The autophagic process triggered by <i>Serratia</i>.

<p><b>A) Autophagy triggered by </b><b><i>Serratia</i></b><b> requires Atg5</b>. MEF <i>Atg5</i>^+/+ and MEF <i>Atg5</i>^−/− cells were infected for 60 min with wild-type <i>S. marcescenes</i>. Thereafter, extracellular bacteria were eliminated with gentamicin. At the indicated times, cells were washed with PBS and lysed with 0.05% Triton X-100. The CFUs on LB agar plates were determined. Units were calculated relative to CFU recovered from MEF <i>Atg5</i>^+/+ cells at 120 min p.i. The media ± S.D. is shown for three independent experiments. <b>B) </b><b><i>S. marcescens</i></b><b> actively induces autophagy from the extracellular media.</b> Overnight <i>S. marcescens</i> cultures were incubated 60 min in LB without antibiotic or supplemented with 20 or 100 µg/ml chloramphenicol (bacteriostatic condition). Subsequently, CHO-EGFP-LC3 cells were infected with bacteria and then extracellular bacteria were eliminated with gentamicin (bacteriostatic treatment was maintained throughout the invasion assay). At 180 min p.i, cells were fixed and bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Cy3. Finally, the percentage of colocalization of vesicles containing <i>Serratia</i> with EGFP-LC3, relative to total infected cells, was determined by fluorescence microscopy. The lower panel shows the percentage of cells, infected and non-infected, with an autophagic phenotype. At least 300 infected cells were counted for each condition. The average ± S.D. is shown for three independent experiments, * and ** p<0.001. <b>C)</b> CHO-EGFP-LC3 cells (green fluorescence) infected with <i>S. marcescens</i> (red fluorescence) untreated (upper panel), treated with 20 µg/ml chloramphenicol (middle panel) or treated with 100 µg/ml chloramphenicol (lower panel) are shown. Bars: 10 µm.