Figure 3.

The NLRX1–Beclin 1–UVARAG complex regulates Group A Streptococcus (GAS) invasion by endocytosis. (A) NLRX1 interacts with the Beclin 1 complex. HeLa cells were transfected with EmGFP-empty vector or EmGFP-tagged Beclin 1 complex proteins (Beclin1, Vps34/PI3KC3, and Atg14) with FLAG-NLRX1. Similarly, cells were transfected with FLAG-empty vector or FLAG-tagged UVARG with EmGFP-NLRX1. Cell lysates were subjected to immunoprecipitations with anti-FLAG or—GFP (for UVRAG) antibody. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP or—FLAG (for UVRAG) antibody. (B,C) Invasion rate of GAS in HeLa wild-type, Beclin 1 KO, UVRAG KO, Rubicon KO, Vps34/PI3KC3 KD, and Atg14 KD cells. ^**P < 0.01. (D,F) Confocal microscopic images of EEA1 (D)- or Rab7 (F)-positive compartments containing GAS in HeLa wild-type, Beclin1, and UVRAG KO cells. Cells were transfected with EmGFP-Rab7, or immunostained with an anti-EEA1 antibody to visualize each endosomal marker. Cellular and bacterial DNA was stained with DAPI (blue). Scale bars, 10 μm. (E,G) The number of cells containing EEA1 (D)-, or Rab7 (F)-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. HeLa wild-type, Beclin 1, and UVRAG KO cells were infected with GAS for the indicated times. The data shown represent results from > 200 infected cells and indicate the mean value ± SD from three independent experiments. ^**P < 0.01.