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Revealing Annexin A2 and ARF-6 enrollment during Trypanosoma cruzi extracellular amastigote-host cell interaction.

Teixeira TL, Cruz L, Mortara RA, Da Silva CV - Parasit Vectors (2015)

Bottom Line: Here we describe the role of Annexin A2 and ARF-6 during extracellular amastigote-mammalian cell interactions.Our results showed ARF-6 accumulation in the amastigote-containing parasitophorous vacuole containing amastigote forms; demonstrated ARF-6 and Annexin A2 critical impact over parasite cell invasion and revealed the effect of Annexin A2 expression on intracellular parasite multiplication.ARF-6 and Annexin A2 are involved in invasion of mammalian cells by T. cruzi amastigotes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, Brasil. thaiselara@yahoo.com.br.

ABSTRACT

Background: Invasion of host cells by Trypanosoma cruzi extracellular amastigotes is host actin polymerization-dependent. However, the role of proteins related to actin dynamics during invasion by amastigotes remains to be investigated. Here we describe the role of Annexin A2 and ARF-6 during extracellular amastigote-mammalian cell interactions.

Findings: Our results showed ARF-6 accumulation in the amastigote-containing parasitophorous vacuole containing amastigote forms; demonstrated ARF-6 and Annexin A2 critical impact over parasite cell invasion and revealed the effect of Annexin A2 expression on intracellular parasite multiplication.

Conclusion: ARF-6 and Annexin A2 are involved in invasion of mammalian cells by T. cruzi amastigotes.

No MeSH data available.


Related in: MedlinePlus

ARF-6 is recruited to T. cruzi EA phagosome and its expression is required during host cell invasion. a: Wild-type MEF cells were treated with control siRNA, ARF-6 siRNA (Santa Cruz Biotechnology) as previously described (Silva et al., 2009). G-strain EAs were allowed to invade cells for space of one hour. Cells were then washed with PBS, fixed with Bouin and stained with Giemsa. The protocol was similar to the one described in Fig. 1. b: Wild-type MEF cell extracts were submitted to Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer into a nitrocellulose membrane for one hour at 250 mA per cm2, incubation with polyclonal antibody anti-ARF-6 [diluted 1:100 in phosphate-buffered saline (PBS)] (Santa Cruz Biotechnology). After washes, membranes were incubated with peroxidase conjugated IgG anti-mouse (diluted 1:5000 in PBS) (Sigma-Aldrich) and developed by chemiluminescence (Silva et al., 2009). c: 5x105 wild-type MEF cells/well were transfected with HA-ARF-6 plasmid (a gift from Prof. Dr. Philippe Chavrier, Department of Cell Biology, Research Center, Institut Curie.) and incubated with G-strain EAs for one hour. Cells were then formaldehyde-fixed and incubated with rabbit polyclonal antibody anti-HA (Santa Cruz Biotechnology) [diluted 1:100 in PBS + 0.02 % gelatin + 0.01 % azide (PGN)] followed by AlexaFluor® 488 conjugated IgG anti-rabbit (Invitrogen) (diluted 1:100 in PGN). Arrows indicate EA phagosome enriched in ARF-6. Bar: 10 μm. (p < 0.01)
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Fig2: ARF-6 is recruited to T. cruzi EA phagosome and its expression is required during host cell invasion. a: Wild-type MEF cells were treated with control siRNA, ARF-6 siRNA (Santa Cruz Biotechnology) as previously described (Silva et al., 2009). G-strain EAs were allowed to invade cells for space of one hour. Cells were then washed with PBS, fixed with Bouin and stained with Giemsa. The protocol was similar to the one described in Fig. 1. b: Wild-type MEF cell extracts were submitted to Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer into a nitrocellulose membrane for one hour at 250 mA per cm2, incubation with polyclonal antibody anti-ARF-6 [diluted 1:100 in phosphate-buffered saline (PBS)] (Santa Cruz Biotechnology). After washes, membranes were incubated with peroxidase conjugated IgG anti-mouse (diluted 1:5000 in PBS) (Sigma-Aldrich) and developed by chemiluminescence (Silva et al., 2009). c: 5x105 wild-type MEF cells/well were transfected with HA-ARF-6 plasmid (a gift from Prof. Dr. Philippe Chavrier, Department of Cell Biology, Research Center, Institut Curie.) and incubated with G-strain EAs for one hour. Cells were then formaldehyde-fixed and incubated with rabbit polyclonal antibody anti-HA (Santa Cruz Biotechnology) [diluted 1:100 in PBS + 0.02 % gelatin + 0.01 % azide (PGN)] followed by AlexaFluor® 488 conjugated IgG anti-rabbit (Invitrogen) (diluted 1:100 in PGN). Arrows indicate EA phagosome enriched in ARF-6. Bar: 10 μm. (p < 0.01)

Mentions: Annexin A2 knockout cells displayed an important reduction in EA cell invasion. In contrast, lack of Annexin A2 expression favored EA intracellular multiplication (Fig. 1a and b). Furthermore, ARF-6 knock-down fibroblasts also showed significantly lower number of internalized parasites compared to control cells (Fig. 2a and b). Also, our results demonstrated the accumulation of ARF-6 (Fig. 2c) around EA parasitophorous vacuole. Unfortunately, due to the high recovery dynamics of ARF-6 turnover, we could not evaluate the impact of ARF-6 reduced expression over parasite intracellular multiplication using siRNA treatment.Fig. 1


Revealing Annexin A2 and ARF-6 enrollment during Trypanosoma cruzi extracellular amastigote-host cell interaction.

