Fig. 1: siRNA screening reveals that Rab8A and Rab41 are critical to maintain the integrity of the autophagolysosomes of GAS.

a, b Screening for Rab GTPases that affect intracellular bacterial viability. HeLa wild-type (WT) (a) or ATG5-knockout (KO) (b) cells transfected with the indicated siRNA were infected with GAS. siNT means non-targeting control siRNA. Colony counting (determination of colony forming unit, CFU) was conducted to determine the number of invading and surviving GAS; the bacterial survival data were calculated as the ratio of “intracellular live GAS at 6 h” to “total intracellular GAS at 2 h”. Data are represented as individual values (circles) and mean ± standard error of the mean (SEM) (n = 3 biologically independent experiments). One-way analysis of variance (ANOVA), Dunnett’s test. Red P values indicated a significant increase and blue ones means a significant decrease. c–e HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS for 4 h, then fixed and immunostained for endogenous LAMP1 (red). Cellular and bacterial DNA were stained with DAPI (cyan). c Shown representative microscopic images are single slice. Scale bar, 10 μm. (d) GcAV formation was quantified as the percentages of cells with GcAVs. Data are represented as individual values and mean ± SEM of more than three independent experiments (200 <cells examined in each independent experiment). One-way ANOVA, Dunnett’s test. e The proportion of the LC3 signal (GcAVs) that colocalized with LAMP1 was quantified by Mander’s coefficient M1. Data are represented as individual values (circles) and mean ± SEM (30 < cells examined over three independent experiments). One-way ANOVA, Dunnett’s test. f, g HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNA, then infected with GAS for the indicated time, and finally fixed. Cells were stained with LysoTracker Red 30 min prior to fixation. Shown confocal images are single slice (f) and the LysoTracker intensity inside GcAVs was quantified (20 < cells examined in each three independent experiments) (g). Scale bar, 10 μm. Data represent mean ± SEM. One-way ANOVA, Dunnett’s test. h, i HeLa WT, Rab8A KO, Rab41 KO cells were infected with GAS for 4 h, and finally fixed. Cells were stained with LysoTracker Red 30 min prior to fixation. Shown confocal images are single slice (h) and the LysoTracker intensity inside GcAVs was quantified (i). Scale bar, 10 μm. Data are individual values (circles) and mean ± SEM (50 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. J HeLa WT, Rab8A KO, and Rab41 KO cells were infected with GAS. CFU was determined to quantify the number of invading and surviving GAS at 2 h and 6 h after infection, respectively. Data are individual values (three independent experiments) and mean ± SEM. One-way ANOVA, Dunnett’s test. Source data are provided as a Source Data file.