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. 2018 Nov 14;8:403. doi: 10.3389/fcimb.2018.00403

Figure 5.

Figure 5

The NACHT domain of NLRX1 interacts with Beclin 1 and is responsible for inhibition of invasion and autophagy during Group A Streptococcus (GAS) infection. (A) NLRX1 interacts with the Beclin 1 complex through its NACHT domain. HeLa cells were transfected with FLAG-empty vector or FLAG-tagged NLRX1 deletion mutants and EmGFP-Beclin 1, and subjected to immunoprecipitation with an anti-FLAG antibody. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with an anti-GFP antibody. (B) Invasion rate of GAS in HeLa wild-type and NLRX1 KO cells transfected with NLRX1 deletion mutants. **P < 0.01. (C) Confocal microscopic images of EEA1- or Rab7-positive compartments containing GAS in HeLa wild-type and NLRX1 KO cells transfected with NLRX1 deletion mutants. Cells were transfected with EmGFP-Rab7 or immunostained with an anti-EEA1 antibody. Cellular and bacterial DNA was stained with DAPI. Scale bars, 10 μm. (D,E) The number of cells containing EEA1-, or Rab7-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. HeLa wild-type and NLRX1 KO cells transfected with NLRX1 deletion mutants were infected with GAS. The data shown represent results from > 200 infected cells and indicate the mean value ± SD from three independent experiments. *P < 0.05. (F) HeLa wild-type or NLRX1 KO cells transfected with both EmGFP-LC3 and NLRX1 deletion mutants were infected with GAS for 240 min, and stained with an antibody against LAMP1 and then with an Alexa 594-conjugated secondary antibody. Cellular and bacterial DNA was stained with DAPI. Scale bars, 10 μm. (G) Quantification of the number of GcAV-positive HeLa wild-type cells and NLRX1 KO cells transfected with NLRX1 deletion mutants. Data are mean ± SD for 500 GAS-infected cells counted per each experiment (n = 3). *P < 0.05. (H) Colocalization rate of GcAVs with lysosomes in HeLa wild-type and NLRX1 KO cells transfected with NLRX1 deletion mutants. Data are mean ± SD of 200 GAS-infected cells counted per each experiment (n = 3). *P < 0.05. (I) The rate of intracellular survival of GAS in HeLa wild-type and NLRX1 KO cells transfected with NLRX1 deletion mutants. The number of intracellular viable GAS bacteria was determined by colony counting and is presented as the ratio of intracellular live GAS bacteria at 240 min to intracellular GAS bacteria at 120 min. Data are representative of three independent experiments.