be reversibly phosphorylated. Approximately 50% of the T. cruzi functional genome encodes cell surface proteins that are grouped in families such as gp63, gp85/trans-sialidase (TS) superfamily, mucins and mucinassociated surface proteins (MASPs) 7-9. These proteins are directly involved in host-parasite interactions (reviewed in 10). TS transfer sialic acid residues to the parasite surface mucins. Also the mucins act protecting the parasite from both the vector's and the host's defensive mechanisms and to ensure the anchorage point and invasion of specific cells and tissues 11. Interactions between host and parasite cell surface proteins trigger signaling cascades in both cells that start the parasite invasion process. Both during infection and parasite differentiation, the intracellular release of Ca2+ is regarded as indispensible for the coordination of molecular events involved in these processes, but the precise molecular mechanisms responsible for controlling this phenomenon are not fully elucidated yet. However, pathways related to this event have already been identified. For instance, the engagement of gp82 with an unknown ligand was shown to trigger in metacyclic trypomastigotes a signaling cascade which involves


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the phosphorylation of a 175 kDa protein 12 and PKC and an inositol-3-phosphate (IP3)-mediated release of Ca2+ reservoirs from the endoplasmatic reticulum 13. Thereby, for pathogen micro-organism such as T. cruzi, studies concerning the characterization of PM subproteome are relevant, not only due to the biological importance of PM proteins, but also because this kind of study may eventually suggest a suitable target for drug intervention, since currently about 70% of all approved pharmaceutical agents act on PM proteins 14. Moreover, a rational approach to elect potential targets for antiparasite chemotherapy is to seek out proteins vital for the parasites and not for the host. Enzymes and receptors can be designated targets for antiparasitic chemotherapy, because their inhibition should bring the parasites under control 15. Other features, however, can also offer opportunity for specifically targeting a parasite enzyme, e.g. when the parasite enzyme has a different location from the host counterpart, with the most accessible situation being if the enzyme is released or surface-located on the pathogen, or if it is involved in a process that is unique to the parasite but at the same time vital, such as the invasion of a host cell 16. This context grounds the goal of surveying and evaluating the enzymatic activity repertoire displayed on the surface of human-hosted life stages of T. cruzi, as a source to provide data for research groups that seek potential targets for drug development. Our group reported the first T. cruzi proteome analysis a decade ago 17, 18, before its genome had been sequenced 9 and also before the landmark work from Atwood and collaborators regarding proteomic shotgun analysis from T. cruzi major life-stages 19. Recently in a previous work using epimastigote life forms, we evaluated and optimized two different methodologies to access the T. cruzi PM subproteome, (i) biotinylation of surface proteins followed by streptavidin affinity chromatography and (ii) cell surface trypsinization (shave) of intact cells and observed that both


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approaches offer together a comprehensive and complementary view of the subproteome 20. Here, we employed these complementary techniques to address the cell surface proteome analysis of human-hosted life stages of T. cruzi. Recently it has been reported a survey of T. cruzi trypomastigote detergent-solubilized membrane proteins enriched by hydrophobic phase partitioning 21, but to our knowledge this is the first report directly addressing the PM subproteome analysis of T. cruzi tissue cultured-derived-trypomastigote and axenic amastigote.

MATERIAL AND METHODS Fetal bovine serum (FBS) was purchased from Sorali Biotecnologia (Campo Grande, Brazil). MicroBCA kit, Sulfo-NHS-LC-BiotinTM and pre-packed 1 mL immobilized Streptavidin columns were from Pierce (Rockford, IL USA). GeLoader tips were from Eppendorf (Hamburg, Germany). AmiconTM filter units with 3 kDa cut-off membrane, were from Millipore (Billerica, MA, USA). Modified trypsin was from Promega (Madison, WI, USA). TPCK treated porcine pancreas trypsin, Dulbecco's Modified Eagle Medium (DMEM) and all others reagents were purchased from Sigma/Aldrich (St. Louis, MO, USA), unless stated otherwise.

Trypomastigote and axenic amastigote in vitro culture Tissue culture-derived trypomastigotes, Y strain, were collected from infected HeLa cell culture monolayers 22 from the second day of outbreak, which released the highest number of parasites that consisted of over 98% trypomastigotes. HeLa cell culture was maintained in DMEM, pH 7.5, supplemented with 5% FBS and 100 µg/mL gentamicin at 37 °C with 5% CO2 atmosphere.


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Axenic amastigotes were generated by incubating trypomastigotes in DMEM, pH 5.0, without FBS for 9 hours at 37 °C and 5% CO2 as previously described 23, 24.

Cell surface trypsinization (Shave) T. cruzi parasites (2 ×109 cells per biological replicate) were washed 3 times with 4 mL DMEM, re-suspended in 8 mL DMEM preheated at 37 °C at a final concentration of 2.5 ×108 parasites/mL and equally divided into 2 tubes. Twenty micrograms of TPCK-treated trypsin were added to one of the tubes (Shave sample) and the other sample was used as a control. Both samples were incubated at 37 °C for 30 min, since as observed with epimastigotes 20, only after about 45 min of incubation there were motionless trypomastigotes and, therefore, we established a maximum of 30 min for incubation for both trypomastigotes and axenic amastigotes. In order to remove cells after incubation, the tubes were centrifuged for 5 min at room temperature in 3 rounds to ensure complete removal of cells and avoid mechanical cell lysis: firstly at 2,000 ×g, then at 4,000 ×g and the last at 6,000 ×g. The supernatants were transferred to new tubes after each centrifugation and the pellets were discarded. Then, 20 µg of TPCK-treated trypsin were added to the Control sample that had been subjected to the centrifugation steps followed by incubation at 37 °C for 30 min. Both Control and Shave samples were vacuum-dried and stored at -20 °C.

