Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
J Extracell Vesicles
2018 Jan 01;71:1463779. doi: 10.1080/20013078.2018.1463779.
Show Gene links
Show Anatomy links
Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells.
Ribeiro KS
,
Vasconcellos CI
,
Soares RP
,
Mendes MT
,
Ellis CC
,
Aguilera-Flores M
,
de Almeida IC
,
Schenkman S
,
Iwai LK
,
Torrecilhas AC
.
???displayArticle.abstract???
Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host-parasite interaction.
Figure 1. Workflow employed for the production, fractionation, and characterization of Trypanosoma cruzi EVs and vesicle-free fraction from Y and YuYu strains. The details of each step are explained in the Materials and Methods section. DMEM, Dulbecco’s Modified Eagle’s Medium; NTA, nanoparticle tracking analysis; SEM, scanning electron microscopy; SEC, size-exclusion chromatography.
Figure 2. SEM of T. cruzi trypomastigotes from Y and YuYu strains showing shedding of EVs. Each panel shows trypomastigotes pre-incubated in DMEM with 2% glucose and attached to glass coverslips containing poly-lysine obtained from the Y (a) and YuYu strains (b), fixed and processed for SEM. The bar sizes are indicated in each image.
Figure 3. Sepharose 4B elution profile of EVs released by T. cruzi trypomastigotes. Total shed vesicles obtained from trypomastigotes (Y, broken lines and YuYu, full line) and control (DMEM and 2% glucose, dotted line) were submitted to gel filtration chromatography using Sepharose CL-4B column and 1 mL fractions were collected. (a) Reactivity of each fraction to antibody 460 was determined by ELISA, and the results are expressed by absorbance at 450 nm. (b) Size (nm) and (c) concentration (particles/mL) of EVs from each fraction as determined by NTA analysis. This figure represents a typical analysis of at least three independent experiments.
Figure 4. The protein expression profile in EVs is different in Y and YuYu strains of the parasite. Immunoblot of the pooled fractions of EVs released by the Y and YuYu strains probed with antibody 460 (a), ant-TS (b), and anti-cruzipain (c). In the right are indicated the position of mass standards in kDaltons.
Figure 5. Venn diagrams and cluster analysis of the peptides and proteins detected in EVs from Y and YuYu strains. (a) Analysis of common individual peptides detected in the soluble (VF) and membrane bound fractions (EVs) of the material obtained after gel filtration of the Y (top left) and YuYu strain (bottom left), or between the EVs (top right) and soluble fractions (VF) of the two strains. (b) The same comparison was made using identified proteins.
Figure 6. Functional annotation of peptide sequences identified in EVs isolated from Y and YuYu strains of T. cruzi using Blast2go software. (a) Biological processes, (b) Cellular components.
Figure 7. Effect of EVs on macrophages. J774 macrophages (105) were pre-incubated for 1 h with EVs from Y and YuYu strains containing each 1 or 10 µg of protein per mL, equivalent to 106 and 107 or just RPMI medium used as control. Cells were then incubated for 2 h with trypomastigotes (10 parasites/cell), washed and the incubation proceeded for more 24 h. The cells were fixed and stained with DAPI and the number of intracellular parasites determined in cells containing from 0 to 5 parasites/cells (a), 6 to 10 parasites/cells (b), 11 to 25 parasites/cells (c), and 26 to 50 parasites/cells (d). The values are means of duplicate experiments.
Figure 8. Effect of EVs on macrophages. LLC-MK2 (105) were pre-incubated 1 h with EVs from Y and YuYu strains containing each 1 or 10 µg of protein per mL, equivalent to 106 and 107, or just DMEM medium used as control. Cells were then incubated for 2 h with trypomastigotes (10 parasites/cell), washed and the incubation proceeded for more 24 h. The cells were fixed and stained with DAPI and the number of intracellular parasites determined in cells containing from 0 to 5 parasites/cells (a), 6 to 10 parasites/cells (b), and 11 to 25 parasites/cells (c). The values are means of duplicate experiments.
Acosta-Serrano,
The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles.
2001, Pubmed
Acosta-Serrano,
The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles.
2001,
Pubmed
Affranchino,
Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease.
1989,
Pubmed
Almeida,
Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents.
2000,
Pubmed
Almeida,
Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi.
1994,
Pubmed
Alves,
Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi.
1986,
Pubmed
Alves,
Comprehensive glycoprofiling of the epimastigote and trypomastigote stages of Trypanosoma cruzi.
2017,
Pubmed
Andrade,
The Trypanosoma cruzi-host-cell interplay: location, invasion, retention.
2005,
Pubmed
Andrade,
Lysosomal fusion is essential for the retention of Trypanosoma cruzi inside host cells.
2004,
Pubmed
Andrews,
Adhesion and interiorization of Trypanosoma cruzi in mammalian cells.
1982,
Pubmed
Aparicio,
A new cruzipain-mediated pathway of human cell invasion by Trypanosoma cruzi requires trypomastigote membranes.
2004,
Pubmed
Atwood,
The Trypanosoma cruzi proteome.
2005,
Pubmed
Baida,
Molecular characterization of serine-, alanine-, and proline-rich proteins of Trypanosoma cruzi and their possible role in host cell infection.
2006,
Pubmed
Bayer-Santos,
Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins.
