Bcl-xL Affects Group A Streptococcus-Induced Autophagy Directly, by Inhibiting Fusion between Autophagosomes and Lysosomes, and Indirectly, by Inhibiting Bacterial Internalization via Interaction with Beclin 1-UVRAG - PubMed (original) (raw)

Bcl-xL Affects Group A Streptococcus-Induced Autophagy Directly, by Inhibiting Fusion between Autophagosomes and Lysosomes, and Indirectly, by Inhibiting Bacterial Internalization via Interaction with Beclin 1-UVRAG

Shintaro Nakajima et al. PLoS One. 2017.

Abstract

Anti-apoptotic Bcl-2 and Bcl-xL are proposed to regulate starvation-induced autophagy by directly interacting with Beclin 1. Beclin 1 is also thought to be involved in multiple vesicle trafficking pathways such as endocytosis by binding to Atg14L and UVRAG. However, how the interaction of Bcl-2 family proteins and Beclin 1 regulates anti-bacterial autophagy (xenophagy) is still unclear. In this study, we analyzed these interactions using Group A Streptococcus (GAS; Streptococcus pyogenes) infection as a model. GAS is internalized into epithelial cells through endocytosis, while the intracellular fate of GAS is degradation by autophagy. Here, we found that Bcl-xL but not Bcl-2 regulates GAS-induced autophagy. Autophagosome-lysosome fusion and the internalization process during GAS infection were promoted in Bcl-xL knockout cells. In addition, knockout of Beclin 1 phenocopied the internalization defect of GAS. Furthermore, UVRAG interacts not only with Beclin 1 but also with Bcl-xL, and overexpression of UVRAG partially rescued the internalization defect of Beclin 1 knockout cells during GAS infection. Thus, our results indicate that Bcl-xL inhibits GAS-induced autophagy directly by suppressing autophagosome-lysosome fusion and indirectly by suppressing GAS internalization via interaction with Beclin 1-UVRAG.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Bcl-xL suppresses GAS internalization.

(A) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with FLAG-control, -Bcl-2, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represents result from >200 infected cells in terms of the mean value ± SD from three independent experiments. * P < 0.05. (B) Confocal microscopic images of GcAV in cells expressing either FLAG-control, -Bcl-2, or–Bcl-xL. Insets show expansion of the boxed areas. Scale bars, 10 μm. (C) The accumulation of LC3-II during GAS infection. HeLa cells were transfected with FLAG-control, -Bcl-2, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Expression of LC3 was analyzed by western blotting using anti-LC3 antibody. (D) Co-localization efficiencies of GcAV and lysosomes were calculated as the percentage of the total number of GcAV. HeLa cells stably expressing GFP-LC3 were transfected with FLAG-control, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-LAMP1 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 GcAVs and each percentage represents the mean value ±SD from three independent experiments. (E) Confocal microscopic images of GcAV associated with lysosomes. White arrows show autophagosomes, white arrow heads show autolysosomes. Scale bars, 10 μm. (F) Intracellular survival rate of GAS. HeLa cells were transfected with FLAG-control, or -Bcl-xL, and infected with GAS (MOI = 100). After 1 h of infection, cells were washed with PBS and further incubated for 1 h with DMEM/10% FCS with gentamicin (100 μg/ml) to kill the extracellular bacteria. Cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments. (G) Invasion rate of GAS. HeLa Cells were transfected and infected as in (F). The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. * P < 0.05. (H) The number of cells containing Galectin-3-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with either FLAG-control, -Bcl-2, or–Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. * P < 0.05. (I) Confocal microscopic images of Galectin-3-positive GAS. White arrow heads show Galectin-3-positive GAS. Scale bars, 10 μm. (J) Co-localization efficiencies of Galectin-3-positive GAS and GcAV were calculated as the percentage of the total number of Galectin-3-positive GAS. HeLa cells stably expressing GFP-LC3 were transfected with either FLAG-control, -Bcl-2, or–Bcl-xL, and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 Galectin-3-positive GAS and each percentage represents the mean value ± SD from three independent experiments. * P < 0.05. (K) Confocal microscopic images of LC3-positive GAS in Galectin-3-positive GAS. Insets show expansion of the boxed areas. Scale bars, 10 μm.

Fig 2

Fig 2. Endogenous Bcl-xL regulates GAS internalization and autophagosome-lysosome fusion during GAS infection.

