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Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication.

Opota O, Gauthier NC, Doye A, Berry C, Gounon P, Lemichez E, Pauron D - PLoS ONE (2011)

Bottom Line: In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Recherche Agronomique, UMR Interactions Biotiques et Santé Végétale, INRA 1301-CNRS 6243-Université de Nice Sophia Antipolis, Sophia Antipolis, France. onya.opota@epfl.ch

ABSTRACT
Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

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Autophagy is enhanced in Bin treated cells and contributes to vacuolation reversion.(A) MDCK-Cpm1 cells were grown for 6 h in complete medium in the absence (CM) or the presence of Bin (CM + Bin), or in nutrient-free medium in the absence of the toxin (EBSS), and then processed for EM analysis. Pictures from randomly chosen EM sections were analyzed for the presence of autophagosomes as defined in figure 2. The results are expressed as the number of autophagosomes per cell. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (B) MDCK-Cpm1 cells expressing GFP-LC3 were grown under the conditions described above. At the indicated time, the number of GFP-LC3 positive cells was scored. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (C) Cells were grown in complete medium or in nutrient-free medium in the presence of toxin. At the indicated period of time, the percentage of vacuolating cells was determined. Mean values ± SD, n  =  3. Asterisks indicate statistically significant differences; P<0.05. (D) Images selected from time lapse videomicroscopy of Bin-treated cells in nutrient-free medium displaying the post-mitotic vacuolation phenomenon, the selection starts 20 h after intoxication (Video S3). Black and white arrows pinpoint two dividing cells that will display this phenomenon. Bars, 10 µm. (E) MDCK-Cpm1 cells were grown in complete medium for 3 h (Δ, ⧫, •). Then the toxin was washed out with either complete medium (•), or nutrient-free medium (Δ, ⧫). Two hours after washing, the cells were returned to CM (⧫). Mean values ± SD, n  =  3. (F) Representative pictures illustrating the various patterns of vacuolation corresponding to each medium condition.
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pone-0014682-g004: Autophagy is enhanced in Bin treated cells and contributes to vacuolation reversion.(A) MDCK-Cpm1 cells were grown for 6 h in complete medium in the absence (CM) or the presence of Bin (CM + Bin), or in nutrient-free medium in the absence of the toxin (EBSS), and then processed for EM analysis. Pictures from randomly chosen EM sections were analyzed for the presence of autophagosomes as defined in figure 2. The results are expressed as the number of autophagosomes per cell. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (B) MDCK-Cpm1 cells expressing GFP-LC3 were grown under the conditions described above. At the indicated time, the number of GFP-LC3 positive cells was scored. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (C) Cells were grown in complete medium or in nutrient-free medium in the presence of toxin. At the indicated period of time, the percentage of vacuolating cells was determined. Mean values ± SD, n  =  3. Asterisks indicate statistically significant differences; P<0.05. (D) Images selected from time lapse videomicroscopy of Bin-treated cells in nutrient-free medium displaying the post-mitotic vacuolation phenomenon, the selection starts 20 h after intoxication (Video S3). Black and white arrows pinpoint two dividing cells that will display this phenomenon. Bars, 10 µm. (E) MDCK-Cpm1 cells were grown in complete medium for 3 h (Δ, ⧫, •). Then the toxin was washed out with either complete medium (•), or nutrient-free medium (Δ, ⧫). Two hours after washing, the cells were returned to CM (⧫). Mean values ± SD, n  =  3. (F) Representative pictures illustrating the various patterns of vacuolation corresponding to each medium condition.

