title: Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells.
abstract: Group A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells.
  
  
  OBJECTIVE
  Previous reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.
authors.affiliation: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Shiou-Ling
authors.initials: SL
authors.lastname: Lu
authors.affiliation: Department of Nursing, I-Shou University, Kaohsiung, Taiwan.
authors.forename: Chih-Feng
authors.initials: CF
authors.lastname: Kuo
authors.affiliation: Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Hao-Wen
authors.initials: HW
authors.lastname: Chen
authors.affiliation: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Yi-Shuan
authors.initials: YS
authors.lastname: Yang
authors.affiliation: Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Ching-Chuan
authors.initials: CC
authors.lastname: Liu
authors.affiliation: Departments of Microbiology & Immunology and Pediatrics, Dalhousie University, Halifax, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Anderson
authors.affiliation: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan jjwu@mail.ncku.edu.tw yslin1@mail.ncku.edu.tw.
authors.forename: Jiunn-Jong
authors.initials: JJ
authors.lastname: Wu
authors.affiliation: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan jjwu@mail.ncku.edu.tw yslin1@mail.ncku.edu.tw.
authors.forename: Yee-Shin
authors.initials: YS
authors.lastname: Lin
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000382:microbiology*; D004847:Epithelial Cells / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability; D010588:Phagosomes / Q000737:chemistry* / Q000382:microbiology*; D013297:Streptococcus pyogenes / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D013301:Streptolysins / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013301:Streptolysins; D037521:Virulence Factors; C025964:streptolysin O