Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.029272
ISSN1535-9484
AutoresXue Yan Yam, Cecilia Birago, Federica Fratini, Francesco Girolamo, Carla Raggi, Massimo Sargiacomo, Angela Bachi, Laurence Berry, Gamou Fall, Chiara Currà, Elisabetta Pizzi, Catherine Braun Breton, Marta Ponzi,
Tópico(s)Mosquito-borne diseases and control
ResumoIntracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking.To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. Plasmodium falciparum, the most deadly agent of human malaria, caused around 216 million infections and 655,000 deaths in 2010. The complex parasite life cycle involves the development in a mosquito vector of the Anopheles genus and eventual migration to a human host. In this host, asymptomatic multiplication in the liver cells is followed by parasite release into the bloodstream and erythrocyte invasion. Inside the erythrocytes, parasites grow (trophozoite stage) and multiply asexually (schizont stage), developing into highly specialized invasive forms (merozoites). A fraction of parasites differentiate into gametocytes, the gamete precursors necessary to complete the transmission cycle. Parasite blood stages, responsible for malaria pathogenesis and transmission, actively remodel the host erythrocyte, generating novel membrane compartments to sustain the export and sorting of proteins to the host cell cytosol, membrane skeleton, and plasma membrane. The parasitophorous vacuole membrane (PVM), 1The abbreviations used are:DRMdetergent-resistant membraneERendoplasmic reticulumGPIglycosylphosphatidyl inositoliRBCinfected red blood cellMCMaurer's cleftPBSphosphate-buffered salinePMplasmepsinPVMparasitophorous vacuole membraneRBCred blood cellSPsignal peptideTMtransmembrane domain. 1The abbreviations used are:DRMdetergent-resistant membraneERendoplasmic reticulumGPIglycosylphosphatidyl inositoliRBCinfected red blood cellMCMaurer's cleftPBSphosphate-buffered salinePMplasmepsinPVMparasitophorous vacuole membraneRBCred blood cellSPsignal peptideTMtransmembrane domain. which surrounds the parasite throughout the erythrocytic cycle, is the site where exported proteins are translocated into the erythrocyte cytosol (1Maier A.G. Cooke B.M. Cowman A.F. Tilley L. Malaria parasite proteins that remodel the host erythrocyte.Nat. Rev. Microbiol. 2009; 5: 341-354Crossref Scopus (314) Google Scholar, 2de Koning-Ward T.F. Gilson P.R. Boddey J.A. 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Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2056) Google Scholar). DRM rafts also restrict free diffusion of membrane proteins, thereby directing the trafficking of proteins and lipids to and from cellular compartments. Because of their endocytic and receptor clustering capacity, an increasing number of pathogens, including Plasmodium falciparum, utilize them when interacting with their target cells for invasion (9Vieira F.S. Corrêa G. Einicker-Lamas M. Coutinho-Silva R. Host-cell lipid rafts: a safe door for micro-organisms?.Biol. Cell. 2010; 102: 391-407Crossref PubMed Scopus (69) Google Scholar, 12Mañes S. del Real G. Martínez A.C. Pathogens: raft hijackers.Nat. Rev. Immunol. 2003; 3: 557-568Crossref PubMed Scopus (396) Google Scholar). Even though cholesterol-rich membrane microdomains are implicated in fundamental processes in the parasite life cycle, Plasmodium is unable to synthesize sterols and depends entirely on hosts for its cholesterol supply. During merozoite invasion, lipid and protein components of the erythrocyte rafts are selectively recruited and incorporated into the nascent PVM (13Murphy S.C. Samuel B.U. Harrison T. Speicher K.D. Speicher D.W. Reid M.E. Prohaska R. Low P.S. Tanner M.J. Mohandas N. Haldar K. Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.Blood. 2004; 103: 1920-1928Crossref PubMed Scopus (127) Google Scholar, 14Murphy S.C. Fernandez-Pol S. Chung P.H. Prasanna Murthy S.N. Milne S.B. Salomao M. Brown H.A. Lomasney J.W. Mohandas N. Haldar K. Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum.Blood. 