Teixeira TL, Cruz L, Mortara RA, Da Silva CV - Parasit Vectors (2015)

ARF-6 is recruited to T. cruzi EA phagosome and its expression is required during host cell invasion. a: Wild-type MEF cells were treated with control siRNA, ARF-6 siRNA (Santa Cruz Biotechnology) as previously described (Silva et al., 2009). G-strain EAs were allowed to invade cells for space of one hour. Cells were then washed with PBS, fixed with Bouin and stained with Giemsa. The protocol was similar to the one described in Fig. 1. b: Wild-type MEF cell extracts were submitted to Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer into a nitrocellulose membrane for one hour at 250 mA per cm2, incubation with polyclonal antibody anti-ARF-6 [diluted 1:100 in phosphate-buffered saline (PBS)] (Santa Cruz Biotechnology). After washes, membranes were incubated with peroxidase conjugated IgG anti-mouse (diluted 1:5000 in PBS) (Sigma-Aldrich) and developed by chemiluminescence (Silva et al., 2009). c: 5x105 wild-type MEF cells/well were transfected with HA-ARF-6 plasmid (a gift from Prof. Dr. Philippe Chavrier, Department of Cell Biology, Research Center, Institut Curie.) and incubated with G-strain EAs for one hour. Cells were then formaldehyde-fixed and incubated with rabbit polyclonal antibody anti-HA (Santa Cruz Biotechnology) [diluted 1:100 in PBS + 0.02 % gelatin + 0.01 % azide (PGN)] followed by AlexaFluor® 488 conjugated IgG anti-rabbit (Invitrogen) (diluted 1:100 in PGN). Arrows indicate EA phagosome enriched in ARF-6. Bar: 10 μm. (p < 0.01)
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Fig2: ARF-6 is recruited to T. cruzi EA phagosome and its expression is required during host cell invasion. a: Wild-type MEF cells were treated with control siRNA, ARF-6 siRNA (Santa Cruz Biotechnology) as previously described (Silva et al., 2009). G-strain EAs were allowed to invade cells for space of one hour. Cells were then washed with PBS, fixed with Bouin and stained with Giemsa. The protocol was similar to the one described in Fig. 1. b: Wild-type MEF cell extracts were submitted to Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer into a nitrocellulose membrane for one hour at 250 mA per cm2, incubation with polyclonal antibody anti-ARF-6 [diluted 1:100 in phosphate-buffered saline (PBS)] (Santa Cruz Biotechnology). After washes, membranes were incubated with peroxidase conjugated IgG anti-mouse (diluted 1:5000 in PBS) (Sigma-Aldrich) and developed by chemiluminescence (Silva et al., 2009). c: 5x105 wild-type MEF cells/well were transfected with HA-ARF-6 plasmid (a gift from Prof. Dr. Philippe Chavrier, Department of Cell Biology, Research Center, Institut Curie.) and incubated with G-strain EAs for one hour. Cells were then formaldehyde-fixed and incubated with rabbit polyclonal antibody anti-HA (Santa Cruz Biotechnology) [diluted 1:100 in PBS + 0.02 % gelatin + 0.01 % azide (PGN)] followed by AlexaFluor® 488 conjugated IgG anti-rabbit (Invitrogen) (diluted 1:100 in PGN). Arrows indicate EA phagosome enriched in ARF-6. Bar: 10 μm. (p < 0.01)
Mentions: Annexin A2 knockout cells displayed an important reduction in EA cell invasion. In contrast, lack of Annexin A2 expression favored EA intracellular multiplication (Fig. 1a and b). Furthermore, ARF-6 knock-down fibroblasts also showed significantly lower number of internalized parasites compared to control cells (Fig. 2a and b). Also, our results demonstrated the accumulation of ARF-6 (Fig. 2c) around EA parasitophorous vacuole. Unfortunately, due to the high recovery dynamics of ARF-6 turnover, we could not evaluate the impact of ARF-6 reduced expression over parasite intracellular multiplication using siRNA treatment.Fig. 1

Bottom Line: Here we describe the role of Annexin A2 and ARF-6 during extracellular amastigote-mammalian cell interactions.Our results showed ARF-6 accumulation in the amastigote-containing parasitophorous vacuole containing amastigote forms; demonstrated ARF-6 and Annexin A2 critical impact over parasite cell invasion and revealed the effect of Annexin A2 expression on intracellular parasite multiplication.ARF-6 and Annexin A2 are involved in invasion of mammalian cells by T. cruzi amastigotes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, Brasil. thaiselara@yahoo.com.br.

ABSTRACT

Background: Invasion of host cells by Trypanosoma cruzi extracellular amastigotes is host actin polymerization-dependent. However, the role of proteins related to actin dynamics during invasion by amastigotes remains to be investigated. Here we describe the role of Annexin A2 and ARF-6 during extracellular amastigote-mammalian cell interactions.

Findings: Our results showed ARF-6 accumulation in the amastigote-containing parasitophorous vacuole containing amastigote forms; demonstrated ARF-6 and Annexin A2 critical impact over parasite cell invasion and revealed the effect of Annexin A2 expression on intracellular parasite multiplication.

Conclusion: ARF-6 and Annexin A2 are involved in invasion of mammalian cells by T. cruzi amastigotes.

No MeSH data available.


Related in: MedlinePlus