Cell surface biotinylation and Streptavidin affinity chromatography


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T. cruzi trypomastigote or axenic amastigote forms (1 × 109 cells) were washed 3 times with Phosphate Buffered Saline pH 8.0 (PBS, containing 80 g/L NaCl, 2 g/L KCl, 26.8 g/L Na2HPO4– 7 H2O and 2.4 g/L KH2PO4) and re-suspended in 5 mL of 250 µg/mL Sulfo-NHS-LC-BiotinTM in PBS, pH 8.0, for 10 min at 37 °C. Subsequently, cells were washed once with TBS, to quench the reaction, and re-suspended in 1 mL Milli-Q water containing a complete cocktail of protease and phosphatase inhibitors (Roche) and lysed though 3 cycles of freeze-thawing in liquid N2. Each sample was then centrifuged at 15,000 ×g for 20 min and the resulting pellet washed by centrifugation (15,000 × g, 20 min) with Milli-Q water to remove excess of water-soluble proteins. The pellet was re-suspended in a solution of 2 mL 2% Triton X-100 and incubated for 1 hour in ice with several vortexing homogenizations and then centrifuged at 10,000 × g for 20 min at 4 °C to remove debris. For affinity chromatography, each extract containing biotinylated proteins was passed through a pre-packed 1 mL immobilized Streptavidin column (PIERCE, Rockford, IL USA) for 1 hour at room temperature. Each column was washed with 15 mL of 1% Triton X-100 in TBS and then 10 mL of 1% Triton X-100 in 1 M NaCl to remove non-specifically bound material. Biotinylated proteins were eluted with 5 mL of 8 M guanidine-hydrochloride, pH 1.5. The guanidine-HCl eluted fraction was immediately neutralized by dilution with 5 mL of 250 mM Tris pH 7.0, in order to ultrafiltrate with 3kDa AmiconTM centrifugal filters. Each sample was concentrated by ultrafiltration to a volume of approximately 200 µL, dried and stored at -80 °C.

Sample preparation for LC-MS/MS


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Protein samples were first quantified using amino acid analysis using a Biochrom 30 amino acid analyzer (Biochrom, Cambridge, U.K.) following the protocol from the manufacturer. Shave and Control samples from 1 ×109 parasites each were re-suspended in 80 µL of 20 mM triethylammonium bicarbonate (TEAB), reduced with 20 mM DTT at 56 °C for 45 min, alkylated with 40 mM IAA at room temperature in the dark for 60 min and further digested overnight at 37 °C with 1 µg modified trypsin (Promega Madison, USA). After digestion, the samples were acidified to a 0.1% TFA final concentration, and desalted on homemade microcolumns of Poros Oligo R3 resin (PerSeptive Biosystems, Framingham, USA) packed (1 cm long) in p200 tips (adapted from 25). To remove the pH indicator present in DMEM, the desalted samples were re-suspended in 90% ACN/0.1% TFA and passed through ZIC-HILIC resin (10 µm particle size and 100 Å, SeQuant, Umeå, Sweden) packed into GeLoader tips and eluted with 0.1% TFA. To ensure minimal sample loss due to overloading, the flow-through was passed through another ZIC HILIC microcolumn and both eluates combined before vacuum dried. Biotinylated cell surface proteins were submitted to acetone/ethanol precipitation to remove guanidine-HCl and traces of Triton X-100. Briefly, the dried sample was re-suspended in 100 µL of 20 mM TEAB diluted 4 times with ice cold ethanol and vortexed. Then the same volume of ice-cold acetone was added, vortexed vigorously and incubated overnight at -20 °C. After incubation, the material was centrifuged at 20,000 ×g at 4 °C for 15 min and the supernatant discarded. The pellet was washed another 3 times with ice cold 40% ethanol/ 40% acetone solution. Finally, the sample was re-suspended in 20 mM TEAB, reduced with 20 mM DTT at 56 °C for 45 min, alkylated with 40 mM IAA at room temperature in the dark for 60 min and digested overnight at 37 °C with 2 µg modified trypsin. After digestion, the sample was acidified


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to 0.1% TFA final concentration, and desalted on homemade microcolumns of Poros Oligo R3 resin (PerSeptive Biosystems, Framingham, MA, USA) packed (1 cm long) in p200 tips (adapted from 25).

LC-MS/MS and data analysis Samples were analyzed using an EASY-nano LC system (Proxeon Biosystems, Odense, Denmark) coupled online with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Waltham, USA). For nanoLC, 3 µg of each sample were loaded onto a 18 cm fused silica emitter (75 µm inner diameter) packed in-house with reverse phase capillary column ResiproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH, Germany) and eluted using a gradient from 100% solvent A (0.1% formic acid) to 26% solvent B (0.1% formic acid, 95% acetonitrile) for 180 min, 26% to 100% B for 5 min and 100% B for 8 min at a flow rate of 200 nL/min). After each run, the column was washed with 90% solvent B and re-equilibrated with solvent A. Mass spectra were acquired in positive ion mode applying data-dependent automatic survey MS scan and tandem mass spectra (MS/MS) acquisition mode. Each MS scan in the Orbitrap analyzer (mass range of m/z 350-1,800 and resolution 100,000) was followed by MS/MS of the fifteen most intense ions in the LTQ. Fragmentation in the LTQ was performed by collision-induced dissociation and selected sequenced ions were dynamically excluded for 30 s. Raw data were viewed in Xcalibur v.2.1 (Thermo Scientific Waltham, USA) and data processing was performed using Proteome Discoverer v.1.3 beta (Thermo Scientific, Waltham, USA). The Raw files were submitted to searching using Proteome Discoverer with in-house Mascot v.2.3 algorithm against Trypanosoma cruzi database downloaded (early 2012) using Database on Demand tool 26


