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Ameobal pathogen mimivirus infects macrophages through phagocytosis.

Ghigo E, Kartenbeck J, Lien P, Pelkmans L, Capo C, Mege JL, Raoult D - PLoS Pathog. (2008)

Bottom Line: Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry.Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry.This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

View Article: PubMed Central - PubMed

Affiliation: URMITE CNRS UMR 6236 - IRD 3R198, Université de la Méditerranée, Marseille, France.

ABSTRACT
Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

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Role of dynamin-II in APMV internalization.RAW 264.7 macrophages (A), macrophages transiently transfected with GFP (B), GFP-tagged active (C) or dominant-negative (D) dynamin-II were infected with APMV (50 PFU/cell) for 6 hours. Viral particles were visualized by immunofluorescence (middle panels). Viral particles were not associated with macrophages transfected with the dominant-negative mutant of dynamin-II. (E) The number of APMV particles internalized was scored. The results, expressed as the percentage of APMV uptake relative to the control, are the mean±SD of 3 experiments. Scale bars represent 50 µm. *p<0.05.
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ppat-1000087-g013: Role of dynamin-II in APMV internalization.RAW 264.7 macrophages (A), macrophages transiently transfected with GFP (B), GFP-tagged active (C) or dominant-negative (D) dynamin-II were infected with APMV (50 PFU/cell) for 6 hours. Viral particles were visualized by immunofluorescence (middle panels). Viral particles were not associated with macrophages transfected with the dominant-negative mutant of dynamin-II. (E) The number of APMV particles internalized was scored. The results, expressed as the percentage of APMV uptake relative to the control, are the mean±SD of 3 experiments. Scale bars represent 50 µm. *p<0.05.

Mentions: Since phagocytosis, but not macropinocytosis, requires dynamin-II, we investigated the role of dynamin-II in APMV uptake by immunofluorescence. RAW 264.7 macrophages were transfected with GFP-tagged dynamin-II (dynII-wt), a dominant-negative variant of dynamin-II (dynII-K44A), and GFP as a control. First, we controlled that dynamin-II was not involved in other endocytic pathways than phagocytosis [26]. Indeed, we investigated the involvement of dynamin-II in clathrin-mediated endocytosis through the transferrin uptake inhibition (Figure S2). The expression of GFP had no effect on the ability of macrophages to internalize transferrin (Figure S2A and Figure S2B). Macrophages transfected with dynII-wt (Figure S2C) or dynII-K44A (Figure S2D) also internalized transferrin (Figure S2E), demonstrating that dynamin-II is not required for clathrin-mediated endocytosis. We also investigated the role of dynamin-II in macropinocytosis through the inhibition of dextran uptake (Figure S3). The expression of GFP had no effect on the ability of macrophages to internalize fluorescent dextran (Figure S3A and Figure S3B). Macrophages transfected with functional dynamin-II (Figure S3C) or dynII-K44A (Figure S3D) internalized dextran in a similar way (Figure S3E) demonstrating that dynamin-II is not required for macropinocytosis. Second, the role of dynamin-II in the phagocytosis process was checked using Mycobacterium avium, a bacterium known to enter macrophages through phagocytosis [27]. The expression of GFP had no effect on the ability of macrophages to internalize M. avium (Figure 12A and Figure 12B): 89±7% of organisms were internalized by GFP-transfected macrophages compared to untransfected cells. Macrophages transfected with dynII-wt also internalized M. avium (Figure 12C). In contrast, M. avium internalization was significantly (p<0.05) decreased in macrophages transfected with dynII-K44A (Figure 12D) as compared to macrophages expressing a functional dynamin-II, since only 18±6% of bacteria were internalized by macrophages transfected with dynII-K44A (Figure 12E). These results clearly show that dynamin-II was required for phagocytosis. Third, the role of dynamin-II in APMV entry was investigated. The expression of GFP had no effect on the ability of macrophages to internalize APMV (Figure 13A and Figure 13B): 90±5% of APMV particles were internalized by GFP-expressing macrophages as compared to untransfected cells. Macrophages transfected with functional dynamin-II also internalized APMV (Figure 13C). In contrast, macrophages transfected with the dominant-negative variant of dynamin-II did not internalize APMV (Figure 13D). In these cells, only 13±4% of APMV particles were associated with macrophages as compared to macrophages that expressed a functional dynamin-II (p<0.05) (Figure 13E). These results clearly show that the entry of APMV within macrophages occurs through phagocytosis.