2007; 110: 2132-2139Crossref PubMed Scopus (46) Google Scholar). Plasmodium liver stages utilize cholesterol internalized by low-density lipoprotein and synthesized by hepatocytes (15Labaied M. Jayabalasingham B. Bano N. Cha S.J. Sandoval J. Guan G. Coppens I. Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver.Cell. Microbiol. 2011; 13: 569-586Crossref PubMed Scopus (82) Google Scholar). To shed light on the organization and dynamics of these assemblies during parasite development inside the infected cell, we identified and validated the DRM-raft proteome of the P. falciparum trophozoite/early schizont. Detected proteins only partially overlap with DRM components of the P. falciparum late schizonts (16Sanders P.R. Gilson P.R. Cantin G.T. Greenbaum D.C. Nebl T. Carucci D.J. McConville M.J. Schofield L. Hodder A.N. Yates 3rd, J.R. Crabb B.S. Distinct protein classes including novel merozoite surface antigens in raft-like membranes of Plasmodium falciparum.J. Biol. Chem. 2005; 280: 40169-40176Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 17Sanders P.R. Cantin G.T. Greenbaum D.C. Gilson P.R. Nebl T. Moritz R.L. Yates 3rd, J.R. Hodder A.N. Crabb B.S. Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.Mol. Biochem. Parasitol. 2007; 154: 148-157Crossref PubMed Scopus (71) Google Scholar) or the mixed blood stages of the rodent malaria agent P. berghei (18Di Girolamo F. Raggi C. Birago C. Pizzi E. Lalle M. Picci L. Pace T. Bachi A. de Jong J. Janse C.J. Waters A.P. Sargiacomo M. Ponzi M. Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.Proteomics. 2008; 8: 2500-2513Crossref PubMed Scopus (33) Google Scholar). Immunolocalization of selected DRM-associated proteins indicated that these assemblies may reside in both exported compartments (PVM, MCs) and intracellular membranes/organelles. The analysis of DRM lipids suggested that distinct microdomains exist in the infected erythrocyte that differ in their relative abundance of cholesterol and phospholipids. P. falciparum 3D7 strain was maintained in continuous in vitro culture (19Trager W. Jensen J. Human malaria parasites in continuous culture.Science. 1976; 193: 673-675Crossref PubMed Scopus (6161) Google Scholar) in the presence of human erythrocytes at 5% hematocrit in RMPI 1640 medium containing 25 mm Hepes, 0.5% AlbuMAX II, 200 μm hypoxanthine, and 20 μg/ml gentamycin and incubated at 37 °C in a tri-gas mix of 5% O2, 5% CO2, and 90% N2. The parasite culture pellet was incubated with five volumes of sterile pre-warmed 5% sorbitol at 37 °C for 10 min and then centrifuged at 2000 rpm for 5 min at room temperature. Sorbitol-treated parasites were washed once with culture medium and put back in culture. Enrichment of late trophozoites and schizonts was accomplished via gelatin flotation with either Plasmion (Laboratoire Fresenius Kabi, Sèvres, France) or Gelofusine. The parasite pellet was incubated with 1.4 volumes of prewarmed culture medium and 2.4 volumes of prewarmed Plasmion or Gelofusine for 30 min at 37 °C. The upper phase containing the enriched late-stage parasites was collected and centrifuged at 2000 rpm for 5 min. The parasite pellet was washed once to remove the Plasmion or Gelofusine. DRMs from Plasmion/Gelofusine-enriched erythrocytes infected with P. falciparum trophozoites/early schizonts were prepared as described elsewhere (18Di Girolamo F. Raggi C. Birago C. Pizzi E. Lalle M. Picci L. Pace T. Bachi A. de Jong J. Janse C.J. Waters A.P. Sargiacomo M. Ponzi M. Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.Proteomics. 2008; 8: 2500-2513Crossref PubMed Scopus (33) Google Scholar). All procedures were performed at 4 °C with the addition of protease mixture inhibitors and PhosStop phosphatase inhibitor (Roche). Infected erythrocytes were lysed in 0.15 m NH4Cl, 0.01 m KHCO3, 1 mm EDTA. The parasite pellet (2 × 108 parasites) was suspended in 0.75 ml of MES-buffered saline (25 mm MES, pH 6.5, 0.15 m NaCl) containing 1% Triton X-100 (v/v) and homogenized with a Potter-Elvehjem glass homogenizer. Parasite extract was adjusted to 40% sucrose by the addition of 0.75 ml of 80% sucrose, prepared in MES-buffered saline, placed at the bottom of an ultracentrifuge tube (4.5 ml, 13 × 15 mm, Beckman) and overlaid with 1.5 ml of 30% sucrose and 1.5 ml of 5% sucrose. Samples were centrifuged (45,000 rpm for 16 h at 4 °C) in an SW60Ti rotor (Beckman Instruments). 