2013,
Pubmed
Brossas,
Secretome analysis of Trypanosoma cruzi by proteomics studies.
2017,
Pubmed
Brunoro,
Reevaluating the Trypanosoma cruzi proteomic map: The shotgun description of bloodstream trypomastigotes.
2015,
Pubmed
Buscaglia,
Characterization of an aldolase-binding site in the Wiskott-Aldrich syndrome protein.
2006,
Pubmed
Buscaglia,
Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood.
1999,
Pubmed
Buscaglia,
Trypanosoma cruzi surface mucins: host-dependent coat diversity.
2006,
Pubmed
Buscaglia,
The surface coat of the mammal-dwelling infective trypomastigote stage of Trypanosoma cruzi is formed by highly diverse immunogenic mucins.
2004,
Pubmed
Campo,
Immunocharacterization of the mucin-type proteins from the intracellular stage of Trypanosoma cruzi.
2006,
Pubmed
Campos,
Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.
2001,
Pubmed
Campos,
Extracellular Vesicles: Role in Inflammatory Responses and Potential Uses in Vaccination in Cancer and Infectious Diseases.
2015,
Pubmed
,
Echinobase
Cestari,
Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles.
2012,
Pubmed
Chuenkova,
Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas' disease.
1995,
Pubmed
Couper,
Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.
2010,
Pubmed
Cuevas,
gp63 homologues in Trypanosoma cruzi: surface antigens with metalloprotease activity and a possible role in host cell infection.
2003,
Pubmed
Dc-Rubin,
T rypanosoma cruzi trans-sialidase as a multifunctional enzyme in Chagas' disease.
2012,
Pubmed
El-Sayed,
The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.
2005,
Pubmed
Filardi,
Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease.
1987,
Pubmed
Frasch,
Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi.
2000,
Pubmed
Freitas,
Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.
2011,
Pubmed
Geiger,
Exocytosis and protein secretion in Trypanosoma.
2010,
Pubmed
Giordano,
Cloning of a surface membrane glycoprotein specific for the infective form of Trypanosoma cruzi having adhesive properties to laminin.
1999,
Pubmed
Gonçalves,
Trypanosoma cruzi: shedding of surface antigens as membrane vesicles.
1991,
Pubmed
Gonçalves,
NLRP3 controls Trypanosoma cruzi infection through a caspase-1-dependent IL-1R-independent NO production.
2013,
Pubmed
Hassani,
Immunomodulatory impact of leishmania-induced macrophage exosomes: a comparative proteomic and functional analysis.
2013,
Pubmed
Ishihama,
Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.
2005,
Pubmed
Lantos,
Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.
2016,
Pubmed
Magdesian,
Infection by Trypanosoma cruzi. Identification of a parasite ligand and its host cell receptor.
2001,
Pubmed
Magdesian,
A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection.
2007,
Pubmed
Mattos,
The Gp85 surface glycoproteins from Trypanosoma cruzi.
2014,
Pubmed
Nogueira,
Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses.
2015,
Pubmed
,
Echinobase
Pedrosa,
The effect of iron deficiency and iron overload on the evolution of Chagas disease produced by three strains of Trypanosoma cruzi in CFW mice.
1990,
Pubmed
Ramirez,
Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.
2017,
Pubmed
Regev-Rudzki,
Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles.
2013,
Pubmed
Rosenberg,
Stage-specific phospholipase C-mediated release of Trypanosoma cruzi neuraminidase.
1991,
Pubmed
Saavedra,
The Trypanosoma cruzi trans-sialidase, through its COOH-terminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells.
1999,
Pubmed
Sant'Anna,
Subcellular proteomics of Trypanosoma cruzi reservosomes.
2009,
Pubmed
Schenkman,
Attachment of Trypanosoma cruzi to mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton.
1991,
Pubmed
Schenkman,
Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes.
1992,
Pubmed
Schenkman,
Structural and functional properties of Trypanosoma trans-sialidase.
1994,
Pubmed
Silverman,
Proteomic analysis of the secretome of Leishmania donovani.
2008,
Pubmed
Silverman,
Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes.
2011,
Pubmed
Silverman,
An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.
2010,
Pubmed
Soares,
Intraspecies variation in Trypanosoma cruzi GPI-mucins: biological activities and differential expression of α-galactosyl residues.
2012,
Pubmed
Tatakihara,
Nitric oxide-releasing indomethacin enhances susceptibility to Trypanosoma cruzi infection acting in the cell invasion and oxidative stress associated with anemia.
2015,
Pubmed
Teixeira,
Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins.
2015,
Pubmed
Trocoli Torrecilhas,
Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response.
2009,
Pubmed
Uphoff,
Detection of mycoplasma contaminations.
2005,
Pubmed
Wiśniewski,
Universal sample preparation method for proteome analysis.
2009,
Pubmed
Yao,
The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function.
2003,
Pubmed
Yokoyama-Yasunaka,
Trypanosoma cruzi: identification of proteinases in shed components of trypomastigote forms.
1994,
Pubmed
Zanforlin,
Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.
2013,
Pubmed
Zingales,
Trypanosoma cruzi genetic diversity: Something new for something known about Chagas disease manifestations, serodiagnosis and drug sensitivity.
2018,
Pubmed
Zingales,
A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.
2009,
Pubmed
