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Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins Ethel Bayer-Santos, Clemente Aguilar-Bonavides, Silas Pessini Rodrigues, Esteban Maurício Cordero, Alexandre F Marques, Armando Varela-Ramirez, Hyungwon Choi, Nobuko Yoshida, José Franco da Silveira, and Igor C Almeida J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr300947g • Publication Date (Web): 08 Dec 2012 Downloaded from http://pubs.acs.org on December 14, 2012

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Proteome Research

Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins Ethel Bayer-Santos†, Clemente Aguilar-Bonavides§,‡, Silas Pessini Rodrigues§, Esteban Maurício Cordero§, Alexandre Ferreira Marques§, Armando Varela-Ramirez§, Hyungwon Choi¶, Nobuko Yoshida†, José Franco da Silveira†,* and Igor C. Almeida§,*

†

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São

Paulo, São Paulo, SP 04023-062, Brazil §

The Border Biomedical Research Center, Department of Biological Sciences, University of

Texas at El Paso, El Paso, TX 79968, USA ‡

Computational Science Program, The Border Biomedical Research Center, University of Texas

at El Paso, El Paso, TX 79968, USA ¶

Saw Swee Hock School of Public Health, National University of Singapore, Singapore

KEYWORDS: Trypanosoma cruzi, protozoan parasite, Chagas disease, secretion pathways, secretome, extracellular vesicles, exosomes, ectosomes, proteomic analysis

ACS Paragon Plus Environment


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ABSTRACT

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes; the second enriched in smaller vesicles resembling exosomes; and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.


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1. INTRODUCTION

Microorganisms such as bacteria, fungi, and parasites release bioactive molecules that modulate host-parasite interaction enabling pathogen survival and replication within the host. In eukaryotes, secreted proteins bear an N-terminal signal sequence that targets protein to the endoplasmic reticulum (ER)/Golgi-dependent classical secretion pathway. On the other hand, secreted proteins that do not bear an N-terminal signal sequence use non-classical secretory pathways and different routes of transportation to outside the plasma membrane.1 Different mechanisms of intracellular trafficking have been proposed to mediate unconventional secretion, and one alternative route is through association with vesicles. There are currently three known mechanisms by which membrane vesicles are released into the extracellular environment: (i) exocytic fusion of multivesicular bodies (MVBs), resulting in exosomes; (ii) budding of vesicles directly from the plasma membrane, resulting in ectosomes or plasma membrane-derived vesicles or microparticles, and (iii) cell death, leading to apoptotic blebs.2 Exosomes are small vesicles usually ranging around 20-100 nm in diameter, with a cup-shaped morphology, whereas ectosomes or plasma membrane-derived vesicles or microparticles are large vesicles ranging around 100-1000 nm in diameter, with a heterogeneous morphology.3, 4 Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical infection endemic in Latin America. Chagas disease affects almost 8 million people,5 with an increasing number of cases being reported in non-endemic regions, including the United States and Europe.6, 7 T. cruzi has four developmental stages in its life cycle; two stages are present in the hematophagous insect vector (a triatomine or reduviid bug, popularly known as the kissing bug) and two in the mammalian host. Epimastigotes are noninfective replicative forms present in


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the vector’s gut that later differentiate into metacyclic trypomastigotes, which are non-replicative infective forms transmitted to mammalians by the insect’s excrements during its bloodmeal or by the oral route.8 Metacyclic forms invade host cells by triggering signaling cascades, with a transient increase in host-cell intracellular Ca2+ concentration and actin cytoskeleton disruption,9 leading to the recruitment of lysosomes to the site of entry,10 an event required for the biogenesis of parasitophorous vacuole.11, 12 A few days later, metacyclic forms promote vacuole disruption and escape to the cell cytoplasm, where they transform into amastigote forms that replicate inside the cell. After a few rounds of replication, intracellular amastigotes transform into infective trypomastigotes that are released into the bloodstream upon cell disruption. It has been reported that mammalian tissue culture cell-derived trypomastigotes (TCTs) release membranous vesicles carrying virulence factors such as members of the trans-sialidase glycoprotein multigene family,13-16 and that these extracellular vesicles were involved in Chagas disease pathogenesis by increasing heart parasitism and inflammation.17 Also, a factor secreted by TCTs was shown to inhibit mitogen-activated protein (MAP) kinase activation and to modify host-cell gene expression.18 In addition, it has been shown that exosome-like vesicles released by Leishmania major and L. donovani inside macrophages were able to modify host cytokine profile.19 Even though several studies identified and characterized specific effector proteins excreted/secreted by T. cruzi,13-16, 20-26 a comprehensive proteomic analysis of T. cruzi secretome and the mechanism(s) by which this parasite releases these bioactive molecules remain elusive. Here, we present evidence suggesting that both noninfective epimastigote and infective metacyclic trypomastigote forms of T. cruzi release proteins to the extracellular milieu in a manner associated or not with vesicles. Parasites use at least two different mechanisms for vesicle formation and release: budding from the plasma membrane and exocytose of MVBs’


