Fig 1: Tollip is required for autophagosome and autophagolysosome formation during GAS infection. (A) Tollip-knockout (KO) cells were generated using CRISPR/Cas9-mediated gene editing. Immunoblotting was used to detect Tollip protein expression in wild-type (WT) and Tollip-knockout cells. (B) HeLa cells transfected with EmGFP-LC3 were infected with GAS, and at 2 hpi, immunofluorescence analysis was performed to examine the formation of bacterium-containing autophagosomes. (C) Cells harboring GAS-containing autophagosomes (left) and cells containing LC3-positive fragments (right) were manually counted using immunofluorescence confocal microscopy. In each experiment, >100 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. P values less than 0.05 were considered to indicate statistical significance, and are marked * for P < 0.05, ** for P < 0.01. (D) LC3-positive fragments in cells were manually counted and examined using confocal microscopy. In each experiment, 100 LC3-positive cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (E) HeLa cells transfected with EmGFP-LC3 were infected with GAS, and at 4 hpi, immunofluorescence analysis was used to examine the localization of EmGFP-LC3 and endogenous LAMP1. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 µm. (F) LAMP1-positive GAS-containing autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, 100 LC3-positive cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (G) The adhesion level of GAS in wild-type and Tollip-knockout HeLa cells. (H) Invasion rate and survival rate of GAS in wild-type and Tollip-knockout HeLa cells.
Fig 2: Model depicting Tollip function in xenophagy. Tollip localizes on the inner surface of the cell membrane by binding to PtdIns3P through its C2 domain. After GAS invasion through endocytosis, Tollip surrounds GAS before GAS damages the endosomal membrane and escapes into the cytosol. After GAS damages the endosomal membrane, galectin-1 and -7 are recruited to the membrane fragments and associate with Tollip, and then other autophagy receptors, including NBR1, TAX1BP1, p62, and NDP52, are recruited.
Fig 3: Tollip is recruited to GAS-containing vacuoles before GAS damages the endosomal membrane. (A) HeLa cells transfected with mCherry-Tollip and EmGFP-LC3 were infected with Group A Streptococcus (GAS) for 4 h, and immunostained with anti-Group A Streptococcus carbohydrate (GAC) to stain GAS. Immunofluorescence analysis was performed to determine the localization of mCherry-Tollip and EmGFP-LC3. Cellular and bacterial DNA were stained with DAPI. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 µm. (B) Percentages of cells containing Tollip-positive GAS and cells containing Tollip-positive autophagosomes; cells were manually counted using immunofluorescence confocal microscopy. In each experiment, >60 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. (C) HeLa cells were transfected with mCherry-Tollip and infected with GAS or SLO-deletion-mutant (?SLO) GAS for 4 h, and then stained for endogenous galectin-3. Immunofluorescence microscopy was used to examine the localization of mCherry-Tollip and endogenous galectin-3. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 µm.
Fig 4: Tollip is required for the recruitment of NBR1, TAX1BP1, and NDP52 to GAS-containing LC3 vacuoles. (A) Wild-type and Tollip-knockout HeLa cells were transfected with EmGFP-LC3 or co-transfected with EmGFP-LC3 and mCherry-NDP52. After infecting with GAS for 4 h, endogenous NBR1, TAX1BP1, and p62 were labeled with antibodies. Insets: enlarged boxed areas; top: EmGFP-LC3; middle: autophagy receptors; bottom: merged image. All images are representative of 3 independent experiments. Scale bar, 10 µm. (B) Percentages of recruitment of autophagy receptors to GAS-containing autophagosome-like vacuoles: NBR1, TAX1BP1, p62, and mCherry-NDP52 (from left to right). In each experiment, >50 LC3-positive vacuoles were evaluated per sample; bars: means ± SD of 3 independent experiments. *P < 0.02, **P < 0.01.
Fig 5: Tollip is involved in the recruitment of galectin-1 and -7 to GAS-containing LC3-vacuoles. (A) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-galectin-7 and mCherry-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the localization of the expressed proteins. (B) EmGFP-galectin-positive autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, >50 GAS-containing LC3-vacuoles were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (C) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was used to examine the localization of endogenous galectin-1 and EmGFP-LC3; galectin-1 was labeled using a rabbit anti-galectin-1 antibody. (D) Cells containing galectin-1-positive GAS-containing autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, >50 GAS-containing autophagosomes were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (E) Wild-type and Tollip-knockout HeLa cells were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the SLO protein expression from GAS. Scale bar, 10 µm. (F) Cells containing SLO-positive GAS were manually counted using immunofluorescence confocal microscopy. In each experiment, >200 GAS-infected cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (G) Tollip interacts with galectin-7. HeLa cells were co-transfected with EmGFP-galectin-7 and FLAG-Tollip for 48 h, after which immunoprecipitation was performed using anti-FLAG. The immunoprecipitates were immunoblotted with anti-GFP to detect EmGFP-galectin-7. Experiments were repeated more than three times. *P < 0.05.
Supplier Page from Abcam for Anti-Tollip antibody