375-μl fractions were collected from the top of each gradient. DRMs containing flotillin, a lipid raft marker, appeared as an opaque band at 10% to 20% sucrose (fractions 4 and 5). For proteomic analyses, these fractions were pooled, diluted 1:5, and centrifuged at 30,000 × g for 30 min at 4 °C. The membrane pellet was subjected to a second sucrose gradient in order to minimize the presence of spurious contaminants. After centrifugation, proteins contained in fractions 4 and 5 were identified via mass spectrometry (see below). Coding regions of the selected genes were amplified and cloned in frame with the glutathione S-transferase (GST) tag of the pGEX vector. Recombinant proteins were expressed in C41 competent cells. Soluble GST fusions were purified on glutathione-Sepharose beads. Insoluble GST fusions were subjected to SDS-PAGE, and gel slices containing Coomassie Blue–stained polypeptides were electro-eluted using a BioTrap electro-elution device (Schleicher & Schuell, Dassel, Germany). Primers used for PCR amplification and gel-separated recombinant proteins are shown in the supplemental "Materials and Methods" section. Antibodies raised against DRM-associated proteins were induced via subcutaneous immunization of 6- to 8-week-old BALB/c mice with 20 to 50 μg of purified protein (first injection in Freund's complete adjuvant; two further injections, performed at 2-week intervals, in Freund's incomplete adjuvant). Mice were bled 1 week after the third immunization. Sources of other antibodies used in this study and the dilutions adopted are detailed in the supplemental "Materials and Methods" section. P. falciparum–infected RBCs (iRBCs) were fixed in 4% paraformaldehyde with 0.0075% glutaraldehyde (EMS Science, Hatfield, PA) in phosphate-buffered saline (PBS) overnight at 4 °C. The next steps were all conducted at room temperature. Fixed cells were washed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and treated with 0.1 m glycine in PBS for 10 min to block free reactive aldehyde groups. iRBCs were incubated in succession with 3% fetal calf serum (FCS) in PBS for 1 h and the primary antibody diluted with 3% FCS in PBS for 1 h. After several washes using 1% FCS in PBS, fixed cells were incubated for 1 h with the secondary antibody conjugated to Alexa 488 or 594 (molecular probe, 1:2000 dilution in 3% FCS in PBS) and washed again. Nuclei were stained with Hoechst 33342 (1:10,000 dilution in PBS). Samples were layered on poly-L lysine-coated immunofluorescence slides (Sigma P8920) and mounted with VECTASHIELD. Slides were viewed under a Zeiss Axioimager epifluorescence microscope (Axio Imager 2). When Z-stack acquisition was performed, images with a slice distance of 0.3 μm were presented. For mitochondria labeling, parasite cultures were incubated for 30 min with MitoTracker® probe (M7512, Invitrogen) at a final concentration of 20 nm in Hank's balanced salt solution. Samples were then washed once with Hank's balanced salt solution medium, fixed in 4% paraformaldehyde, 0.0075% glutaraldehyde in PBS overnight at 4 °C, and further processed as described above. DRM-raft proteins (fractions 4 and 5) were precipitated, solubilized in Laemmli sample buffer at 65 °C for 1 h, loaded on SDS-PAGE (homemade 5% stacking/12% resolving), and run just long enough to allow the protein marker (BenchMark™ Pre-Stained Protein Ladder, Invitrogen) to enter the resolving gel. After Coomassie staining (Novex, Colloidal Blue staining gel, Invitrogen), unseparated bands were excised and in-gel tryptic digestion was performed as described below. Supernatants were used directly for LC-MS/MS analysis. Proteins contained in fractions 4 and 5 were separated on Invitrogen precast 4–20% Bis-Tris Gels and stained with Coomassie Colloidal Blue. After visualization, each sample lane was cut into sequential slices. Gel slices were destained in 50 mm NH4CO3/CH3CN 1:1 and covered with acetonitrile until