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,Atsuko Minowa-Nozawa,Chihiro Aikawa,Ichiro Nakagawa,Hirotaka Toh,Takashi Nozawa,Vladimir Trajkovic

doi:10.1371/journal.pone.0170138

Shintaro Nakajima, Atsuko Minowa-Nozawa + Show 5 more

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https://doi.org/10.1371/journal.pone.0170138

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Journal: PloS one	Publication Date: Jan 13, 2017
Citations: 18	License type: CC BY 4.0

Affiliation: Kyoto University

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.

Highlights

Group A Streptococcus (GAS; Streptococcus pyogenes), a clinically important bacterial pathogen, causes mild and severe human diseases [1]
To confirm the inhibitory function of Bcl-2 and Bcl-xL in starvation-induced autophagy, HeLa cells stably expressing green fluorescent protein-fused LC3 (GFP-LC3) were transfected with either empty FLAG control vector (FLAG-Control), FLAG-Bcl-2, or FLAG-Bcl-xL, and the number of lipid-conjugated forms of LC3 (LC3-II) puncta were counted under starvation conditions
Western blotting analysis showed that the conversion ratio of LC3-II/LC3-I in both Bcl-2- and Bcl-xL-overexpressing cells was lower than that of cells transfected with control vector after a 2 h starvation period (S1C Fig)

Summary

Introduction

Group A Streptococcus (GAS; Streptococcus pyogenes), a clinically important bacterial pathogen, causes mild and severe human diseases [1]. Bcl-xL Regulates GAS Internalization and Autophagosome-Lysosome Fusion to eliminate the invading bacterium such as autophagy, an intracellular degradation system and apoptosis for removing infected cells themselves. Apoptosis in response to GAS internalization is triggered by actin rearrangement and small GTPase Rac1-mediated ROS production and prevents the spread of GAS infection through programmed suicide [2, 3], and this apoptotic cell death is inhibited by the overexpression of Bcl-2 [2]. GAS strains are classified under serotypes associated with human disease. Strain JRS4 is a serotype M6 clone of GAS and is degraded by autophagy efficiently, so this infection model is widely used to prove the induction mechanism of GAS-induced selective autophagy. GAS JRS4 is not representative of all GAS strains because it is much less prevalent associated with human disease [6]. Barnett et al reported that the globally disseminated serotype M1T1 clone of GAS (strain 5448) can avoid autophagy pathway [7]

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