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. 2019 Oct 1;10(5):e02148-19. doi: 10.1128/mBio.02148-19

FIG 1.

FIG 1

GAS-containing LC3-positive compartments show characteristics of LAP which cause bacterial multiplication in HMEC-1 cells. HMEC-1 cells were infected with GAS at an MOI of 5 for 30 min, and gentamicin was added to kill extracellular bacteria. (A) Cells stably expressing LC3-GFP were fixed and stained with Hoechst for cell nuclear and bacterial DNA staining at 1 h postinfection. Stained specimens were examined by confocal microscopy. After marking the location of cells of interest, the specimen was further fixed and embedded, followed by TEM to analyze the membrane structure of the LC3-positive GAS-containing vacuole in the same cell. (B to D) Cells were collected at 1 h postinfection and stained with anti-NOX2 (B), anti-ULK1 (C), and anti-LC3 antibodies. DAPI was used for cell nuclear and bacterial DNA staining. Images were obtained by confocal microscopy. LC3-positive GAS surrounded with NOX2 or ULK1 (D) was relative to total intracellular LC3-positive GAS. Data represent the means ± SD from three independent experiments, and over 100 cells were counted in each sample. (E and F) Cells were pretreated with diphenyleneiodonium (DPI) (E) and N-acetyl-l-cysteine (NAC) (F) for 1 h and infected with GAS at an MOI of 5 for 30 min after removal of the above-named inhibitors and washing with PBS for 2 times. Then gentamicin was added to kill extracellular bacteria in the presence or absence of DPI or NAC. Cells were collected at 1 and 6 h postinfection. The colony forming assay was performed to quantify the amounts of bacteria, and the fold values of GAS replication were calculated by normalizing the GAS count at 6 h with that at 1 h postinfection. Data represent the means ± SD from three independent experiments. ***, P < 0.001 compared to GAS-infected group without inhibitor. (G and H) NOX2 (G) and ULK1 (H) expression was silenced in HMEC-1 cells by using three lentivirus-based shRNAs. Luciferase shRNA (shLuc) was used as a negative control. The expression of NOX2 and ULK1 was detected by Western blot analysis (upper panel). Cells were further infected with GAS at an MOI of 5 for 30 min, and gentamicin was added to kill extracellular bacteria. The colony forming assay was performed to quantify the numbers of bacteria, and the fold values of GAS replication were calculated by normalizing the GAS count at 6 h with that at 1 h postinfection (lower panel). Data represent the means ± SD from three independent experiments. **, P < 0.01 compared to shLuc-transfected cells. ns, not significant.