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containing the proteins of the parasite found in UniProt/SWISS-PROT and UniProt/TrEMBL. Contaminant proteins (several types of human keratins, BSA and porcine trypsin) were also added to the database and all contaminant proteins identified were manually removed from the result lists. The searches were performed with the following parameters: MS accuracy 10 ppm, MS/MS accuracy 0.5 Da, 2 missed cleavage sites allowed, carbamidomethylation of cysteine as fixed modification and oxidation of methionine and protein N-terminal acetylation as variable modifications. For the biotinylated sample search peptide N-terminus NHS-LC-biotinylation and NHS-LC-biotinylated lysine were also added as variable modifications (increment of 339.16 Da). Number of proteins, protein groups and number of peptides were filtered for False Discovery Rates (FRD) less than 1% and only peptides with rank 1 and minimal of 2 peptides per protein were accepted for identification using Proteome Discoverer. ProteinCenter software (Thermo Scientific Waltham, USA) was used to interpret the results at protein level, e.g, statistical comparison of Gene Ontology terms between data sets, number of proteins with transmembrane domains. Better annotation of the identified proteins and enzyme activity prediction and classification (Enzyme Commission numbers) were acquired using Blast2GO software (http://www.blast2go.com/b2ghome) using default parameters. The glycosylphosphatidylinositol (GPI) prediction was performed using FragAnchor (http://navet.ics.hawaii.edu/∼fraganchor/NNHMM/NNHMM.html) 27 and only the sequences with high probability were accepted as potential GPI-anchored, as described elsewhere 28, 29. SignalP v.4.0 (http://www.cbs.dtu.dk/services/SignalP/) software was used to predict proteins secreted by classical ER/Golgi pathway.

Repository data


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Reference protein sequence database, raw files as well peptide and protein identification files (MGF files) have been deposited in a public repository - The PeptideAtlas database (http://www.peptideatlas.org/PASS/PASS00403) under the datasetTag Tcruzi_Surface_T_A and database identifier PASS00403.

RESULTS AND DISCUSSIONS

Comparison of trypomastigote and axenic amastigote cell surface subproteome The previously optimized and validated techniques for enrichment of T. cruzi epimastigote surface proteins 20 were successfully employed to isolate plasma membrane proteins from both mammalian-hosted life stages. Altogether 1049 and 1118 protein groups were identified in trypomastigotes and axenic amastigotes, respectively, by cell surface trypsinization (Shave samples) and in the Control samples (that comprises proteins released under Shave conditions without trypsin), 679 and 626 protein groups, respectively. Analyses of biological duplicates were performed for Shave and Control samples from both trypomastigotes and axenic amastigotes. Biotinylation followed by affinity chromatography yielded 576 and 693 unambiguous identifications from a single biological replicate from each life stage respectively. (Supplementary Tables S4 and S5) Similarly to the previous observation with the epimastigote life stage 20, each enrichment procedure yielded several specific proteins (fig. 1 and Supplementary Table S1). GO Slim


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annotation obtained with ProteinCenter software also from trypomastigotes and axenic amastigotes displayed a great proportion of membrane proteins evidencing the successful enrichment of PM proteins (fig. 2). In addition, the percentage of proteins with predicted transmembrane domains was substantial (table 1). This high percentage in the Control sample corroborates the literature, which reports the release of membrane vesicles by metacyclic trypomastigotes and tissue culture-derived trypomastigotes. These vesicles are carriers of virulence factors such as members of TS superfamily and increase inflammation and parasitism 30-32

. Regarding the amastigote stage, it is still to be confirmed whether or not this life stage

releases such vesicles in vivo, intra- or extracellularly, but the notable proportion of predicted transmembrane proteins identified here is a strong indication for that. In agreement with previous reports from detergent-solubilized membrane proteins enriched by hydrophobic phase partitioning 21, a rather small percentage (lower than 10%) of the identifications were predicted to have GPI-anchor and/or ER/Golgi signal peptide (table 1) in our enriched samples as well as in the control. Although this shows that the diversity of proteins bound to the PM through that manner is not noteworthy, the data does not inform about their abundance in copies per cell. For instance, GPI-anchored proteins are held to extensively coat T. cruzi parasites and such proteins will be further discussed. Since the scope of the present work was no longer to compare the methodologies used for PM protein isolation, as was the previous work with the epimastigote life stage, we gathered all protein groups identified in Biotinylated and Shave samples together in a single set from now on named surface/exposed proteins, regardless if some of these proteins were also found in the Control, as we understand that these proteins may also be part of or associated to the plasma


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membrane subproteome and they are still relevant potential targets for drug intervention due to their accessible localization. As depicted in figure 3A, tissue culture-derived trypomastigotes and axenic amastigotes shared the majority (61%) of the protein groups identified and none of the GO Slim categories had any term over-represented between the human-hosted life stages. Among the annotated proteins, the terms membrane and metabolic process were the most abundant. Besides regulating eukaryotic cellular processes, in parasites such T. cruzi, cell surface proteins are involved in breakdown of extracellular components and adhesion to host tissues to guarantee access to the host cell surface and to increase infection efficiency. The prominent abundance of proteins classified with the GO Slim molecular function terms protein binding and catalytic activity (fig. 3D) reflects the engagement of the parasite cell surface proteome in the abovementioned processes. Moreover, the amount of these proteins also annotated with nucleotide binding term (fig. 3D) suggests their dependence on nucleotides, such as ATP or GTP, to perform their activities. As mentioned before, proteins found only in the parasite are potential good drug targets for therapeutic intervention especially if they are located on the cell surface or are secreted/excreted 16

. Thus, surface proteins named Hypothetical in the database could fit in this category. Overall

we identified in trypomastigotes and axenic amastigotes respectively 251 and 245 hypothetical proteins, from which 72 and 66 were exclusively detected in one life stage and 92 and 101 presented at least one predicted transmembrane domain. Hypothetical proteins annotated with GO slim catalytic activity term had 30 representatives in trypomastigotes, from which 7 were


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stage specific, while axenic amastigotes had 32 hypothetical proteins, 9 being stage specific (Supplementary Table S3). Due to the significant percentage of unannotated proteins (around 50%), Blast2GO software was also employed to obtain a sharper and more comprehensive detailing of our identifications, including Enzyme Commission number (EC number) to classify the enzymes, which will be addressed in the following.

