FIG 1.

S. marcescens ShlA induces autophagy in CHO cells. (A) S. marcescens or E. coli strains were grown ON, without shaking, at 30°C or 37°C, respectively. The saturated cultures were centrifuged, and the supernatant was filtered with 0.2 μm acetate-cellulose filters, precipitated with 12% trichloroacetic acid, and centrifuged for 30 min at 30,000 × g. The precipitated proteins were resuspended in protein sample buffer and loaded onto SDS-PAGE gels. Hemolysin levels were determined by Western blotting using S. marcescens anti-ShlA rabbit polyclonal antibodies. To determine the hemolytic activity, 200 μl of the bacterial suspension was incubated with 200 μl of a human red blood cell suspension for 1 h at 30°C. The amount of released hemoglobin in the supernatant was determined by measuring absorbance at 562 nm. The absorbance of each sample was normalized to the OD₆₀₀ of the corresponding culture. Relative activity was plotted by considering the wild-type strain hemolytic activity value as the unit. The averages and standard deviations (SD) are shown for four independent experiments performed in duplicate in each case. Statistical analysis was performed using one-way ANOVA and the Tukey-Kramer multiple-comparison test. **, P < 0.01; ***, P < 0.001; statistically significantly different from wild-type S. marcescens. At the top are shown representative images of the immunodetection assay; each lane corresponds to the sample analyzed for hemolytic activity in the plot below. (B and C) CHO-EGFP-LC3 cells were infected with wild-type S. marcescens (c and d) or with noninvasive E. coli W3110/pT7 (e and f), E. coli W3110/pES14 (g and h), E. coli W3110/pBB2 (i and j), or E. coli W3110/pphlAB (k and l) and fixed at 120 min p.i. (B) or treated with gentamicin and fixed at 300 min p.i. (C). Noninfected cells are shown as a control (a and b). The cells were visualized by confocal microscopy. Representative differential interference contrast (DIC) and green fluorescence from EGFP-LC3 images are shown. Bars, 10 μm. (D) Quantitative measurement of the autophagy assay. CHO-EGFP-LC3 cells subjected to the assay depicted in panels B and C were visualized by confocal microscopy. At least 300 infected cells were counted for each condition. The averages and SD are shown for three independent experiments performed in duplicate in each case. Statistical analysis was performed using one-way ANOVA and the Tukey-Kramer multiple-comparison test. ***, P < 0.001.