Figure 1. S. marcescens invades and replicates inside non- phagocytic cells. A) CHO cells or B) CHO, HeLa, MEF Atg5+/+ and T24 cells were infected for 60 min with wild-type S. marcescens and then 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 were determined on LB agar plates, percentages were calculated relative to CFUs in the inoculum. In the plot shown in (B) fold change in CFUs were calculated relative to the values obtained at 120 min p.i. The average 6 S.D. for two independent experiments is shown (* p,0.001). doi:10.1371/journal.pone.0024054.g001 An identical invasion (gentamicin protection) assay to the one performed with CHO cells was also carried out using HeLa, MEF or T24 epithelial cell lines. Intracellular CFUs were determined at 120, 240 and 360 min p.i. and CFUs fold change relative to the value obtained at 120 min p.i was plotted. As shown in Fig. 1B, Serratia invasion and intracellular replication was sustained by the four cell lines assayed. Upon 360 min p.i. T24 cells microscop- ically showed signals of cellular damage (rounding and detaching, not shown) that correlated with the intracellular CFUs decrease observed at this time point. Together, these results indicate that the capacity of S. marcescens to invade and proliferate intracellularly is not restricted to one specific epithelial cell type. The autophagy system targets the Serratia-containing vacuole To characterize the intracellular structures that harbor Serratia, that we will hereafter call Serratia-containing vacuoles (SeCVs), and address whether they interact with the autophagy pathway, we used a stably transfected CHO cell line expressing the EGFP-LC3 fusion protein. We previously verified that the Serratia infection Serratia Invasion to Epithelial Cells time-course using EGFP-LC3-expressing CHO cells was identical to the one determined for non-transfected CHO cells (not shown). Fig. 2A shows representative images obtained from colocalization assays at 120 or 360 min p.i.; in this assay SeCVs were observed decorated by EGFP-LC3. Colocalization was calculated as the ratio between the number of SeCVs that colocalize with the fluorescently labeled marker and the total number of infected cells. After bacterial entry, a gradual increase in Serratia colocalization with EGFP-LC3 was detected, reaching 47.962.9% colocalization 180 min p.i., with no detectable decay up to 360 min p.i. (Fig. 2C). Maturation of autophagosomes is a multi-step process that involves fusion events with vesicles originating from the endo/ lysosomal compartment. Rab7 is a GTPase involved in coordi- nating tethering/docking of vesicles to late endosomes/lysosomes, either in homotypic or heterotypic fusion events [27]. In this context, it has been shown that a functional Rab7 GTPase is targeted to autophagic vacuoles, being its acquisition involved in the maturation of autophagosomes engaged in bacterial elimina- tion [16–18]. To further characterize the autophagic-like vacuoles associated with internalized Serratia, we tracked bacterial coloca- lization with Rab7 following infection of transiently transfected CHO cells overexpressing EGFP-Rab7. Representative images of Serratia colocalization with Rab7 acquired at 120 min and 360 min p.i. are shown in Fig. 2B. Rab7 can be observed decorating the SeCVs. Colocalization with Rab7 increased after bacterial internalization reaching 53.7 6 2.0% at 180 min p.i., a score that was in average sustained up to 360 min p.i. (Fig. 2C). It is worth highlighting that a comparable acquisition kinetic pattern and percentages of colocalization were obtained for LC3 and Rab7. This observation strongly suggests that both markers are recruited to the Serratia-containing compartment early on after bacterial internalization and remain associated throughout its progression to become a large crowded vacuole. In addition, the induction of the autophagic process evidenced microscopically as the punctate LC3 pattern was also observed when HeLa, MEF or T24 cells transiently expressing ERFP-LC3 were challenged with Serratia following the same infection protocol as used for CHO-EGFP-LC3 cells without addition of gentamicin (Fig. 3, panels a–d and g–h). This result demonstrates that the autophagic induction in response to Serratia invasion is not limited to CHO cells. SeCVs population is predominantly composed of non- acidic, non-degradative autophagic compartments During the course of its maturation process, a canonical autophagocytic vacuole fuses with compartments from the endo/ lysosomal pathway, undergoing acidification and the acquisition of hydrolytic enzymes that become active in this environment. Consequently, we analyzed whether the LC3-decorated SeCVs underwent acidification. For this purpose, Lysotracker, a fluores- cent marker that labels acidic compartments, was added to the cell cultures 60 min p.i. At 240 min p.i. co-localization of SeCVs and LC3 was estimated in 66.2%. However, 60% were LC3-decorated but did not co-localize with Lysotracker, indicating that they were non- acidic autophagic compartments. An average of 10% were found to be SeCVs that were labeled by the acidotropic marker, while they did not colocalize with LC3 (quantitation of the Serratia- containing vesicles colocalization with EGFP-LC3 relative to total infected cells and Lysotracker assay at 240 and 360 min p.i. is given in Fig. 4A; representative captured images from 360 min p.i. of the colocalization assay, indicating both acidic –arrows- and non-acidic vacuoles –inset-, are shown in Fig. 4B). PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e24054