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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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Bin-induced vacuoles originate from autolysosomes.(A) MDCK-Cpm1 cells transfected with GFP-LC3 and grown in complete medium (CM) or in nutrient-free medium (EBSS). Diffuse GFP-LC3 pattern seen in CM-grown cells is representative of non-autophagic cells. In contrast, GFP-LC3 punctated pattern seen in EBSS-grown cells is characteristic of autophagic cells. (B) Localization of the autophagic marker GFP-LC3 in cells treated with Bin for 6 h. To allow the detection of GFP-LC3 associated with autolysosomes NH4Cl (5 mM, final concentration) was added to the culture medium during the last hour of intoxication. A similar treatment with NH4Cl in the absence of toxin had no toxic effect on the cells and did not induce any change in GFP-LC3 pattern. (C) Bin-treated cells were processed for EM analysis as described in Experimental procedures. Vacuoles induced by Bin are electron-clear matrices enclosed by a single membrane (black arrows). In contrast, autophagosomes are double membrane vesicles (white arrows). Bin-induced vacuoles often contained vesicle-like structure (arrow-head) and partially degraded material (large black arrows). Bars, 5 µm (A and B); 2 µm (C).
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pone-0014682-g002: Bin-induced vacuoles originate from autolysosomes.(A) MDCK-Cpm1 cells transfected with GFP-LC3 and grown in complete medium (CM) or in nutrient-free medium (EBSS). Diffuse GFP-LC3 pattern seen in CM-grown cells is representative of non-autophagic cells. In contrast, GFP-LC3 punctated pattern seen in EBSS-grown cells is characteristic of autophagic cells. (B) Localization of the autophagic marker GFP-LC3 in cells treated with Bin for 6 h. To allow the detection of GFP-LC3 associated with autolysosomes NH4Cl (5 mM, final concentration) was added to the culture medium during the last hour of intoxication. A similar treatment with NH4Cl in the absence of toxin had no toxic effect on the cells and did not induce any change in GFP-LC3 pattern. (C) Bin-treated cells were processed for EM analysis as described in Experimental procedures. Vacuoles induced by Bin are electron-clear matrices enclosed by a single membrane (black arrows). In contrast, autophagosomes are double membrane vesicles (white arrows). Bin-induced vacuoles often contained vesicle-like structure (arrow-head) and partially degraded material (large black arrows). Bars, 5 µm (A and B); 2 µm (C).

Mentions: Because of the convergence between the endocytic and the autophagic pathways [18], [19], we investigated the possibility that Bin-induced vacuoles displayed both autophagic and late endocytic/lysosomal features. The microtubule-associated protein light chain 3 (LC3), is a mammalian homologue of Atg8 which is a constituent of the autophagic vesicles in yeast [20], [21]. When autophagy is induced, LC3 is processed from a cytosolic form, LC3-I, to the LC3-II form that is associated with the autophagic vesicle membranes: elongating membranes and autophagosomes. In autophagic cells expressing GFP-LC3, these structures can be detected by fluorescence microscopy as dots (also named GFP-LC3 positive cells), while in non-autophagic cells GFP-LC3 presents a diffuse cytosolic pattern [20], [22] (Fig. 2 A). While we could find Bin treated cells presenting an autophagic pattern, we could not detect any GFP-LC3 signal that could be related to an association with the membrane of the giant vacuoles (Fig. 2B).


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)

Bin-induced vacuoles originate from autolysosomes.(A) MDCK-Cpm1 cells transfected with GFP-LC3 and grown in complete medium (CM) or in nutrient-free medium (EBSS). Diffuse GFP-LC3 pattern seen in CM-grown cells is representative of non-autophagic cells. In contrast, GFP-LC3 punctated pattern seen in EBSS-grown cells is characteristic of autophagic cells. (B) Localization of the autophagic marker GFP-LC3 in cells treated with Bin for 6 h. To allow the detection of GFP-LC3 associated with autolysosomes NH4Cl (5 mM, final concentration) was added to the culture medium during the last hour of intoxication. A similar treatment with NH4Cl in the absence of toxin had no toxic effect on the cells and did not induce any change in GFP-LC3 pattern. (C) Bin-treated cells were processed for EM analysis as described in Experimental procedures. Vacuoles induced by Bin are electron-clear matrices enclosed by a single membrane (black arrows). In contrast, autophagosomes are double membrane vesicles (white arrows). Bin-induced vacuoles often contained vesicle-like structure (arrow-head) and partially degraded material (large black arrows). Bars, 5 µm (A and B); 2 µm (C).
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Related In: Results  -  Collection

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pone-0014682-g002: Bin-induced vacuoles originate from autolysosomes.(A) MDCK-Cpm1 cells transfected with GFP-LC3 and grown in complete medium (CM) or in nutrient-free medium (EBSS). Diffuse GFP-LC3 pattern seen in CM-grown cells is representative of non-autophagic cells. In contrast, GFP-LC3 punctated pattern seen in EBSS-grown cells is characteristic of autophagic cells. (B) Localization of the autophagic marker GFP-LC3 in cells treated with Bin for 6 h. To allow the detection of GFP-LC3 associated with autolysosomes NH4Cl (5 mM, final concentration) was added to the culture medium during the last hour of intoxication. A similar treatment with NH4Cl in the absence of toxin had no toxic effect on the cells and did not induce any change in GFP-LC3 pattern. (C) Bin-treated cells were processed for EM analysis as described in Experimental procedures. Vacuoles induced by Bin are electron-clear matrices enclosed by a single membrane (black arrows). In contrast, autophagosomes are double membrane vesicles (white arrows). Bin-induced vacuoles often contained vesicle-like structure (arrow-head) and partially degraded material (large black arrows). Bars, 5 µm (A and B); 2 µm (C).
Mentions: Because of the convergence between the endocytic and the autophagic pathways [18], [19], we investigated the possibility that Bin-induced vacuoles displayed both autophagic and late endocytic/lysosomal features. The microtubule-associated protein light chain 3 (LC3), is a mammalian homologue of Atg8 which is a constituent of the autophagic vesicles in yeast [20], [21]. When autophagy is induced, LC3 is processed from a cytosolic form, LC3-I, to the LC3-II form that is associated with the autophagic vesicle membranes: elongating membranes and autophagosomes. In autophagic cells expressing GFP-LC3, these structures can be detected by fluorescence microscopy as dots (also named GFP-LC3 positive cells), while in non-autophagic cells GFP-LC3 presents a diffuse cytosolic pattern [20], [22] (Fig. 2 A). While we could find Bin treated cells presenting an autophagic pattern, we could not detect any GFP-LC3 signal that could be related to an association with the membrane of the giant vacuoles (Fig. 2B).

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