Predicted enzyme activities of surface/exposed proteins (cell surface-located and/or released proteins) The set of surface/exposed proteins (surface-located or released) from both human-hosted life stages of T. cruzi contained several representatives of each of the six main enzyme activity groups (fig. 4 and Supplementary Table S2) classified according to the Enzyme Commission number, axenic amastigotes having the greatest number of activity classes and representatives of each catalytic group. As a whole, hydrolytic enzymes (EC: 3) is the most represented group in axenic amastigote and trypomastigote with 134 and 94 members, respectively, in 29 and 21 different classes, in which the most frequent is the exo-α-sialidase (EC:3.2.1.18). Transferases (EC: 2) were the most diverse group with 50 and 47 different classes in axenic amastigotes and trypomastigotes, respectively. Within this group, Ser/Thr protein kinases were the most abundant with 14 and 13 members in axenic amastigotes and trypomastigotes, of which 4 and 3 proteins were identified in our data exclusively in axenic amastigotes and trypomastigotes, respectively. Adenylate kinases (EC:2.7.4.3), which have an important role in


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cell energetic homeostasis, had 5 representatives identified in both parasite life stages. Tyrprotein kinases (EC:2.7.10) whose activity regulates primordial cellular functions like division and signal transduction, presented 4 members identified in the surface proteins, of which the putative MAPK (gi|407834743) was here only detected in axenic amastigotes. It had one predicted transmembrane domain, and was also identified in Control. In fact, these predicted Tyr-protein kinases were also predicted as Ser/Thr-protein kinases. No dedicated T. cruzi Tyrkinase was predicted which is in agreement with the literature 33. In our conditions, several catalytic classes were identified in only one of the analyzed parasite life stages, with 39 unique classes in axenic amastigotes and 13 classes in trypomastigotes (table 2). Indeed, our data cannot prove that these enzymes exist in the surface of just one of the life stages. However, when an enzyme of a specific predicted activity was detected only in one life stage, this means either that there is no representative of these enzymes in the other or that they are much less expressed. Most of these supposed stage specific enzymes catalyze reactions from metabolic pathways, particularly of lipids and carbohydrates. Arising from the great diversity of enzymes, from axenic amastigotes, involved in several points of metabolic pathways, we can infer that the parasite is prepared to actively interfere within the metabolism of a host cell to maintain itself intracellularly, but it is not confirmed if those features are also shared with intracellular amastigotes. By this means, it is probably provided with several nutritional sources. It is also important noting that enzymes related to lipid metabolism have been described as key participants of the amastigogenesis, e.g. a phospholipase C anchored to the cell membrane and specifically to phosphatidyl inositol that participates in the Ca2+ activation pathway releasing the intracellular messengers diacylglycerol and IP3 34, 35. Also, major changes in the parasite lipidic


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profile occur during the differentiation process. It has been reported the increase in production and remodeling of inositol phosphoceramide and phosphatidyl inositol, through the action of phospholipases and transferases, as well as the increase of free ceramide level in amastigotes 36, 37

.

Trypomastigotes presented membrane-derived/associated enzymes involved in glycolysis and phospholipid biosynthesis such as glycerol-3-phosphate dehydrogenase, putative (gi|407859729, EC:1.1.5.3). They also displayed exposed enzymes like superoxide dismutase (EC:1.15.1.1) which have already been reported as soroprevalent excreted antigens of T. cruzi 38. This enzyme protects the parasite against oxidative stress, the attack of superoxide radicals from host blood cells 39 and represents a virulence factor and potential target for anti-parasitic drugs 40. Proteases are also accepted as good targets for chemotherapeutic intervention in cancer and autoimmune diseases as well as for infections caused by viruses such as HIV and bacteria 41-44. Indeed, in parasites, proteases are generally regarded as potential targets 45, although few have been fully validated from any parasite, indicating that a single protease is essential for its survival 15, 16. But still, released and surface proteases could offer attractive drug targets, both because mammalian counterparts may be less exposed and drug delivery problems are likely to be less severe. Here proteins classified as Hydrolases (EC: 3) and also, more specifically, Proteases (EC: 3.4.) were analyzed more deeply due to the previously mentioned importance in parasite-host interaction and in infection. The axenic amastigotes displayed the greatest amount and diversity of hydrolases. Among the most represented classes (fig. 5), threonine endopeptidase class (EC:3.4.25.0) presented many