the gel pieces shrank. The acetonitrile was removed and the gel particles were dried by centrifugation under vacuum. Proteins were reduced (10 mm DTT, 25 mm NH4CO3) for 30 min at 56 °C, shrunk with acetonitrile, and alkylated (55 mm iodoacetamide, 25 mm NH4CO3) for 30 min in the dark at room temperature. Gel pieces were washed in 50 mm NH4CO3/CH3CN 1:1 for 15 min and covered with acetonitrile until they shrank. Acetonitrile was removed and gel particles were dried by centrifugation under vacuum. In-gel digestion was performed by adding 12.5 ng/μl of trypsin (Promega) in 25 mm NH4CO3 at 37 °C overnight under stirring. Supernatants were directly used for mass spectrometry analysis. Nano-RPLC was performed using a nano-HPLC 3000 Ultimate (Dionex, Sunnyvale, CA) connected online to an LTQ-XL linear ion trap (Thermo Fisher). Tryptic digests (20 μl) were first loaded on a C18 reversed-phase pre-column (300 μm inner diameter × 5 mm; 5-μm particle size; 100-Å pore size; LC Packings-Dionex) and washed by the loading pump at 20 μl/min with buffer A (5% CH3CN, 0.1% HCOOH) for 5 min and then on a homemade 13 cm × 75 μm inner diameter Silica PicoTip (8 ± 1 μm) column (PicoTip Emitter, New Objective, Woburn, MA) packed with Magic C18AQ (5-μm particle size; 200-Å pore size; Michrom Bioresouces Inc., Auburn, CA) for chromatographic separations. Peptides were eluted at 0.3 μl/min along a 120-min linear gradient from 20% to 60% buffer B (95% CH3CN, 0.1% HCOOH) and electro-sprayed directly into the mass spectrometer with a spray voltage of 1.60 to 1.65 kV and a capillary temperature of 180 °C. Data acquisition was performed in Top5 data-dependent mode to automatically switch between MS and MS2. Full-scan MS parameter settings were as follows: automatic gain control value of 30,000 ions, maximum injection time of 50 ms, and m/z 400–2000 mass range. The five most intense ions were sequentially selected and fragmented in collision-induced dissociation mode with the following parameter settings: isolation width of 2.0, automatic gain control value of 10,000 ions, maximum injection time of 100 ms, m/z 50–2000 mass range, collision energy of 35%, minimum signal threshold of 200 counts, and wide band activation on. A dynamic exclusion of ions previously sequenced within 60 s was applied. Samples (5 μl) were mixed with an equal volume of 3% acetonitrile, 1% formic acid and analyzed via nano-LC electrospray ionization MS/MS. Chromatographic separations were carried out using a 75-μm inner diameter, 15-cm-long fused silica capillary column (LC Packings PepMap, 100 A). Nano-reversed-phase LC was performed using an Agilent 1100 system (Agilent Technologies, Santa Clara, CA). After sample injection, the column was washed for 5 min with 90% mobile phase A (2% CH3CN, 0.1% HCOOH) and peptides eluted using a linear gradient from 10% to 90% mobile phase B (98% CH3CN, 0.1% HCOOH) over 75 min with a constant flow rate of 0.2 μl/min. The nano-reversed-phase LC column was coupled online to a hybrid quadrupole TOF mass spectrometer (API Q-STAR pulsar, PE SCIEX, Toronto, ON, Canada) using a nano-electrospray source (Proxeon Biosystems, Odense, Denmark) with an applied electrospray potential of 1800 V; the m/z range was 350–1400 for MS and 100–2000 for MS/MS. Spectra were recorded using external calibration. Raw MS/MS data were analyzed using Analyst QS 1.1 (Applied Biosystems, Darmstadt, Germany) as provided by the manufacturer and were reported as monoisotopic masses. Spectra files from both experiments were analyzed by the Sequest search engine with Proteome Discoverer 1.4 (ThermoFisher) using a homemade database constructed with the Human UniProt–Swiss-Prot database (released June 2012) and P. falciparum 3D7 (PlasmoDB 9.2, released November 2012) and containing a decoy database (150,994 total entries). Carbamidomethylation of cysteines was specified as a fixed modification, and the oxidation of methionine was set as a variable modification. The mass tolerance was set to 0.8 Da for precursor ions and 0.4 Da for fragment ions, and a maximum of two missed cleavages was allowed. The Percolator tool was used for peptide validation based on the q-value, and high confidence was chosen, corresponding to a false discovery rate of ≤1% on the peptide level. Proteins were identified with a minimum of two peptides (rank = 1) in both experiments. A manually curated functional classification was performed on the basis of the Gene Ontology annotation available in PlasmoDB. Gene expression profiling of the DRM-associated proteins was reconstructed from RNA sequencing data (20López-Barragán M.J. Lemieux J. Quiñones M. Williamson K.C. Molina-Cruz A. Cui K. Barillas-Mury C. Zhao K. Su X.Z. