Serratia Invasion to Epithelial Cells Figure 9. The autophagic process triggered by Serratia. A) Autophagy triggered by Serratia requires Atg5. MEF Atg5+/+ and MEF Atg52/2 cells were infected for 60 min with wild-type S. marcescenes. 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 Atg5+/+ cells at 120 min p.i. The media 6 S.D. is shown for three independent experiments. B) S. marcescens actively induces autophagy from the extracellular media. Overnight S. marcescens cultures were incubated 60 min in LB without antibiotic or supple- mented with 20 or 100 mg/ml chloramphenicol (bacteriostatic condi- tion). 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-Serratia coupled with a secondary antibody labeled with Cy3. Finally, the percentage of colocalization of vesicles containing Serratia 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 6 S.D. is shown for three independent experiments, * and ** p,0.001. C) CHO-EGFP-LC3 cells (green fluorescence) infected with S. marcescens (red fluorescence) untreated (upper panel), treated with 20 mg/ml chloramphenicol (middle panel) or treated with 100 mg/ml chloram- phenicol (lower panel) are shown. Bars: 10 mm. doi:10.1371/journal.pone.0024054.g009 vacuole) were observed at early times after infection, while large compartments packed with bacteria (.20 bacteria/vacuole) were predominantly visualized at later infection times. These results indicate that Serratia manages to counteract or delay the eradication processes that are expected to be physiologically triggered inside the host cell to get rid of the intruder. Upon cell invasion, autophagy is a key component of the innate host defence that intracellular bacteria must deal with. However, Figure 10. S. marcescens flagellum expression is involved in adhesion to CHO cells. A) CHO cells were infected for 60 min with S. marcescens wild-type, flhD and flhD/pflhD strains (dark gray, light gray or black bars, respectively). Cells were washed with PBS and lysed with 0.05% Triton X-100 (left graphic); or incubated 120 o 240 min p.i with medium supplemented with gentamicin to eliminate extracellular bacteria. Finally, cells were lysed with 0.05% Triton X-100 (right graphic) and the CFUs were determined on LB agar plates. Adherence and intracellular CFUs percentages were calculated relative to initial inoculums. The average 6 S.D. is shown for four independent experiments, * p,0.001. B) CHO-EGFP-LC3 cells (green fluorescence) were infected with S. marcescens wild-type (b and f), flhD (c and g) and phlA (d and h) strains. Non infected cells are shown as control (a and e). Then, cells were not treated (a-d) or treated with gentamicin (e-h) for 120 min. Cells were fixed and bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Cy3 (red fluorescence). Representative confocal Z-slices are shown. Bars: 10 mm. doi:10.1371/journal.pone.0024054.g010 numerous pathogens such as Coxiella burnetii [23,32], Staphylococcus aureus [33], or Group A Streptococcus (GAS) [10] display sophisticated strategies to evade, hamper or even manipulate the normal progression of the autophagic process to establish a persistent infection. Remarkably, there is accumulating evidence that, within an invasion event, populations of internalized bacteria can manage to differentially interact with the vesicular traffic pathways, resulting in diverse destinies. This is nicely exemplified by the work of Birgmingham et al. [12] where they provide evidences that at least four distinct Salmonella enterica intracellular populations coexist: bacteria that reside and proliferate in a modified endosomal compartment avoiding maturation; a population in which the Salmonella-containing vacuole (SCV) is damaged by action of bacterial effectors either promoting progression to degrading endolysosomes or being targeted by the autophagy system, and a last population that is able to escape from the damaged SCV to the cytosol. PLoS ONE | www.plosone.org 13 August 2011 | Volume 6 | Issue 8 | e24054