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more components identified in amastigotes, whereas ubiquitin tiolesterases (EC:3.1.2.15) and proton transporting ATPases (EC:3.6.3.6) dominated in trypomastigotes. Exo-α-sialidases (EC:3.2.1.18) were the most represented enzyme class in the surface/exposed proteins of both life stages. This is largely due to the high number of TS and other glycoproteins with sialidase activity site identified. Moreover, axenic amastigotes displayed almost twice the number of sialidases compared to trypomastigotes. The ATPases (EC:3.6.1.3) are also abundant, although almost all are shared by both stages, except the Ribonuclease L inhibitor, putative (gi|322830647) detected only in axenic amastigotes. The surface/exposed proteins detected exclusively on axenic amastigotes and trypomastigotes (table 3) are mostly TS and glycoproteins, which possibly consist of the known stage-exclusive antigens, but several of these proteins do not have any predicted transmembrane domain or have predicted GPI anchors. Moreover, some of them were also identified in the Control samples; thereafter they may comprise released proteins that are not necessarily associated to the PM, although some of these proteins may present in the microvesicles and exosomes and also be present in the PM. Interestingly, axenic amastigotes presented putative ribonuclease L inhibitor (gi|322830647) probably attached to the PM by a single transmembrane domain. Ribonuclease L is one of the components of the innate immune defense, strongly induced by interferon molecules and usually thought of in terms of its antiviral functions, but is also protective in mice against infections by Bacillus anthracis and Escherichia coli 46, 47, perform anti-proliferative and tumor suppression functions and is involved in cell senescence and aging 48. In fact, ribonuclease L-null mice had a significantly prolonged lifespan by about 20 weeks compared with wild-type mice 48, thus we


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can hypothesize that T. cruzi parasites could modulate host-cells senescence upon infection and intracellular replication by means of inhibiting this enzyme. Regarding the peptidases identified (table 4), both life forms have threonine endopeptidases (EC:3.4.25.0), aminopeptidases (EC:3.4.11.0), metalloendopeptidases (EC:3.4.24.0), omega peptidases (EC:3.4.19.0) and cysteine endopeptidases (EC:3.4.22.0), which is the activity performed by the only peptidase exclusively identified in trypomastigotes (gi|70887345), but this activity is also performed by other enzymes in the axenic amastigote form. Beside the abovementioned activities, axenic amastigotes also displayed on their surface a metallocarboxypeptidase (EC:3.4.17.0), which has a predicted transmembrane domain and was also detected in the Control. The surface-located or released components identified also include proteins that potentially act in host cell metabolic pathways to avoid parasite degradation and/or exposure of antigens to the host immune system. For example, there were putative thimet oligopeptidases (TOP), known to act in the degradation of peptides released by proteasomes, thereby limiting presentation of parasite antigens by MHC class I 49. Also enzymes with ubiquitin hydrolase activity or deubiquitinases were detected. They have more than a role in post-translational regulation of protein expression and have been reported to inhibit the production of NF-κB in cells infected by virus 50 and in macrophages 51. NF-κB has frequently been referred to as a central mediator of the immune response, since a variety of pathogens leads to their activation which in turn controls the expression of various proinflammatory cytokines, chemokines, immune receptors and cell surface adhesion molecules 52.


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In fact, we could observe a greater diversity of proteins with ubiquitin hydrolase activity in trypomastigotes and more representatives of TOP enzymes in amastigotes. For what it appears, during the initial stage of cell infection while the trypomastigote is not differentiated yet, the parasite is prone to defend itself from protein degradation, whereas replicative amastigotes are committed to avoid antigen exposure in MHC class I molecules. Recently, it was reported that T. cruzi infection does down-modulate the immunoproteasome biosynthesis and the MHC class I cell surface expression 53. GPI-anchored proteins Glycosylphosphatidylinositol (GPI)-anchored proteins are held to extensively coat the parasite PM and are involved in a variety of host-parasite interaction aspects such as adhesion, invasion of host cells, pathogenesis and modulation of host immune response 11, 54-57. A genome-wise prediction study have reported that about 11.9% of T. cruzi genes possibly encode GPI-anchored proteins 28. The same group, working with T. cruzi trypomastigotes (Y strain), published the prediction of GPI-anchored proteins among those identified by a proteomic approach without previous PM enrichment 29. Overall the reported GPI-anchored proteins comprise products encoded by multigene families like mucins, MASPs, TSs and other surface glycoproteins. Using bioinformatic tools we were able to detect 37 protein groups with high probability to be GPI-anchored in trypomastigote and amastigote surface/exposed subproteomes (supplementary table S1). Despite this being significantly lower than the 178 protein groups predicted previously in trypomastigotes 29, they comprise mostly the same protein families (MASPs, TS/gp63 and gp85, c71, surface antigens TASV, TolT, and some uncharacterized proteins). This discrepancy may be due to the differences of stringency in protein identification and grouping, but the


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similarities in the protein descriptions ensure the good recover of such molecules by our enrichment methods. It is also worth mentioning that the number of protein groups identified do not correlate with the actual abundance of proteins expressed in the surface and different proteins from the same family are likely to be designated in a few or even a single protein group as those from multigene families. We could observe that trypomastigotes and axenic amastigotes shared the majority of predicted GPI-anchored protein groups (supplementary table S1). There were nine GPI-anchored proteins exclusively detected in axenic amastigote (a few uncharacterized proteins, some TS, one amastigote cytoplasmic antigen, one TASV surface protein and one gp63) and only two exclusively detected in trypomastigotes, a TS family member and a putative syntaxin (gi|322818853). The later has not been previously reported in any proteomic study. The diversity in number of protein groups attached to the PM by transmembrane domains was significantly higher than those by GPI anchor (table 1), which does not necessarily reflect the absolute number of proteins expressed in a cell. We observed a higher proportion of GPIanchored proteins in the axenic amastigote control sample which further suggests release of vesicles in this life-stage, but the prediction showed that most GPI-anchored proteins were also detected in the control sample of trypomastigotes.

