FIG 3.

Macrophages expressing Mtb PPE51 inhibited autophagy and enhanced intracellular bacterial survival and phagocytosis. (A) THP-1 cells transduced with empty vector (Mϕ::EV) or HA-tagged PPE51 (Mϕ::51) were induced for 16 h with aTCN at 0, 125, 250, 500, or 1,000 ng/mL. Lysates were analyzed by Western blotting using an anti-HA antibody to detect PPE51 as a 40-kDa band. (B) Immunoblot of LC3B accumulation in Mϕ::EV and Mϕ::51 at 3 h after treatment with 10 μM Torin-1 or Msm (MOI 10) or uninfected (UI) macrophages. A representative blot of 3 independent assays is shown. (C) The densitometric summary analysis was calculated by LC3B-II density normalized to GAPDH density. (D) Msm survival was determined in Mϕ::EV and Mϕ::51 at 20 h postinfection. (E) Msm uptake by Mϕ::EV and Mϕ::51 (MOI 10) at 4 h postinfection was calculated by the ratio of intracellular bacteria to the inoculum. (F) Msm uptake by Mϕ::EV and Mϕ::51 (MOI, 10) with or without 100 ng/mL Pam₃CSK₄ at 4 h postinfection was determined. All graphs represent one of three independent experiments, with data expressed as means and SD. Significance was calculated by two-way ANOVA (corrected by Dunnett's test for multiple comparisons (C and F) or unpaired t test (D and E). *, P < 0.05; **, P < 0.01; ***, P < 0.001.