Mentions: In Bin-treated MDCK-Cpm1 cells we could detect many autophagosomes that were either isolated or initiating fusion with the enlarged autolysosomes induced by Bin, which raised the hypothesis of an autophagy induction. A quantitative analysis confirmed that the number of autophagosomes was significantly higher in Bin-treated cells than in untreated cells grown in complete medium (Fig. 4A). Autophagy can also be monitored by scoring the fraction of GFP-LC3 positive cells [20], [27]. Under starvation, the percentage of GFP-LC3 positive cells is dramatically enhanced as a result of the induction of autophagy (Fig. 4B). In Bin treated cells, the percentage of GFP-LC3 positive cells was similarly enhanced (Fig. 4B). The increase in both the number of autophagosomes and the number of GFP-LC3 positive cells strongly suggested that autophagy is induced in MDCK-Cpm1 cells exposed to Bin. We next probed the effect of the induction of autophagy before Bin treatment. Figure 4C shows that when Bin was added to cells cultured in nutrient-free medium, vacuolation was inhibited. However, the percentage of vacuolating cells increased in a time-dependent manner and ultimately affected there again a subset of the cell population as in figure 4C. Videomicroscopy analysis indicated that this late vacuolation induced by Bin in nutrient-free medium (Fig. 4D and Video S3), was almost exclusively the post-mitotic phenomenon described in cells intoxicated in complete medium (Fig. 3B and Video S1). These results demonstrate that induction of autophagy prior to intoxication protects cells from vacuolation but does not inhibit the post-mitotic vacuolation. Because autophagy has been shown to be inhibited in dividing cells we hypothesised that this could explain the recurrence of post-mitotic vacuolation [28]. To challenge this hypothesis MDCK-Cpm1 cells were cultured in complete medium with Bin such that, after 3 h, approximately 50% of the cells were vacuolated (Figs. 4E and F). When the toxin was removed by washing with complete medium, the number of vacuolated cells continued to increase until it reached a maximum about 2 h after washing. Thereafter the vacuolation slowly decreased and was completely absent by 48 h after washing (unpublished data). In contrast, when the cells were washed with a nutrient-free medium, the vacuoles disappeared within 2 h (Figs. 4E and F). In order to confirm that this reversion was due to starvation alone, cells were grown again in toxin-free complete medium and the vacuolation rapidly reappeared (Figs. 4E and F). These results demonstrate that up-regulation of autophagy inhibited Bin-induced autolysosomal enlargement and in contrast, the consequence of a down-regulation of autophagy, similar to that which occurs in dividing cells, is the reappearance of the vacuolation.


Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication.

Opota O, Gauthier NC, Doye A, Berry C, Gounon P, Lemichez E, Pauron D - PLoS ONE (2011)