CONCLUSIONS The analysis of the cell surface subproteome of the human-hosted life stages, by biotinylation of exposed surface proteins followed by streptavidin affinity chromatography and by surface


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trypsinization of intact cells, has provided a comprehensive view of the PM proteins, just as observed in our previous work with the epimastigote life stage 20. Regardless the subcellular localization of the identified proteins, although the vast majority is proven to be membrane-derived and/or to have transmembrane domains, our approach revealed an extensive protein repertoire involved in host cell infection, protein adhesion, modulators of mammalian host immune response, several parasite virulence factors and proteins potentially capable of acting at several metabolic pathways of the host and also regulate and enable cell differentiation of the parasite itself. Emerging from the comparison between surface-located and/or released proteins present on the parasites in its different forms as well as the surface enzyme repertoire, we can list new potential drug targets, especially with the potentially stage-specific enzyme classes. In sum, by the approaches here employed, one can have a broad overview of the extensive protein inventory used by the parasite in infection, defense against the host immune system and interference in the host metabolism. The future use of the same approaches with parasites treated with drugs or enzyme-specific inhibitors will provide more insights about the T. cruzi biology. FIGURES


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Figure 1. Venn diagram of protein groups identified in each set of enriched samples from axenic amastigotes (A) and trypomastigotes (B).


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Figure 2. GO slim cell component terms of protein groups identified in axenic amastigotes (A) and trypomastigotes (B). Y-axis represents the number of protein groups identified.


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Figure 3. Comparison between cell surface subproteomes of trypomastigotes and axenic amastigotes. Venn diagram of protein groups identified in each life stage (A) and GO slim terms (ProteinCenter) of cell component (B), biological process (C) and molecular function (D). Y-axis represents the number of protein groups.


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Figure 4. Comparison of the number of proteins displayed on the cell surface in each humanhosted life stage classified within the six main enzymatic activity groups.

Figure 5. Comparison of the most represented hydrolytic enzyme activities (EC 3) predicted in the surface subproteome of trypomastigotes and axenic amastigotes.


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TABLES. Table 1. Number of protein groups with predicted transmembrane domains, GPI-anchored or ER signal peptide.

Axenic amastigote proteins Sample Total TMa GPIa SignalPb Biotinylated 268 38.7% 1.1% 9.4% Shave 455 40.7% 2.2% 8.4% Control 213 34.0% 12.2% 8.0%

Trypomastigote proteins Total 200 427 202

TMa 34.7% 40.7% 29.7%

GPIa SignalPb 1.0% 7.3% 8.9% 7.9% 5.8% 8.5%

a percentage of protein groups with predicted transmembrane domains (TM) or GPI-anchored (GPI) related to the total number of proteins identified in each sample. b percentage of protein groups with predicted ER/Golgi signal peptide

Table 2. Stage-specific enzyme activities predicted in the surface/released subproteome.

Enzyme Nomenclature

Enzyme Codes

Life stage

Number of elements

phosphogluconate dehydrogenase (decarboxylating)

EC:1.1.1.44

A

2

glucose-6-phosphate dehydrogenase

EC:1.1.1.49

A

1

squalenemonooxygenase

EC:1.14.99.7

A

1

acyl-CoA dehydrogenase

EC:1.3.99.3

A

1

pyrroline-5-carboxylate reductase

EC:1.5.1.2

A

1

peptide-methionine (S)-S-oxide reductase

EC:1.8.4.11

A

1

transferring groups other than amino-acyl groups

EC:2.3.1.0

A

2


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fatty-acid synthase

EC:2.3.1.85

A

1

adenine phosphoribosyltransferase

EC:2.4.2.7

A

1

cysteine synthase

EC:2.5.1.47

A

2

methionine adenosyltransferase

EC:2.5.1.6

A

1

tyrosine transaminase

EC:2.6.1.5

A

1

Glucokinase

EC:2.7.1.2

A

1

pyruvate kinase

EC:2.7.1.40

A

1

mannose-1-phosphate guanylyltransferase

EC:2.7.7.13

A

1

mannose-1-phosphate guanylyltransferase (GDP)

EC:2.7.7.22

A

1

calf thymus ribonuclease H

EC:3.1.26.4

A

1

fructose-bisphosphatase

EC:3.1.3.11

A

1

sulfuric ester hydrolases

EC:3.1.6.0

A

1

metallocarboxypeptidases

EC:3.4.17.0

A

1

serine proteases

EC:3.4.21.0

A

2

acetylornithine deacetylase

EC:3.5.1.16

A

2

guanine deaminase

EC:3.5.4.3

A

1

Kynureninase

EC:3.7.1.3

A

1

cystathionine b-synthase

EC:4.2.1.22

A

1

Urocanatehydratase

EC:4.2.1.49

A

1

DNA-(apurinic or apyrimidinic site) lyase

EC:4.2.99.18

A

1

histidine ammonia-lyase

EC:4.3.1.3

A

1

UDP-glucose 4-epimerase

EC:5.1.3.2

A

1

triose-phosphate isomerase

EC:5.3.1.1

A

1

ribose-5-phosphate isomerase

EC:5.3.1.6

A

1

glucose-6-phosphate isomerase

EC:5.3.1.9

A

1


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Phosphoglucomutase

EC:5.4.2.2

A

1

Phosphomannomutase

EC:5.4.2.8

A

1

phosphoenolpyruvate mutase

EC:5.4.2.9

A

1

aspartate-tRNA ligase

EC:6.1.1.12

A

1

acetate-CoA ligase

EC:6.2.1.1

A

1

biotin carboxylase

EC:6.3.4.14

A

1

acetyl-CoA carboxylase

EC:6.4.1.2

A

1

hydroxymethylglutaryl-CoA reductase (NADPH)

EC:1.1.1.34

T

1

glycerol-3-phosphate dehydrogenase (quinone)

EC:1.1.5.3

T

1

glutathione peroxidase

EC:1.11.1.9

T

1

sterol 14-demethylase

EC:1.14.13.70 T

2

superoxide dismutase

EC:1.15.1.1

T

1

aldehyde dehydrogenase (NAD(P)+)

EC:1.2.1.5

T

1

hexaprenyldihydroxybenzoate methyltransferase

EC:2.1.1.114

T

1

3-demethylubiquinone-9 3-Omethyltransferase

EC:2.1.1.64

T

1

glycylpeptide N-tetradecanoyltransferase

EC:2.3.1.97

T

1

uracil phosphoribosyltransferase

EC:2.4.2.9

T

1

Nucleotidyltransferases

EC:2.7.7.0

T

1

diacylglycerol ethanolaminephosphotransferase

EC:2.7.8.1

T

1

DNA topoisomerase

EC:5.99.1.2

T

1

a present in the surface of trypomastigotes (T) or axenic amastigotes (A).