(A) Invasion rate of GAS in Bcl-xL KO cells. Both wild-type and Bcl-xL KO cells were infected with GAS (MOI = 100). At 0.5 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 0.5 h post-infection”. Data are representative of ≥ three independent experiments. * P < 0.05. (B) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Bcl-xL KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. ** P < 0.01. (C) Quantification of the number of GcAV per GcAV-positive cell. Wild-type and Bcl-xL KO cells stably expressing GFP-LC3 were infected and treated as in (B). The data shown represent results from >50 GcAV-positive cells in terms of the mean value ± SD from three independent experiments. * P < 0.05. (D) Confocal microscopic images of GcAV in Bcl-xL KO cells. White arrowheads show GcAV. Scale bars, 10 μm. (E) The accumulation of LC3-II during GAS infection. Wild-type and Bcl-xL KO cells were cultured and infected with GAS (MOI = 100) for 4 h. Expression of LC3 was analyzed by western blotting using anti-LC3 antibody. (F) The number of cells containing Galectin-3-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Bcl-xL KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for indicated times. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. * P < 0.05. (G) Confocal microscopic images of Galectin-3-positive GAS at 1 h post-infection. White arrow heads show Galectin-3-positive GAS. Scale bars, 10 μm. (H) Co-localization efficiencies of Galectin-3-positive GAS and GcAV were calculated as the percentage of total number of Galectin-3-positive GAS. Wild-type and Bcl-xL KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 Galectin-3-positive GAS and each percentage represents the mean value ± SD from three independent experiments. (I) Confocal microscopic images of LC3-positive GAS in Galectin-3-positive GAS in Bcl-xL KO cells. Insets show expansion of the boxed areas. Scale bars, 10 μm. (J) Co-localization efficiencies of GcAV and lysosomes in Bcl-xL KO cells. Wild-type and Bcl-xL KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for indicated times. Cells were then immunostained with an anti-LAMP1 antibody. Cellular and bacterial DNA was stained with DAPI. Co-localization efficiencies of GcAV and LAMP1 were calculated as the percentage of total number of GcAV. The data shown represent results of >50 GcAVs and each percentage represents the mean value ± SD from three independent experiments. * P < 0.05. (K) Confocal microscopic images of GcAV associated with lysosomes in Bcl-xL KO cells. White arrows show autophagosomes, white arrow heads show autolysosomes. Scale bars, 10 μm. (L) Intracellular survival rate of GAS in Bcl-xL KO cells. Both wild-type and Bcl-xL KO cells were infected and treated as in (A). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥three independent experiments.

Fig 3

Fig 3. Knockout of Beclin 1 inhibits GAS internalization.

(A) Invasion rate of GAS in Beclin 1 KO cells. Both wild-type and Beclin 1 KO cells were infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. ** P < 0.01. (B) The number of cells containing Galectin-3-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for the indicated times. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. * P < 0.05, ** _P_ < 0.01. (C) Confocal microscopic images of Galectin-3-positive GAS at 4 h after infection. White arrow heads show Galectin-3-positive GAS. Scale bars, 10 μm. (D) Co-localization efficiencies of Galectin-3-positive GAS and GcAV were calculated as the percentage of total number of Galectin-3-positive GAS. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 Galectin-3-positive GAS and each percentage represents the mean value ± SD from three independent experiments. (E) Confocal microscopic images of LC3-positive GAS in Galectin-3-positive GAS in Beclin 1 KO cells. Insets show expansion of the boxed areas. Scale bars, 10 μm. (F) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. (G) Confocal microscopic images of GcAV in Beclin 1 KO cells. White arrowheads show GcAVs. Scale bars, 10 μm. (H) The accumulation of LC3-II in Beclin 1 KO cells. Wild-type and Beclin 1 KO cells were infected with GAS (MOI = 100) for 4 h. Expression of LC3 was analyzed by western blotting using anti-LC3 antibody. (I) Co-localization efficiencies of GcAV and lysosomes in Beclin 1 KO cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-LAMP1 antibody. Cellular and bacterial DNA was stained with DAPI. Co-localization efficiencies of GcAV and LAMP1 were calculated as the percentage of total number of GcAV. The data shown represent results of >50 GcAVs and each percentage represents the mean value ± SD from three independent experiments. (J) Confocal microscopic images of GcAV associated with lysosomes in Beclin 1 KO cells. Insets show expansion of the boxed areas. Scale bars, 10 μm. (K) Survival rate of GAS in Beclin 1 KO cells. Both wild-type and Beclin 1 KO cells were infected and treated as in (A). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments.

Fig 4

Fig 4. UVRAG but not Atg14 regulates GAS internalization.

(A) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA or Atg14 siRNA and infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. (B) Confocal microscopic images of GcAV in Atg14L knockdown cells. White arrowheads show GcAVs. Scale bars, 10 μm. (C) Invasion rate of GAS in Atg14L knockdown cells. HeLa cells were transfected with a control siRNA or Atg14 siRNA and infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. (D) Intracellular survival rate of GAS in Atg14L knockdown cells. HeLa cells were transfected and infected as in (C). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments. (E) UVRAG interacts with Bcl-xL. HEK293T cells transfected with FLAG-control or -Bcl-xL and EmGFP-UVRAG and cultured under nutrient-rich and starvation conditions for 2 h, or were infected with GAS for 4 h, and then subjected to immunoprecipitations with an anti-FLAG antibody. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP antibody. (F) Invasion rate of GAS in UVRAG-overexpressing Beclin 1 KO cells. Wild-type HeLa cells transfected with FLAG-control and Beclin 1 KO cells transfected with FLAG-control or FLAG-UVRAG were infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. * P < 0.05. ** P < 0.01. (G) Intracellular survival rate of GAS in UVRAG-overexpressing Beclin 1 KO cells. Wild-type HeLa cells and Beclin 1 KO cells were transfected and infected as in (F). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments.

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