Autophagy is enhanced in Bin treated cells and contributes to vacuolation reversion.(A) MDCK-Cpm1 cells were grown for 6 h in complete medium in the absence (CM) or the presence of Bin (CM + Bin), or in nutrient-free medium in the absence of the toxin (EBSS), and then processed for EM analysis. Pictures from randomly chosen EM sections were analyzed for the presence of autophagosomes as defined in figure 2. The results are expressed as the number of autophagosomes per cell. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (B) MDCK-Cpm1 cells expressing GFP-LC3 were grown under the conditions described above. At the indicated time, the number of GFP-LC3 positive cells was scored. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (C) Cells were grown in complete medium or in nutrient-free medium in the presence of toxin. At the indicated period of time, the percentage of vacuolating cells was determined. Mean values ± SD, n  =  3. Asterisks indicate statistically significant differences; P<0.05. (D) Images selected from time lapse videomicroscopy of Bin-treated cells in nutrient-free medium displaying the post-mitotic vacuolation phenomenon, the selection starts 20 h after intoxication (Video S3). Black and white arrows pinpoint two dividing cells that will display this phenomenon. Bars, 10 µm. (E) MDCK-Cpm1 cells were grown in complete medium for 3 h (Δ, ⧫, •). Then the toxin was washed out with either complete medium (•), or nutrient-free medium (Δ, ⧫). Two hours after washing, the cells were returned to CM (⧫). Mean values ± SD, n  =  3. (F) Representative pictures illustrating the various patterns of vacuolation corresponding to each medium condition.
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pone-0014682-g004: Autophagy is enhanced in Bin treated cells and contributes to vacuolation reversion.(A) MDCK-Cpm1 cells were grown for 6 h in complete medium in the absence (CM) or the presence of Bin (CM + Bin), or in nutrient-free medium in the absence of the toxin (EBSS), and then processed for EM analysis. Pictures from randomly chosen EM sections were analyzed for the presence of autophagosomes as defined in figure 2. The results are expressed as the number of autophagosomes per cell. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (B) MDCK-Cpm1 cells expressing GFP-LC3 were grown under the conditions described above. At the indicated time, the number of GFP-LC3 positive cells was scored. Mean values ± SD, n  =  3. Different letters indicate statistically significant differences; P<0.05. (C) Cells were grown in complete medium or in nutrient-free medium in the presence of toxin. At the indicated period of time, the percentage of vacuolating cells was determined. Mean values ± SD, n  =  3. Asterisks indicate statistically significant differences; P<0.05. (D) Images selected from time lapse videomicroscopy of Bin-treated cells in nutrient-free medium displaying the post-mitotic vacuolation phenomenon, the selection starts 20 h after intoxication (Video S3). Black and white arrows pinpoint two dividing cells that will display this phenomenon. Bars, 10 µm. (E) MDCK-Cpm1 cells were grown in complete medium for 3 h (Δ, ⧫, •). Then the toxin was washed out with either complete medium (•), or nutrient-free medium (Δ, ⧫). Two hours after washing, the cells were returned to CM (⧫). Mean values ± SD, n  =  3. (F) Representative pictures illustrating the various patterns of vacuolation corresponding to each medium condition.
Mentions: In Bin-treated MDCK-Cpm1 cells we could detect many autophagosomes that were either isolated or initiating fusion with the enlarged autolysosomes induced by Bin, which raised the hypothesis of an autophagy induction. A quantitative analysis confirmed that the number of autophagosomes was significantly higher in Bin-treated cells than in untreated cells grown in complete medium (Fig. 4A). Autophagy can also be monitored by scoring the fraction of GFP-LC3 positive cells [20], [27]. Under starvation, the percentage of GFP-LC3 positive cells is dramatically enhanced as a result of the induction of autophagy (Fig. 4B). In Bin treated cells, the percentage of GFP-LC3 positive cells was similarly enhanced (Fig. 4B). The increase in both the number of autophagosomes and the number of GFP-LC3 positive cells strongly suggested that autophagy is induced in MDCK-Cpm1 cells exposed to Bin. We next probed the effect of the induction of autophagy before Bin treatment. Figure 4C shows that when Bin was added to cells cultured in nutrient-free medium, vacuolation was inhibited. However, the percentage of vacuolating cells increased in a time-dependent manner and ultimately affected there again a subset of the cell population as in figure 4C. Videomicroscopy analysis indicated that this late vacuolation induced by Bin in nutrient-free medium (Fig. 4D and Video S3), was almost exclusively the post-mitotic phenomenon described in cells intoxicated in complete medium (Fig. 3B and Video S1). These results demonstrate that induction of autophagy prior to intoxication protects cells from vacuolation but does not inhibit the post-mitotic vacuolation. Because autophagy has been shown to be inhibited in dividing cells we hypothesised that this could explain the recurrence of post-mitotic vacuolation [28]. To challenge this hypothesis MDCK-Cpm1 cells were cultured in complete medium with Bin such that, after 3 h, approximately 50% of the cells were vacuolated (Figs. 4E and F). When the toxin was removed by washing with complete medium, the number of vacuolated cells continued to increase until it reached a maximum about 2 h after washing. Thereafter the vacuolation slowly decreased and was completely absent by 48 h after washing (unpublished data). In contrast, when the cells were washed with a nutrient-free medium, the vacuoles disappeared within 2 h (Figs. 4E and F). In order to confirm that this reversion was due to starvation alone, cells were grown again in toxin-free complete medium and the vacuolation rapidly reappeared (Figs. 4E and F). These results demonstrate that up-regulation of autophagy inhibited Bin-induced autolysosomal enlargement and in contrast, the consequence of a down-regulation of autophagy, similar to that which occurs in dividing cells, is the reappearance of the vacuolation.

Bottom Line: In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Recherche Agronomique, UMR Interactions Biotiques et Santé Végétale, INRA 1301-CNRS 6243-Université de Nice Sophia Antipolis, Sophia Antipolis, France. onya.opota@epfl.ch

ABSTRACT
Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

Show MeSH
Related in: MedlinePlus