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Table 3. Stage-specific hydrolases identified in the cell surface/released subproteome.

ID (Uniprot)

Description (Blast2GO)

Life stage

Enzyme Codes

Q71MD6

80 kDa prolyl oligopeptidase

0/-

A*

EC:3.4.21.0

A0PGB9

85 kDa surface glycoprotein

1/-

A*

EC:3.2.1.18

E7L0Q3

acetylornithine putative

0/-

A

EC:3.5.1.16

Q8ITG1

amastigote cytoplasmic antigen

1/+

A

EC:3.2.1.18

E7LIQ6

aminopeptidase, putative,metallopeptidase, Clan MG, Family M24, putative

0/-

A*

EC:3.4.11.0

P25779

Cruzipain

0/-

A

EC:3.4.22.0

O61109

cysteine peptidase, putative

1/-

T

EC:3.4.22.0

E7L0T6

glutamamyl putative

0/-

A

EC:3.5.1.16

A7BI39

glycoprotein 82 kDa

1/-

T

EC:3.2.1.18

E7LCS9

guanine deaminase, putative

1/-

A*

EC:3.5.4.3

E7LJR0

hypothetical TCSYLVIO_006396

1/-

A

EC:3.1.3.16

O61097

hypothetical protein, conserved

4/-

A

EC:3.1.6.0

E7LA29

kynureninase, putative

2/-

A*

EC:3.7.1.3

Q2VLK7

metacaspase 3

2/-

A

EC:3.4.22.0

Q6ZXC0

metallocarboxypeptidase

1/-

A*

EC:3.4.17.0

E7LB45

mitochondrial ATP-dependent zinc metallopeptidase

2/-

A

EC:3.6.1.15

E7L7V0

peptidase, putative

2/-

A

EC:3.4.24.0

E7KZH2

phosphatase-like protein, putative

0/-

T

EC:3.1.3.0

E7LF15

phosphomannomutase, putative

0/-

A*

EC:3.1.3.11, EC:5.4.2.8

TM/GPIa

deacetylase-like,

carboxypeptidase,

protein


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E7LH12

proteasome alpha 1 subunit, putative

0/-

A*

EC:3.4.25.0

E7KWU5


0/-

A*

EC:3.4.25.0, EC:3.4.24.0

E7KWV2


0/-

A*

EC:3.4.25.0

E7KYQ4


0/-

A*

EC:3.4.25.0

E7LLV5

proteasome beta-1 subunit, putative

1/-

A*

EC:3.4.25.0

E7KWY5

proteasome subunit alpha type

1/-

A*

EC:3.4.25.0

E7L503


0/-

A*

EC:3.4.25.0

P92188

proteasome subunit alpha type-1

0/-

A*

EC:3.4.25.0

E7L5A7

putative uncharacterized protein

2/-

A*

EC:3.1.3.0

E7L129

ribonuclease HII, putative

0/-

A

EC:3.1.26.4, EC:3.4.21.0

E7KWF2

ribonuclease L inhibitor, putative

1/-

A

EC:3.6.1.3

Q03625

sialidase, partial

2/+

A

EC:3.2.1.18

Q26853

surface glycoprotein

1/-

A

EC:3.2.1.18

Q2VYD0

surface glycoprotein Tc-85/16

1/-

A

EC:3.2.1.18

Q2VYC9

surface glycoprotein Tc-85/19

0/-

A

EC:3.2.1.18

Q9NG33

surface protease GP63, putative

0/+

A

EC:3.4.24.0

Q86DL8

surface protein-2

1/-

A

EC:3.2.1.18

A5JUX4

surface protein-2

1/-

T

EC:3.2.1.18

Q8T2W4

Tcc1i14-2.1

4/-

A

EC:3.4.24.0

E7L3G9

thimet oligopeptidase, putative

1/-

A

EC:3.4.24.0

F1D695

trans-sialidase

0/-

A

EC:3.2.1.18

B3VSM7

trans-sialidase

1/-

T

EC:3.2.1.18

Q26968

trans-sialidase

1/-

T*

EC:3.2.1.18

E7KY86

trans-sialidase, putative

4/+

A

EC:3.2.1.18

E7KZ25


0/-

A*

EC:3.2.1.18

E7L092


0/-

A

EC:3.2.1.18


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E7L0A8


0/-

A

EC:3.2.1.18

E7L0B8


0/-

A

EC:3.2.1.18

E7L3F5


1/+

A

EC:3.2.1.18

E7L3S5


0/-

A

EC:3.2.1.18

E7L3T3


0/-

A

EC:3.2.1.18

E7L5S1


0/-

A

EC:3.2.1.18

E7L6N9


1/-

A

EC:3.2.1.18

E7L652


0/-

T*

EC:3.2.1.18

E7L8U7

ubiquitin hydrolase, putative, cysteine peptidase, Clan CA, family C19, putative

2/-

ubiquitin hydrolase, putative, cysteine peptidase, Clan CA, family C19, putative

0/-

vacuolar ATP synthase subunit B

0/-

E7LAD4 G4WJZ8

EC:3.1.2.15 T EC:3.1.2.15 T T

EC:3.6.3.6

a number of predicted transmembrane domains/presence(+) or absence(-) of predicted GPI anchor. b present in the surface of trypomastigotes (T) or axenic amastigotes (A) and/or detected in e control sample (*).

