Serratia Invasion to Epithelial Cells labeled with Alexa Fluor 647. The percentages of colocalization of bacteria with EGFP-LC3 and/or with DQ-BSA were determined by fluorescence microscopy. At least 300 infected cells were counted for each condition. The average 6 S.D. is shown for two independent experiments, *and **p,0.001. D) Representative confocal laser captured images of CHO-EGFP-LC3 cells (green fluorescence, d) pre-incubated with DQ-BSA (red fluorescence,c) and infected with S. marcescens (blue fluorescence, b) at 360 min p.i. are shown. Inset image (f) shows higher magnification of the boxed area in the merged image (e) and highlights a non-degradative, autophagic SeCV. Arrows point at degradative vesicles. Bars: 10 mm. doi:10.1371/journal.pone.0024054.g004 process. We also analyzed the effect of the addition of one of the best-characterized autophagy inhibitors, wortmannin. When we induced autophagy by starving the cells in EBSS medium (see Materials and Methods for details), no increase in intracellular CFUs was detected either at 120 or 360 min p.i. when compared to non-starved cells (Fig. 5B, aMEM and Starv). On the other hand, when wortmannin was used to block autophagy by pre- treatment of the non-starved or starved cells with the inhibitor, intracellular CFUs were drastically diminished in comparison to non-treated cells, both at 120 and 360 min p.i. (Fig. 5B, aMEM + Wn and Starv + Wn). Microscopical inspection of the CHO cells assayed revealed that, when wortmannin was used, the scarce intracellular compartments observed harboring Serratia were packed with bacteria and decorated with LC3, exhibiting identical phenotype to the SeCVs observed in the profusely invaded, non- treated cells (Fig. 8, compare a-MEM, panels a–d to a-MEM + Wn, panels a–h, the percentage of CHO cells invaded with Serratia that displayed each phenotype is indicated to the right of the images). Representative starved invaded cells are shown either when cells were treated with or without wortmannin (Fig. 8, Starvation, panels a–d, and Starvation + Wn, panels a–h, respectively). It is worth pointing out that the control assay showing the inhibition of autophagy induction by wortmannin pre-treatment in starved non-invaded cells corroborated the expected action of the inhibitor examined microscopically and by immunodetection of LC3 conversion (Fig. 6, compare control assay, a-MEM, Starv and Starv+Wn lanes, and Fig. 8, non- invaded cells, compare panels a–c). These results indicate that treatment with the inhibitor diminished the invasion process, although it was not able to prevent the induction of the autophagic process promoted by Serratia as well as the formation of large LC3- decorated SeCV in the few invaded cells observed. Wortmannin suppresses canonical autophagy by its phosphati- dylinositol 3-kinase (PI3K) inhibitory activity [9], therefore, we wondered whether this inhibitor was somehow preventing Serratia entry into the CHO cells. To examine this possibility, we added wortmannin after allowing internalization of Serratia into CHO cells (60 min p.i.); no detectable difference was obtained in the invasion percentage at 360 min p.i. with the one obtained in non- treated cells (to avoid inactivation of the inhibitor through the assay, fresh medium with wortmannin was replenished at 210 min p.i., Fig.5B, aMEM + Wn p.i.). In accordance with these results, the LC3-II band was detected in all Serratia invaded cells irrespective of the treatment applied (i.e. in presence or absence of wortmannin, in either starved or non starved cells; Fig. 6). A lower LC3-II/LC3-I ratio was detected in those lanes that correspond to wortmannin treatment when compared to those from non-treated, invaded cells, reflecting the reduced number of LC3–recruiting SeCVs (Fig. 6, and see also Fig. 5B and Fig. 8). Together, these results show that wortmannin is unable to inhibit the autophagic process triggered by Serratia, although it can hinder the bacterial internalization process. Atg5 (autophagy related factor 5) is essential for the early stages of canonical autophagosome formation, previous to the recruitment of LC3. To further explore the autophagic cascade activated by Serratia, wild-type (MEF Atg5+/+) and Atg5 knock-out (MEF Atg52/2) cells were used for the bacterial infection assays. The autophagic phenotype was observed in wild-type RFP-LC3-MEF cells, while no punctate pattern was detected in RFP-LC3-MEF Atg52/2 cells challenged with Serratia (see Fig. 3, panels c–f). In addition, whereas in Atg5+/+ cells bacteria augmented its intracellular population in the 120–360 min lapse p.i., no changes were observed for the CFUs recovered at this time period from the knock-out cells (Fig. 9A). In sum, these results indicate the involvement of Atg5, but not of PI3K, in the autophagic route induced by Serratia, and suggest that the induction of a non-canonical autophagic pathway is required for the development of the invasion process in epithelial cells. Autophagy is activated by a bacterial factor from outside the host cell We next analyzed whether active bacterial protein synthesis is involved in the invasion process. With this aim, we halted de novo protein synthesis by the addition of chloramphenicol (Cm) to the bacterial inoculum, maintaining the action of the bacteriostatic agent throughout the invasion assay. We verified that Cm has bacteriostatic but not bactericidal action at the concentrations and time-lapses used in the assay (determined by recovered CFUs counted before and after Cm treatment of the bacterial cultures, data not shown). The treatment with increasing concentrations of the antibiotic drastically reduced Serratia intracellular CFUs (4-fold reduction when 100 mg/ml Cm were used), as well as colocaliza- tion of the bacteria with EGFP-LC3 (7-fold decrease when 100 mg/ml Cm were used) when compared with non-treated bacteria (Figs. 9B and 9C, invasion and LC3 co-localization percentages were calculated relative to total scored cells). Remarkably, when we scored the total amount of cells that showed LC3 puncta (Fig. 9B, lower inset) it became evident that not only invaded but also non-invaded cells displayed this autophagic phenotype (also compare phenotypes of invaded to non-invaded cells, Fig. 9C, untreated cells). In addition, when the assay was carried out using heat-killed bacteria (as detailed in Materials and Methods) no differences from non-invaded cells were detected in the intracellular CFUs or in the autophagic phenotype (not shown). These results indicate that metabolically active bacteria are required for an efficient invasion process and for autophagy induction. Additionally, they strongly suggest that the autophagic response can be promoted by Serratia by a heat- labile factor from outside the host cell. Flagellar expression is involved in the early steps of bacterial interaction with the host cell In a variety of bacterial pathogens such as Burkholderia, Aeromonas or enterotoxigenic E. coli the flagellum was shown to be involved in initial adherence to the target cell [28–30]. In search for bacterial factors that could be involved in the first steps of Serratia extracellular interaction with the epithelial cells, we analyzed a Serratia flhD mutant strain, impeded in flagellum biogenesis. In S. marcescens the flagellum biogenesis depends on a regulatory cascade mastered by the FlhD2C2 transcriptional factor. FlhD2C2- modulated cascade also couples flagellar biogenesis to the expression of PhlA, a phospholipase of unknown function that is exported through the flagellar secretome [31]. As shown in Fig. 10A, a flhD mutant strain was severely impaired both in adherence (8-fold reduction) and in invasion (4-fold or 8-fold PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e24054