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum.BMC Genomics. 2011; 12: 587Crossref PubMed Scopus (213) Google Scholar) available in PlasmoDB. For each of the six time points, rings, early trophozoites, late trophozoites, schizonts, early gametocytes of stage II, and late gametocytes of stage V were analyzed. RNA abundance values (x) were normalized according to Z=(x−m)stdvEq. 1 where m is the mean value and stdv is the standard deviation. The self-organizing tree algorithm was used to group normalized gene expression profiles. The Pearson correlation coefficient was chosen as a metric, and the "max cycle" parameter was fixed at 7 to obtain six clusters. Transmembrane helices were predicted using TMHMM 2.0. GPI-SOM was used to predict the GPI-anchor addition. plasmodium export element–containing Plasmodium exported proteins were predicted using dedicated software (21Sargeant T.J. Marti M. Caler E. Carlton J.M. Simpson K. Speed T.P. Cowman A.F. Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites.Genome Biol. 2006; 7: R12Crossref PubMed Scopus (330) Google Scholar). Trophozoite-infected RBCs (6 × 108) were purified and host cells were subjected to osmotic lysis. Recovered parasites were suspended in cold Triton, and Triton-insoluble material was fractionated by means of discontinuous sucrose gradient centrifugation (see the paragraph on parasite purification and analysis in the preceding section). Fractions 2–6 were diluted 1:5 and centrifuged at 30,000 × g for 30 min at 4 °C. Lipids were extracted from pellets using a chloroform:methanol (2:1, v/v) solution according to the procedure described by Folch et al. (22Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Extracts were dried under nitrogen atmosphere and used for neutral lipid and phospholipid thin layer chromatography (TLC). TLC for neutral lipids was done by eluting lipid extract in a solution of hexane:diethylether:acetic acid (70:30:1, v/v). TLC for phospholipids was performed by running lipid extract in a chloroform:methanol:acetic acid:formic acid:H2O (35:15:6:2:1, v/v) solution in order to separate sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The spots were visualized via copper acid staining (3% w/v). The plate was heated for about 5 min at 180 °C, and the abundance of the different lipid classes in each fraction was estimated in comparison with lipid standards (Sigma-Aldrich Corporation, St. Louis, MO) using a GS-700 imaging densitometer (Bio-Rad). Trophozoite/early schizont-infected RBCs were purified from highly synchronous P. falciparum cultures. Intact parasites and associated membranous structures were recovered after osmotic lysis of the erythrocyte plasma membrane (18Di Girolamo F. Raggi C. Birago C. Pizzi E. Lalle M. Picci L. Pace T. Bachi A. de Jong J. Janse C.J. Waters A.P. Sargiacomo M. Ponzi M. Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.Proteomics. 2008; 8: 2500-2513Crossref PubMed Scopus (33) Google Scholar). DRM rafts were then isolated based on their resistance to Triton X-100 extraction at low temperature and their specific density in discontinuous sucrose gradients (18Di Girolamo F. Raggi C. Birago C. Pizzi E. Lalle M. Picci L. Pace T. Bachi A. de Jong J. Janse C.J. Waters A.P. Sargiacomo M. Ponzi M. Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.Proteomics. 2008; 8: 2500-2513Crossref PubMed Scopus (33) Google Scholar). DRM rafts, detected as an opaque band floating to the 5%-to-30% sucrose boundary (fractions 4 an
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