Table 4. Proteases identified in the T. cruzi surface/released subproteome.

ID

Life-

Enzyme Description (Blast2GO)

(Uniprot)

stagea

Q6ZXC0

A*

E7L126

T* and A* proteasome beta 2 subunit,

b

TM/GPI

Code metallocarboxypeptidase

1/-

EC:3.4.17.0

1/-

EC:3.4.25.0


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putative Q9NG33

A

E7LH12

A*

surface protease GP63, putative

0/+

EC:3.4.24.0

0/-

EC:3.4.25.0

0/-

EC:3.4.11.0

0/-

EC:3.4.25.0

1/-

EC:3.4.24.0

0/-

EC:3.4.24.0

0/-

EC:3.4.25.0

1/-

EC:3.4.25.0

0/-

EC:3.4.25.0

proteasome alpha 1 subunit, putative E7KWH0

T and A

P92188

A*

methionine aminopeptidase proteasome subunit alpha type1

E7L3G8

T* and A* thimet oligopeptidase, putative mitochondrial

E7KYG3

processing

T and A* peptidase alpha subunit proteasome alpha 2 subunit,

E7KWU5

A* putative

E7KWY5

A*

proteasome subunit alpha type proteasome alpha 7 subunit,

E7KYQ4

A* putative

E7L4J2

ubiquitin

carboxyl-terminal

hydrolase,

putative,cysteine

T and A

EC:3.1.2.15; 0/-

peptidase, Clan CA, family

EC:3.4.19.0

C12, putative


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P25779

A*

E7LLD9

T* and A

cruzipain mitochondrial

0/-

EC:3.4.22.0

0/-

EC:3.4.24.0

processing

peptidase alpha subunit Q71MD6

A*

80 kDa prolyl oligopeptidase

0/-

EC:3.4.21.0;

E7L503

A*


0/-

EC:3.4.25.0

B5U6U4

T and A

AAA ATPase-like protein G8

1/-

EC:3.4.24.0; EC:3.6.1.3 Q2VLK7

A

metacaspase 3

2/-

EC:3.4.22.0

E7L7V0

A

peptidase, putative

2/-

EC:3.4.24.0

3/-

EC:3.4.11.0

1/-

EC:3.4.24.0

aminopeptidase, E7LD64

T* and A* putative,metallo-peptidase, clan MA(E), family M1, putative O-

E7KZZ7

T and A

sialoglycoproteinendopeptidase, putative

E7KYK4

T* and A* aminopeptidase, putative

0/-

EC:3.4.11.0

E7L3G9

A*

1/-

EC:3.4.24.0

E7KWV2

A*

0/-

EC:3.4.25.0

thimet oligopeptidase, putative proteasome alpha 5 subunit, putative


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aminopeptidase, E7L2H2

T and A

putative,metallo-peptidase,

2/-

EC:3.4.11.0

1/-

EC:3.4.25.0

4/-

EC:3.4.24.0

0/-

EC:3.4.11.0

Clan MF, Family M17, putative proteasome E7LLV5

beta-1

subunit,

A* putative

Q8T2W4

A

Tcc1i14-2.1 aminopeptidase,

E7LIQ6

A

metallo-peptidase,

putative, clan

MG,

family M24, putative Q2VLK5

T* and A* metacaspase 3

2/-

EC:3.4.22.0

O61109

T

1/-

EC:3.4.22.0

cysteine peptidase, putative

a present in the surface of trypomastigotes (T), axenic amastigotes (A) and/or detected in control samples (*). b number of predicted transmembrane domains/presence(+) or absence(-) of predicted GPI anchor. .

ASSOCIATED CONTENT


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Supporting Information. Supplementary tables; Supplementary Table S1, Comparison of protein identifications by each methodology, Supplementary Table S2, Cell surface enzyme activity prediction, Supplementary Table S3, Hypothetical proteins identified, Supplementary Table S4, Total ID lists from axenic amastigotes, Supplementary Table S5, Total ID lists from trypomastigotes. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Tel: +5561 31073095. Fax: +556131070487. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant nº 563998/2010-5 and PhD fellowship for RMLQ), FAPEG (Fundação de Amparo a Pesquisa do Estado de Goiás), CAPES (Programa Nacional de Incentivo a Pesquisa em Parasitologia Basica grant nº: 23038.005298/2011-83) and FINEP (Financiadora de Estudos e Projetos).

ABBREVIATIONS BSA, bovine serum albumine; DMEM, Dulbecco's Modified Eagle Medium; DTT, dithiothreitol; FBS, fetal bovine serum; FDR, false discovery rate; GO, gene ontology; GPI, glycosylphosphatidylinositol; IAA, iodoacetamide; IP3, inositol-3-phosphate; MASP, mucin-


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associated surface proteins; PBS, phosphate buffered saline; PKC, protein kinase C; PM, plasma membrane; TASV, trypomastigote alanine, serine and valine rich proteins; TBS, tris buffered saline; TEAB, triethylammonium bicarbonate; TFA, trifluoroacetic acid; TOP, thimet oligopeptidases; TS, trans-sialidase; ZIC-HILIC, zwitterionic hydrophilic interaction liquid chromatography

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Cell Surface Proteome Analysis of Human-Hosted Trypanosoma cruzi

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