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content. We demonstrate by a combination of methods, such as morphological, immunochemical, and label-free quantitative proteomic analysis, that distinct proteins are enriched in each vesicle population, revealing a rich collection of excreted/secreted molecules involved in diverse cellular processes. 2. EXPERIMENTAL SECTION 2.1 Reagents Otherwise indicated, reagents used in this study were of molecular biology-, HPLC-, or spectrometry/proteomics-grade from Sigma-Aldrich (St. Louis, MO). 2.2 Ethics Statement This study was carried out in accordance with recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on Animal Experiment Ethics of the Federal University of Sao Paulo (UNIFESP) (Protocol Number: CEP09555-07). 2.3 Parasite cultures and conditioned medium isolation and fractionation T. cruzi Dm28c27 clone was maintained by cyclic passage in mice and axenic cultures in liver-infusion tryptose (LIT) medium containing 10% fetal bovine serum (FBS) at 28 oC.28 Epimastigote forms were transfected with pGFP plasmid described by Bayer-Santos et al.,29 which comprises the pTEX vector carrying the green fluorescent protein (GFP). To obtain the parasite conditioned medium (CM), epimastigotes were harvested from exponentially growing cultures, washed in Dulbecco’s modified Eagle medium (DMEM) without FBS and incubated in


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the same medium at a concentration of 1 × 108 cells/mL for 6 h at 28 oC. Metacyclic trypomastigote forms were obtained by induction of metacyclogenesis according to Contreras et al.,27 purified by anion-exchange chromatography as described by Yoshida 30, and incubated in TAU3AAG medium27 at a concentration of 1 × 108 cells/mL for 6 h at 28 oC. Following 6-h incubation, parasites were removed by centrifugation at 3,000 g for 10 min at 4 oC. The cell-free supernatant was filtered in 0.45-µm syringe filter (Millipore), transferred to 13.2-mL polyallomer tube, and centrifuged at 100,000 g for 2 h at 4 oC to obtain the first pellet, enriched in larger extracellular vesicles (V2). The resulting supernatant was transferred to another polyallomer tube and then centrifuged at 100,000 g for 16 h at 4 oC, to obtain the second pellet, enriched in smaller vesicles (V16) and the vesicle-free supernatant (VF) (see Figure 2A). All ultracentrifugation steps were carried out using a TH-641 titanium swinging-bucket rotor (Thermo Fisher Scientific) in a WX80 ultracentrifuge (Thermo Fisher Scientific). 2.3 Parasite viability After removal of CM, 3 × 107 epimastigote or metacyclic trypomastigote cells were resuspended in 200 µL 10 mM phosphate-buffered saline (PBS), containing 20 µg/mL propidium iodide (PBS-PI), and analyzed in a Cytomics FC500 Beckman counter to assess parasite viability. Controls containing dead cells were obtained by incubating parasites with 50% ice-cold methanol in PBS for 5 min on ice, followed by washing with PBS, and staining with PI. After 6-h incubation, no significant death was detected in either epimastigote or metacyclic forms and over 98% of parasite cells were viable (data not shown). 2.4 Transmission electron microscopy (TEM)


