Figure 11.

Inhibition of ULK1 suppresses Staphylococcus intracellular replication and host cell death. A, ULK1 was targeted in HeLa cells using shRNA (shULK1). WT HeLa or shULK1 cells were treated to bafilomycin A1 (10 nm) ± starvation (St+Baf) in EBSS for 2 h. Cell lysates were analyzed and quantified for LC3B lipidation as in Fig. 1. n = 2 ± range. B, WT HeLa or shULK1 cells were infected with varying multiplicities of infection of S. aureus NCTC8325 and assayed for cell viability as in Fig. 6. Averages of n = 3 ± S.D. ***, p < 0.001; *, p < 0.05 by unpaired t test: shULK1 versus HeLa. C, WT HeLa or shULK1 cells were infected with 1:100 diluted S. enterica sv. typhimurium and incubated for 72 h before staining for cell viability. Averages of n = 3 ± S.D. **, p < 0.01 by unpaired t test. D, HEK293A cells were infected with NCTC8325 (200 m.o.i.). At the time of infection, ULK1 inhibitors MRT68921 or SBI-0206965 were added at 1 or 10 μm. Gentamycin (0.05 mg/ml) was added 1 h post-infection. Cells were incubated for 48 h and assayed for cell viability as in Fig. 6. Averages of n = 3 ± S.D. E, HEK293A cells were infected with 1:100 diluted Salmonella as in C. At the time of infection, ULK1 inhibitors were added at 10 μm. Gentamycin was added 50 min post-infection. Cells were incubated for 24 h and assayed for cell viability. Shown are averages from n = 3 ± S.D. D and E, ***, p < 0.0001; **, p < 0.001; *, p < 0.05 by unpaired t test: infected cells comparing (+) versus (−) ULK1 inhibitor. F, Staphylococcus NCTC8325 or S. enterica sv. typhimurium cultures were diluted 1:100 and grown in the presence of ULK1 inhibitors (10 μm) or gentamicin (0.05 mg/ml). Averages of n = 3 ± S.D. G and H, HeLa cells were incubated in EBSS for 2 h in the presence or absence of ULK1 inhibitors (10 μm). After fixation, cells were stained with anti-LC3B antibody. Scale bars, 10 μm. Average LC3B(+) puncta/cell from n = 40 cells per condition ± S.E. **, p < 0.001 versus starved no-drug control by ANOVA and Tukey's post test. I and J, HeLa/GFP–p62 stable cells were infected with NCTC8325 (100 m.o.i.) for 3 h in the presence or absence of ULK1 inhibitors (10 μm). After fixation, cells were stained with anti-protein A antibody. Scale bars, 10 μm. Arrow, large-sized p62 aggregates induced by NCTC8325 infection. Average large GFP–p62 aggregates/cell from n = 20 cells per condition ± S.E. Data are representative of two experiments. K, HEK293A cells were infected with NCTC8325 (100 m.o.i.) in the presence of MRT68921 (MRT) (1 μm) or SBI-0206965 (SBI) (10 μm). Gentamycin (0.05 mg/ml) was added 1 h post-infection. Cells were further incubated 3, 6 or 24 h before lysis. Bacterial titers in cell lysates were measured by growth on solid media. Average CFU from 3- and 6-h time points (n = 3 ± S.D.) are shown. Bacterial growth changes color of phenol red in bacterial plates. No host cells remained 24 h after infection in the absence of ULK1 inhibitor. Uninf., uninfected.