Ameobal pathogen mimivirus infects macrophages through phagocytosis.

Ghigo E, Kartenbeck J, Lien P, Pelkmans L, Capo C, Mege JL, Raoult D - PLoS Pathog. (2008)

Role of dynamin-II in APMV internalization.RAW 264.7 macrophages (A), macrophages transiently transfected with GFP (B), GFP-tagged active (C) or dominant-negative (D) dynamin-II were infected with APMV (50 PFU/cell) for 6 hours. Viral particles were visualized by immunofluorescence (middle panels). Viral particles were not associated with macrophages transfected with the dominant-negative mutant of dynamin-II. (E) The number of APMV particles internalized was scored. The results, expressed as the percentage of APMV uptake relative to the control, are the mean±SD of 3 experiments. Scale bars represent 50 µm. *p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2398789&req=5

ppat-1000087-g013: Role of dynamin-II in APMV internalization.RAW 264.7 macrophages (A), macrophages transiently transfected with GFP (B), GFP-tagged active (C) or dominant-negative (D) dynamin-II were infected with APMV (50 PFU/cell) for 6 hours. Viral particles were visualized by immunofluorescence (middle panels). Viral particles were not associated with macrophages transfected with the dominant-negative mutant of dynamin-II. (E) The number of APMV particles internalized was scored. The results, expressed as the percentage of APMV uptake relative to the control, are the mean±SD of 3 experiments. Scale bars represent 50 µm. *p<0.05.
Mentions: Since phagocytosis, but not macropinocytosis, requires dynamin-II, we investigated the role of dynamin-II in APMV uptake by immunofluorescence. RAW 264.7 macrophages were transfected with GFP-tagged dynamin-II (dynII-wt), a dominant-negative variant of dynamin-II (dynII-K44A), and GFP as a control. First, we controlled that dynamin-II was not involved in other endocytic pathways than phagocytosis [26]. Indeed, we investigated the involvement of dynamin-II in clathrin-mediated endocytosis through the transferrin uptake inhibition (Figure S2). The expression of GFP had no effect on the ability of macrophages to internalize transferrin (Figure S2A and Figure S2B). Macrophages transfected with dynII-wt (Figure S2C) or dynII-K44A (Figure S2D) also internalized transferrin (Figure S2E), demonstrating that dynamin-II is not required for clathrin-mediated endocytosis. We also investigated the role of dynamin-II in macropinocytosis through the inhibition of dextran uptake (Figure S3). The expression of GFP had no effect on the ability of macrophages to internalize fluorescent dextran (Figure S3A and Figure S3B). Macrophages transfected with functional dynamin-II (Figure S3C) or dynII-K44A (Figure S3D) internalized dextran in a similar way (Figure S3E) demonstrating that dynamin-II is not required for macropinocytosis. Second, the role of dynamin-II in the phagocytosis process was checked using Mycobacterium avium, a bacterium known to enter macrophages through phagocytosis [27]. The expression of GFP had no effect on the ability of macrophages to internalize M. avium (Figure 12A and Figure 12B): 89±7% of organisms were internalized by GFP-transfected macrophages compared to untransfected cells. Macrophages transfected with dynII-wt also internalized M. avium (Figure 12C). In contrast, M. avium internalization was significantly (p<0.05) decreased in macrophages transfected with dynII-K44A (Figure 12D) as compared to macrophages expressing a functional dynamin-II, since only 18±6% of bacteria were internalized by macrophages transfected with dynII-K44A (Figure 12E). These results clearly show that dynamin-II was required for phagocytosis. Third, the role of dynamin-II in APMV entry was investigated. The expression of GFP had no effect on the ability of macrophages to internalize APMV (Figure 13A and Figure 13B): 90±5% of APMV particles were internalized by GFP-expressing macrophages as compared to untransfected cells. Macrophages transfected with functional dynamin-II also internalized APMV (Figure 13C). In contrast, macrophages transfected with the dominant-negative variant of dynamin-II did not internalize APMV (Figure 13D). In these cells, only 13±4% of APMV particles were associated with macrophages as compared to macrophages that expressed a functional dynamin-II (p<0.05) (Figure 13E). These results clearly show that the entry of APMV within macrophages occurs through phagocytosis.

Bottom Line: Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry.Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry.This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

View Article: PubMed Central - PubMed

Affiliation: URMITE CNRS UMR 6236 - IRD 3R198, Université de la Méditerranée, Marseille, France.

ABSTRACT
Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

Show MeSH
Related in: MedlinePlus