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Purified vesicles from epimastigote or metacyclic forms were absorbed onto formvar/carbon-coated copper grids, washed in deionized water, and stained with 1% uranyl acetate for 1 min. Grids were allowed to air dry and viewed on microscope. For ultrastructural analysis, parasites were fixed in 2% paraformaldehyde (PFA), 2.5% glutaraldehyde (GAA), in 100 mM phosphate buffer, pH 7.2, for 1 h at room temperature (RT). Samples were washed in PBS and post-fixed in 1% osmium tetroxide for 1 h. Samples were washed, dehydrated in a graded series of ethanol and embedded in Eponate 12 resin. Sections of 95 nm were stained with uranyl acetate and lead citrate, and observed on microscope. For immunolocalization, parasites were fixed in 4% PFA, 0.05% GAA, in 100 mM phosphate buffer, pH 7.2, for 1 h at 4 oC. Samples were embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose, 20% polyvinylpyrrolidone, in PIPES/MgCl2 at 4 oC. Samples were frozen in liquid nitrogen and sectioned with a cryo-ultramicrotome. Sections of 50 nm were blocked with 5% FBS, 5% normal goat serum, and incubated with mouse monoclonal antibody (mAb) 10D8 (anti-GP35/50),31 followed by 18-nm colloidal gold-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Sections were washed in 0.1 M PIPES buffer, pH 7.2, and stained with 0.3% uranyl acetate, 2% methyl cellulose. Parallel controls omitting the primary antibody were consistently negative at the concentration of colloidal gold-conjugated antibodies used (not shown). All samples were viewed on a JEOL 1200EX transmission electron microscope at an accelerating voltage of 80 kV. All TEM experiments were performed at the Molecular Microbiology Imaging Facility of Washington University School of Medicine (St. Louis, MO). 2.5 Nanoparticle tracking analysis The size of T. cruzi extracellular vesicles from epimastigote (eV2 and eV16) and metacyclic forms (mV2 and mV16) was determined by nanoparticle tracking analysis (NTA), by


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measuring the rate of Brownian motion to particle size, using a NanoSight LM14 system (NanoSight, Wiltshire, UK). Three replicates of diluted vesicles aliquots (500 µL in PBS) were injected into the machine's specimen chamber and vesicles were tracked and measured for 60 s at RT. Data were analyzed with NTA software (version 2.2). 2.6 Polyacrylamide gel electrophoresis and western blotting Two micrograms of protein from each CM fraction from epimastigote (eV2, eV16, and eVF) and metacyclic forms (mV2, mV16, and mVF) were resolved in 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were processed as described elsewhere.32 T. cruzi surface glycoproteins GP82, GP35/50, and flagellar calcium-binding protein (FCaBP) were detected by mAbs 3F6, 10D8, and 25, respectively.31, 33 GFP was detected by rabbit antiGFP polyclonal antibody (Invitrogen, Life Technologies, Grand Island, NY). It is worth pointing out that GP82 glycoprotein is expressed only in metacyclic forms, whereas GP35/50 (mucin-like glycoproteins) and FCaBP are expressed in both epimastigote and metacyclic forms.34 2.7 Immunofluorescence analysis Parasites collected after 6 h of incubation in serum-free medium were washed with PBS, fixed with 4% PFA for 10 min, placed on glass slides pre-treated with poly-lysine and incubated with 50 mM NH4Cl. After wash, cells were blocked and permeabilized with 3% bovine serum albumin (BSA), 0.5% saponin in PBS for 30 min at RT, and incubated with primary antibodies (mAb 10D8) diluted in 1% BSA, 0.1% saponin for 1 h at RT. Cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen) containing 10 mM 4’,6diamidine-2’-phenylindole, dihydrochloride (DAPI, Invitrogen, Carlsbad, CA), for 1 h at RT, protected from light. After washing cells by centrifugation (3,000 g, 10 min, 4 oC) in PBS-1%


