Parasitology Research

, Volume 113, Issue 1, pp 285–304 | Cite as

Extracellular vesicles shed by Trypanosoma cruzi are linked to small RNA pathways, life cycle regulation, and susceptibility to infection of mammalian cells

  • Maria R. Garcia-Silva
  • Roberta Ferreira Cura das Neves
  • Florencia Cabrera-Cabrera
  • Julia Sanguinetti
  • Lia C. Medeiros
  • Carlos Robello
  • Hugo Naya
  • Tamara Fernandez-Calero
  • Thais Souto-Padron
  • Wanderley de Souza
  • Alfonso Cayota
Original Paper

Abstract

The protozoan parasite Trypanosoma cruzi has a complex life cycle characterized by intracellular and extracellular forms alternating between invertebrate and mammals. To cope with these changing environments, T. cruzi undergoes rapid changes in gene expression, which are achieved essentially at the posttranscriptional level. At present, expanding families of small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, T. cruzi lacks canonical small RNA pathways. In a recent work, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by a homogeneous population of tRNA-derived small RNAs (tsRNAs). In T. cruzi epimastigotes submitted to nutrient starvation, tsRNAs colocalized with an argonaute protein distinctive of trypanosomatids (TcPIWI-tryp) and were recruited to particular cytoplasmic granules. Using epifluorescence and electronic microscopy, we observed that tsRNAs and the TcPIWI-tryp protein were recruited mainly to reservosomes and other intracellular vesicles including endosome-like vesicles and vesicular structures resembling the Golgi complex. These data suggested that, in T. cruzi, tsRNA biogenesis is probably part of endocytic/exocytic routes. We also demonstrated that epimastigotes submitted to nutrient starvation shed high levels of vesicles to the extracellular medium, which carry small tRNAs and TcPIWI-tryp proteins as cargo. At least a fraction of extracellular vesicle cargo was transferred between parasites and to mammalian susceptible cells. Our data afford experimental evidence, indicating that extracellular vesicles shed by T. cruzi promote not only life cycle transition of epimastigotes to trypomastigote forms but also infection susceptibility of mammalian cells

Supplementary material

436_2013_3655_Fig10_ESM.jpg (89 kb)
Figure S1

Time-course analysis of tRNA halves accumulation in epimastigotes submitted to nutritional stress. Epimastigotes under optimal growth (unstressed), stressed for 48 h (sE48) and trypomastigotes were fixed and hybridized with probes specific for tRNAGlu 5′ halves (red). Samples were counterstained with DAPI 4,6-diamidino-2-phenylindole (DAPI) at 1 mg ml−1 and merged images were obtained by superimposing the indicated images files. (JPEG 88 kb)

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High Resolution Image (TIFF 1780 kb)
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Figure S2

T. cruzi epimastigotes submitted to nutrient starvation. Epimastigotes at the late-stationary phase of growth in LIT medium were washed and resuspended in serum-free RPMI-1640 medium and further cultured for 48 h (sE48). a Visualization of sE48 parasites by differential interference contrast microscopy. b HeLa cells after 2 days of infection with sE48 parasites where DAPI stained amastigotes are indicated by arrows. c Percentage of trypomastigotes overtime in different culture conditions. Normal growing parasites in LIT medium (LIT), epimastigotes at the late-stationary phase in LIT medium (stationary phase) and epimastigotes submitted to nutrient starvation in RPMI (RPMI). (JPEG 106 kb)

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High Resolution Image (TIFF 3717 kb)
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Figure S3

Reactivity and specificity of TcPIWI-tryp mRNA probes used in FISH assays. Fixed parasites were hybridized for TcPIWI-tryp mRNA with antisense (positive control) or sense (negative control) oligoprobes conjugated to fluorescein isothiocyanate (FITC). Samples were counterstained with DAPI 4,6-diamidino-2-phenylindole (DAPI) at 1 mg ml−1. Merged images were obtained by superimposing the indicated images files. (JPEG 113 kb)

436_2013_3655_MOESM3_ESM.tif (3.5 mb)
High Resolution Image (TIFF 3558 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Maria R. Garcia-Silva
    • 1
  • Roberta Ferreira Cura das Neves
    • 2
  • Florencia Cabrera-Cabrera
    • 1
  • Julia Sanguinetti
    • 1
  • Lia C. Medeiros
    • 3
  • Carlos Robello
    • 4
  • Hugo Naya
    • 5
  • Tamara Fernandez-Calero
    • 5
  • Thais Souto-Padron
    • 2
  • Wanderley de Souza
    • 3
  • Alfonso Cayota
    • 1
    • 6
  1. 1.Functional Genomics UnitInstitut Pasteur de MontevideoMontevideoUruguay
  2. 2.Laboratório de Biologia Celular e Ultraestrutura, Instituto de Microbiologia Paulo de GóesUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  3. 3.Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  4. 4.Molecular Biology UnitInstitut Pasteur de MontevideoMontevideoUruguay
  5. 5.Bioinformatics UnitInstitut Pasteur de MontevideoMontevideoUruguay
  6. 6.Department of MedicineFaculty of MedicineMontevideoUruguay

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