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Parasitology Research

, Volume 113, Issue 6, pp 2323–2333 | Cite as

Structures containing galectin-3 are recruited to the parasitophorous vacuole containing Trypanosoma cruzi in mouse peritoneal macrophages

  • Lissa Catherine Reignault
  • Emile Santos Barrias
  • Lia Carolina Soares Medeiros
  • Wanderley de Souza
  • Tecia Maria Ulisses de Carvalho
Original Paper

Abstract

Trypanosoma cruzi has a complex life cycle where the infective forms for the vertebrate host are trypomastigotes and amastigotes. Both forms invade and lyse their parasitophorous vacuole (PV) membrane, entering into the cytoplasm of its host cells. Galectin-3 (Gal-3) is a protein abundantly distributed in macrophages and epithelial cells. Previous studies demonstrated that Gal-3 binds to a 45KDa mucin of trypomastigotes surface, enhancing its adhesion to the extracellular matrix and even its entry into cells. Gal-3 has another novel cytoplasmic function recently described: a vacuole lyses marker in intracellular bacteria. Considering (1) the importance of Gal-3 during T. cruzi early infection and (2) the importance of T. cruzi PV lyses for parasite differentiation and replication, this study intended to explore a possible recruitment of structures containing Gal-3 (G3CSs) to T. cruzi PVs. Microscopy analyses showed these G3CSs around PVs after 30 and 90 min of amastigotes and trypomastigotes infection, respectively. This recruitment was specific for T. cruzi PVs since we did not observe the same distribution at macrophages vacuoles containing fluorescent microspheres (FM). Concomitantly, this study intended to analyze the participation of actin cytoskeleton in T. cruzi PV maturation. We observed that actin filaments form a “belt-like” structure around trypomastigotes and amastigotes PVs, also labeled for Gal-3. At the time proposed for PV lysis, we observed an actin disassembling while LAMP-1 was recruited to PVs membrane. However, this pattern was maintained in macrophages derived from Gal-3 knockout mice, revealing that the actin belt structure forms independently from Gal-3. Taken together, these data suggest that G3CSs are recruited to vicinity of T. cruzi PV and that actin filaments localize and remain around T. cruzi PVs until the time of its lysis.

Keywords

Trypanosoma cruzi Galectin-3 Actin filaments LAMP-1 and macrophages 

Notes

Acknowledgments

The authors thank Luzinete da Silva, Juliana Fernandes, Noêmia Rodrigues, Rachel Rachid, Thiago Luiz de Barros Moreira for the technical assistance and Ricardo Vilela for operating Leica Sp5 confocal laser scanning microscope (INMETRO). We also thank Dr. Marc Benhamou for kindly supplying the anti-galectin-3 M3/38 antibody. This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Supplementary material

436_2014_3887_MOESM1_ESM.tif (1.1 mb)
Supplementary Fig. S1 G3CS and actin filaments were also observed around vacuoles containing T. cruzi after 240 (A, B) and 300 minutes (C, D) post-infection. (A, B) A punctual Gal-3 labeling was observed surrounding a trypomastigote-containing PV (B - white arrow) and an amastigote-containing PV (B – white arrowhead) at 240 minutes post-infection in macrophages. (C, D) After 300 minutes of infection, G3CS and actin filaments co-localize with a PV containing an amastigote (D – white arrowhead). At this same time, a second amastigote-containing PV (D – asterisk) was surrounded only by Gal-3 in a punctual manner. A trypomastigote was also observed, however, with no G3CS or actin filaments surrounding it (D - white arrow). (AD) Host cells and parasites’ DNA can be visualized with DAPI (Blue). Bars: 10 μm (TIFF 1085 kb)
436_2014_3887_MOESM2_ESM.tif (362 kb)
Supplementary Fig. S2 G3CS and actin filaments were also observed around vacuoles containing opsonized parasites. After 30 (A - B) and 90 minutes (C - F) post-infection, G3CS was found around phagosomes containing opsonized trypomastigotes (B, D and F - white arrowheads) and opsonized amastigotes (B and F - white arrows). Actin filaments were also found around PVs containing amastigotes (A, B – white arrow) or trypomastigotes (D – white arrow). (B, D, F) Host cells and parasites’ DNA can be visualized with DAPI (blue). The samples were examined in a SP5 Leica confocal microscope. Bars: 2.5 μm (A), 5 μm (D), 7.5 μm (B, C) (TIFF 361 kb)
436_2014_3887_MOESM3_ESM.tif (1.1 mb)
Supplementary Fig. S3 G3CS was not observed around vacuoles containing fluorescent microspheres (FM) but an actin belt appeared around them. (A) Actin filaments (white arrowhead) localize close to a phagosome containing two FMs after 30 minutes post-infection. (B) After 90 minutes, phagosomes presented a high concentration of FMs, but actin filaments are no longer seen around them (white arrowhead). (C, D) G3CS was not found around phagosomes containing FMs neither after 30 minutes (white arrow) nor after 90 minutes (white arrow). (AD) Host cell DNA can be visualized with DAPI (blue). The samples were examined in a SP5 Leica confocal microscope. Bars: 5 μm (A, B, C) and 7.5 μm (D) (TIFF 1089 kb)
436_2014_3887_MOESM4_ESM.tif (1.5 mb)
Supplementary Fig. S4 G3CS was not observed around phagosomes containing Tphk (B - D) or Amhk (FH) but actin filaments or LAMP-1 appeared around them at 90 minutes post-infection. (B) Actin filaments surround two phagosomes containing one Tphk each (white arrows), but with no G3CS labeling (B’ – white asterisks). Another Tphk was also observed inside a phagosome not surrounded by actin filaments (arrowhead). (D) LAMP-1 labeling was observed around phagosomes containing Tphk (white arrows). (F) Actin filaments were also found surrounding an Amhk phagosome (white arrowheads). (H) LAMP-1 was also observed around a phagosome containing an Amhk (white arrowhead). A-H: Host cell and parasite DNA can be visualized with DAPI (blue). The samples were examined in a SP5 Leica confocal microscope. Bars: 2.5 μm (E, F), 5 μm (A, B, G, H) and 7.5 μm (C, D) (TIFF 1513 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Lissa Catherine Reignault
    • 1
    • 2
  • Emile Santos Barrias
    • 3
  • Lia Carolina Soares Medeiros
    • 4
  • Wanderley de Souza
    • 1
    • 2
    • 3
  • Tecia Maria Ulisses de Carvalho
    • 1
    • 2
  1. 1.Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, CCS, Bloco GssCidade UniversitáriaRio de JaneiroBrazil
  2. 2.Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e BioimagensUniversidade Federal do Rio de JaneiroIlha do FundãoBrazil
  3. 3.Instituto Nacional de MetrologiaQualidade e Tecnologia—Inmetro, Av. Nossa Senhora das GraçasRio de Janeiro, Brazil
  4. 4.Laboratório de Biologia Celular, Instituto Carlos Chagas—ICC, FIOCRUZ-PRRio de JaneiroBrazil

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