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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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After internalization, Bin reaches recycling endosomes.MDCK-Cpm1 cells were transfected with markers of early and late endocytotic compartments, intoxicated with a mixture of unlabelled BinA and BinB-Al543 for 30 min and processed for confocal microscopy analysis. (A) BinB-Al543 fluorescent signal colocalized with GFP-Rab4, a typical marker of recycling compartments. (B) A significant colocalization was detected with TfR-myc that marks both endocytotic and recycling vesicles. (C - E) No colocalization was found with GFP-Rab7 and GFP-Lamp1 that marks late-endocytotic/lysosomal compartments and GFP-LC3 a marker of autophagic vesicles. Bars, 10 µm.
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pone-0014682-g006: After internalization, Bin reaches recycling endosomes.MDCK-Cpm1 cells were transfected with markers of early and late endocytotic compartments, intoxicated with a mixture of unlabelled BinA and BinB-Al543 for 30 min and processed for confocal microscopy analysis. (A) BinB-Al543 fluorescent signal colocalized with GFP-Rab4, a typical marker of recycling compartments. (B) A significant colocalization was detected with TfR-myc that marks both endocytotic and recycling vesicles. (C - E) No colocalization was found with GFP-Rab7 and GFP-Lamp1 that marks late-endocytotic/lysosomal compartments and GFP-LC3 a marker of autophagic vesicles. Bars, 10 µm.

Mentions: Because Bin was not associated with the vacuoles it induces, we tried to identify the intracellular compartment reached by the toxin. After 10 min of internalization, the vesicles stained with Bin were negative for the early endosomal markers GFP-Rab5 and Caveolin1-GFP, the transferrin receptor that marks both early and recycling endosomes, and GFP-Rab4 a marker of recycling endosomes (Fig. S2). Interestingly after 30 min of internalization, the vesicles containing Bin colocalized with the recycling endosomal marker GFP-Rab4 and the transferrin receptor, which exploits the recycling pathway during its trafficking (Figs. 6A and 6B). In contrast, no association could be observed with the late endosomal marker Rab7, the lysosomal marker Lamp1 or the marker of autophagic vesicles LC3 (Figs. 6C-E). We next investigated the fate of the toxin in dividing cells. The sequential phases of mitosis were identified via immunodetection of β-tubulin and DNA staining. During mitosis, the toxin localized to the microtubule organizing center (Fig. 7A). As shown in figure 7B, and consistent with figure 6A, recycling endosomes stained with GFP-Rab4 en route for partitioning into the daughter cells were located in the same area [31], [32]. Using time lapse microscopy, we confirmed that while cells progressed through mitosis, the internalized toxin was partitioned to the daughter cells and, when post-mitotic vacuolation appeared, the toxin was not associated at any time with the vacuoles but remained clustered in the free space (Fig. 7C; Video S4) as reported above (Fig. 5C). Taken together, these results established that the toxin is associated with recycling endosomes but not with the degradative compartments.


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)

After internalization, Bin reaches recycling endosomes.MDCK-Cpm1 cells were transfected with markers of early and late endocytotic compartments, intoxicated with a mixture of unlabelled BinA and BinB-Al543 for 30 min and processed for confocal microscopy analysis. (A) BinB-Al543 fluorescent signal colocalized with GFP-Rab4, a typical marker of recycling compartments. (B) A significant colocalization was detected with TfR-myc that marks both endocytotic and recycling vesicles. (C - E) No colocalization was found with GFP-Rab7 and GFP-Lamp1 that marks late-endocytotic/lysosomal compartments and GFP-LC3 a marker of autophagic vesicles. Bars, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038859&req=5

pone-0014682-g006: After internalization, Bin reaches recycling endosomes.MDCK-Cpm1 cells were transfected with markers of early and late endocytotic compartments, intoxicated with a mixture of unlabelled BinA and BinB-Al543 for 30 min and processed for confocal microscopy analysis. (A) BinB-Al543 fluorescent signal colocalized with GFP-Rab4, a typical marker of recycling compartments. (B) A significant colocalization was detected with TfR-myc that marks both endocytotic and recycling vesicles. (C - E) No colocalization was found with GFP-Rab7 and GFP-Lamp1 that marks late-endocytotic/lysosomal compartments and GFP-LC3 a marker of autophagic vesicles. Bars, 10 µm.
Mentions: Because Bin was not associated with the vacuoles it induces, we tried to identify the intracellular compartment reached by the toxin. After 10 min of internalization, the vesicles stained with Bin were negative for the early endosomal markers GFP-Rab5 and Caveolin1-GFP, the transferrin receptor that marks both early and recycling endosomes, and GFP-Rab4 a marker of recycling endosomes (Fig. S2). Interestingly after 30 min of internalization, the vesicles containing Bin colocalized with the recycling endosomal marker GFP-Rab4 and the transferrin receptor, which exploits the recycling pathway during its trafficking (Figs. 6A and 6B). In contrast, no association could be observed with the late endosomal marker Rab7, the lysosomal marker Lamp1 or the marker of autophagic vesicles LC3 (Figs. 6C-E). We next investigated the fate of the toxin in dividing cells. The sequential phases of mitosis were identified via immunodetection of β-tubulin and DNA staining. During mitosis, the toxin localized to the microtubule organizing center (Fig. 7A). As shown in figure 7B, and consistent with figure 6A, recycling endosomes stained with GFP-Rab4 en route for partitioning into the daughter cells were located in the same area [31], [32]. Using time lapse microscopy, we confirmed that while cells progressed through mitosis, the internalized toxin was partitioned to the daughter cells and, when post-mitotic vacuolation appeared, the toxin was not associated at any time with the vacuoles but remained clustered in the free space (Fig. 7C; Video S4) as reported above (Fig. 5C). Taken together, these results established that the toxin is associated with recycling endosomes but not with the degradative compartments.

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