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BSA, coverslips were mounted on slides using anti-fading agent (Vectashield, Vector Laboratories, Burlingame, CA) and sealed with nail polish. Images were acquired with a Leica DMI 6000 B epifluorescence microscope. To visualize vesicles released by metacyclic trypomastigotes in contact with HeLa cells, parasites were incubated for 10 min on ice with mAb 3F6, washed with PBS, diluted in DMEM-10% FBS and incubated for 2 h with HeLa cells at 10:1 multiplicity of infection (MOI). Cells were washed to eliminate parasites not attached or internalized, fixed, and processed as described above, except for incubation with secondary antibody conjugated to Alexa Fluor 594 (Invitrogen) and phalloidin-Alexa Fluor 488 (Invitrogen). Images were acquired with a Zeiss LSM 700 confocal microscope and processed with ZEN 2009 software (Zeiss, NY). 2.8 Sucrose-density gradient Vesicles were separated by flotation in a linear sucrose-density gradient as previously described.19 Briefly, pellets containing V2 or V16 vesicles (30 µg of protein, as measured by the BCA kit, Pierce) from either epimastigote or metacyclic form were resuspended in 2.5 M sucrose and overlaid with a step-wise gradient (2.5–0.25 M sucrose, 20 mM HEPES, pH 7.4) followed by centrifugation at 200,000 g for 18 h at 4 oC. Fractions of 1 mL were collected from the bottom of the tube and density was determined in a refractometer. Each fraction was diluted with 10 mL PBS and centrifuged at 200,000 g for 2 h. Pellets were solubilized in reducing Laemmli sample buffer35 and analyzed by 10% SDS-PAGE and western blotting. 2.9 Protein digestion and 2D LC-MS/MS analysis In this study we analyzed six biological samples: three from epimastigotes (i.e., eV2, eV16, and eVF) and three from metacyclic trypomastigotes (i.e., mV2, mV16, and mVF), each


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in technical triplicate, thus totalizing 18 samples that were subjected to 2D LC-MS/MS. To obtain sufficient amount of protein for proteomic analysis, we used two distinct CM preparations that were mixed and filter-concentrated in a 3-kDa MWCO Centricon (Millipore). Twenty micrograms of protein from each fraction (V2, V16, and VF) were solubilized with RapiGest (Waters) and trypsin-digested as specified by Turiak et al.36 Resulting tryptic peptides were desalted using reverse-phase microcolumns as described by Jurado et al.,37 prior to mass spectrometry analysis. Peptide mixtures were separated by two-dimensional liquid chromatography and analyzed on-line by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) using an ESI-linear ion-trap-MS (LTQ XL, Thermo Fisher Scientific, San Jose, CA), as described by Nakayasu et al.38 Briefly, peptides were loaded into the SCX trap column and eluted using the autosampler by injecting increasing salt concentrations (0, 25, 50, 100, 200, and 500 mM NaCl in 5% ACN/0.5% FA, pH 2.5). Eluting peptides were automatically loaded into the C18-trap column, which was then washed with 2% ACN/0.1% FA (solvent A) at 2.5 µL/min flow rate for 130 min. In the second dimension, the peptides were separated in the capillary C18 column at 300 nL/min flow rate using a linear gradient from 5 to 40% solvent B (80% ACN/0.1% FA), over 100 min, followed by 10 min wash with 100% solvent B and 20 min with 5% solvent B. The nanoelectrospray was set to 1.8 kV, and the ion trap was set for a maximum injection time of 100 msec for full scan and 150 msec for MS/MS scan. The 10 most abundant ions were selected for collision-induced dissociation (CID) with an isolation width of 3.0 amu and normalized collision energy of 35%. Dynamic exclusion was set for fragmenting each ion twice and then excluding it for 1 min.


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2.10 Data analysis The 2D LC-MS/MS analysis of each sample from epimastigote or metacyclic form gave rise to eight raw MS/MS data files, thus the 18 samples analyzed in this study generated a total of 144 raw MS/MS data files and 27,728 MS/MS spectra. These spectra were searched using Sequest (version v.27; Thermo Fisher Scientific) and X! Tandem (version 2007.01.01.2; http://www.thegpm.org/tandem/) algorithms against sequences from Trypanosoma ssp., BSA, human keratin, and porcine trypsin (downloaded from GenBank on October 10th, 2011). Sequences (total of 99,536) were used in forward and reverse orientations to calculate the falsediscovery rate (FDR). Parameters for database search were as follows: trypsin cleavage at both termini and two missed cleavage allowed; 1 Da for peptide-mass tolerance; 1 Da for fragmentmass tolerance; cysteine carbamidomethylation and methionine oxidation as fixed and variable modifications, respectively. Scaffold platform (version 3.4.3; Proteome Software, Portland, OR)39 was used to validate peptide and protein identifications, which were accepted if greater than 95% for peptides and 95% for proteins containing at least 2 peptides, according to the PeptideProphet40 and ProteinProphet41 algorithms. Using these parameters, the FDR was estimated to be

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