To understand the events that allow Serratia to establish an intracellular replicative niche, we explored the interaction of this bacterium with the autophagic pathway. The induction of the autophagic process, evidenced by EGFP-LC3 recruitment from the cytoplasm to the membrane of the autophagosomes, was detectable as early as 30 min p.i. EGFP-LC3 colocalization with Serratia raised from 10% to 55% in the 45 min-6 h post-infection period. LC3 I to lipidated-LC3II conversion triggered by the invasion process was also corroborated by immunodetection assays. By confocal microscopy an autophagic phenotype was also observed in other epithelial cell lines challenged with Serratia, showing that it is not a response only restricted to CHO cells. The intracellular bacteria targeted by autophagy are expected to be removed by degradation when the autophagic vesicle containing the unwanted cargo is delivered to the lysosome, forming the autophagolysosome. This process involves a stepwise progression in which the initial phagophore fuses with endocytic vesicles. Rab GTPases are essential in regulating the generation, transport, recognition and fusion of vesicles. In particular, the GTPase Rab7, that catalyzes the homotypic fusion of late endocytic vacuoles, has been demonstrated to be required not only for the maturation of canonical autophagosomes [16,17] but also in the formation of non-canonical autophagosomes that surround intracellular pathogens such as GAS [18]. In the Serratia- containing vacuoles (SeCV), Rab7 followed an acquisition kinetic similar to LC3 recruitment. Rab7 acquisition was detected early after internalization and decorated the SeCV at all times analyzed along the invasion assay. This correlation strongly suggests that this GTPase participates in the biogenesis of the autophagosome- like vacuole that sustains Serratia intracellular life. We can conjecture that the large bacteria-packed autophagosomes ob- served mostly at prolonged times post-infection could be the result not only of active intravacuole bacterial replication but also of autophagosome-autophagosome fusion mediated by Rab7. However, an arrest of the fusion events of the SeCV with late endo/lysosomal compartments was evidenced because in an estimated 85 to 90% of the total SeCV labelled by the LC3 autophagic marker, neither colocalization of the bacteria with the acidotropic Lysotracker marker nor with hydrolyzed DQ-BSA was detected even at six hours post-infection. Therefore, the expected progression to a functional autophagolysosome was somehow blocked or delayed by an intracellular Serratia population. As a consequence, we can conclude that Serratia is able to occupy a niche where the adverse action of acidic pH and lytic enzymes is circumvented. In contrast to the predicted outcome, when autophagy was induced by starving the CHO cells, no enhancement of intracellular survival of Serratia was observed. Moreover, blockage of Serratia-mediated autophagy activation by action of the PI3K inhibitor wortmannin was also ineffective, although this inhibitor drastically diminished the intracellular levels of CFUs recovery when added previous to the bacterial invasion with the aim of blocking the process extracellularly triggered by Serratia. Wort- mannin was, as expected, proficient in blocking the starvation- activated autophagic process, but it was ineffective once Serratia induction has been triggered. The overall effect of wortmannin is to block autophagy because class III PI3Ks act downstream of class I PI3Ks in the autophagic process. However, wortmannin has been found to be an inhibitor of class III (autophagy stimulator) as well as class I PI3K (autophagy negative regulator) [9,34], while also inhibiting other members of the PIK family, such as mTOR and the DNA-dependent protein kinase as well [35]. Taking also into account that PI3Ks are involved in other membrane trafficking processes, including formation of phagocytic cups and Serratia Invasion to Epithelial Cells micropinosomes [36], our results allow us to infer that Serratia is internalized in the CHO cells by a process in which PI3K activity is required and that the autophagic process induced by Serratia is able to bypass the involvement of PI3K. On the other hand, the invasion assays performed with Atg5 knock-down cells showed that Atg5, component of the Atg12-Atg5-Atg16 complex which acts as a ligase for LC3-phosphatidylethanolamine conjugation, is essen- tial for the autophagy activated by Serratia. Mestre et al [33] recently demonstrated that, in the Staphylococcus aureus infection, a- toxin-dependent activation of autophagy is independent of PI3K activity. In addition, a recent work from Kageyama et al [37] shows that LC3 and the Atg16L complex are recruited to the autophagosome-like membrane structure that surrounds Salmonella inside the Salmonella containing-vacuole, in a PI3K-independent way. Together, these observations contribute to indicate that, depending on the specific pathogen, autophagy triggered by bacteria might implicate non-canonical pathways, in a process still barely understood. Additionally, we show that the integrity of the flagellar biogenesis cascade is required for the early interaction of Serratia with CHO cells. As expected, the adhesion impairment of the non- flagellated strain essentially abrogated the subsequent entry of the bacteria into the cells. Nonetheless, neither the lack of flagellum biogenesis nor the inhibited expression of the PhlA phospholipase (the only Serratia exoprotein that is known to depend on the flagellar regulatory cascade for its expression and secretion) prevented the induction of autophagy. This result clearly shows that even when the bacterium-cell interaction stage that precedes invasion is inhibited, autophagy can still be activated by extracellular Serratia. Furthermore, when bacterial protein synthe- sis was halted prior and along the invasion assay, the induction of the autophagic phenotype was prevented. Besides, the autophagic phenotype of non-invaded CHO cells was lost as soon as metabolically active extracellular bacteria were killed by gentami- cin action. This result indicates that the autophagocytic response is induced by metabolically active bacteria, previous to internaliza- tion. Reinforcing this observation, heat-killed bacteria were capable neither to induce autophagy nor to invade the cells, ruling out the involvement of bacterial heat-resistant structures such as the lipopolysaccharide in autophagy activation. In addition, we show that an early Serratia invasion event is severely stalled by preventing vacuole acidification, suggesting that a bacterial determinant that requires a low pH for intracellular action might be implicated. In this context, it has been demonstrated that diverse bacterial toxins that enter their target cell by receptor-mediated endocytosis, require acidification for their processing and activation, being its effect prevented by treatment with bafilomycin A1 or NH4Cl [38,39]. Alternatively or in addition, the increase of the vacuoles luminal pH can secondarily cause changes in eukaryotic receptors turn-over, in the conformation of proteins or in the affinity between receptors and ligands required for invasion (reviewed in [40]). We are currently analyzing the identity and mechanism of action of the virulence factors required for entry and for the promotion of the autophagic process. In light of our results, it is tempting to speculate that the extracellular action of a virulence factor previous to the internal- ization process activates a non-canonical autophagic pathway as a strategy that leads to the subsequent generation of a specialized vacuole that supports the intracellular life of Serratia. Once inside the SeCV, the bacterium would delay or preclude the interaction of the autophagosome-like vacuole with the lysosomal compartment. As a consequence, Serratia alters the normal outcome of the autophago- some, building a propitious environment for survival and PLoS ONE | www.plosone.org 14 August 2011 | Volume 6 | Issue 8 | e24054