population can be targeted by the autophagic system [12,13]. Free cytosolic bacteria can be wrapped by the nascent autophagosomal vesicle (phagophore) into a double membrane vacuole, the autophagosome, which fuses with lysosomal vesicles, finally forming a degradative autophagolysosome. In the case of membrane-bound bacteria, the compartment can be also sequestered by a phagophore, possibly attracted by signals of membrane damage originated by bacterial effectors, and once more, this structure is fused to lysosomes giving rise to an autolysosome. While pathogens like Shigella flexneri and Listeria monocytogenes are able to evade autophagy recognition [14,15], it was shown that others, like Brucella abortus, Coxiella burnetii or Legionella pneumophila, can replicate in autophagosome-like compartments (reviewed in [8]). Therefore, and in spite of the array of host cell protective strategies, numerous pathogens are able to hijack the cellular defensive machinery at different stages, escaping from, avoiding, delaying or blocking its normal progression, to finally establish an intracellular niche where they can survive and proliferate in the host. In this work, we explore the invasion process of S. marcescens to non-phagocytic cells. We demonstrate that, once inside the cell, Serratia is able to inhabit and proliferate inside a large membrane- bound compartment. These vesicles exhibit autophagic-like features as they acquire LC3 and Rab7, markers that have been demonstrated to be recruited throughout the progression of the autophagosome biogenesis in the antibacterial process [16–18]. However, we show that the vast majority of the autophagic vacuole population is non-acidic and has no degradative properties, indicating an alteration of the normal delivery to lysosomal compartments. In addition, our results put forth that non-canonical autophagy, which does not involve phosphatidyli- nositol 3-kinase (PI3K) activity, can be induced by the bacteria before internalization, from outside the target cell. We also show that flagellar expression participates in the early interaction of the bacteria with the host cell and provide evidences indicating that the internalization process engages PI3K activity and requires intravacuolar acidification. These findings shed light on the mechanism that Serratia employs to invade and survive inside non-professional phagocytic cells and also reveal that S. marcescens constitutes an attractive model organism to further explore the strategies that opportunistic pathogens have evolved to succeed in the host. Table 1. Strains and plasmid used in this study. Serratia Invasion to Epithelial Cells Materials and Methods Materials a-MEM cell culture media, fetal calf serum (FCS), Lipofecta- mine 2000 and Earles Balanced Salt Solution (EBSS) were obtained from Invitrogen (Argentina). Antibiotics kanamycin (50 mg/ml), ampicillin (100 mg/ml), chloramphenicol (20 mg/ml), and spectinomycin (100 m/ml), bafilomycin A1, NH4Cl and wortmannin were purchased from Sigma (Argentina). Rabbit anti-S. marcescens polyclonal antibodies were prepared in our laboratory and the secondary goat anti-rabbit IgG (H+L) antibodies conjugated with Cy3 were provided by Zymed Laboratories. Secondary goat anti-rabbit IgG (H+L) antibodies conjugated with Alexa Fluor 647, rabbit anti-GFP polyclonal antibodies, self-quenched red Bodipy dye conjugated to BSA (DQ- BSA), and LysoTracker were purchased from Molecular Probes. Bacterial strains and plasmids The strains and plasmids used in this study are listed in Table 1. pGFP was obtained by sub-cloning gfpmut3.1 gene from pGFPmut3.1 (reppMB1 ApR gfpmut3.1, [19]) into pBBR1-MCS2 between XhoI and HindIII sites. S. marcescens flhD was disrupted by integration of the suicide plasmid pKNOCK-CmR [20]. Briefly, an internal fragment of 317 bp from gen flhD was amplified by PCR using primers FlhD-Nter-b42-pknock and FlhD-Cter-rev- Serratia (59- CGGGATCCTGACATCAATTTGTC-39 and 59- TCCAAGCTTCAGGCTCTTTTCTTCG-39, respectively). The purified PCR product was digested with the BamHI and SalI restriction enzymes and the fragment of 217 bp was ligated into the BamHI and SalI sites of pKNOCK-CmR. The resulting plasmid was introduced into competent E. coli SM10(lpir) cells by electroporation and then mobilized into S. marcescens RM66262 by conjugation. Insertional mutants were confirmed by PCR analysis. For complementing S. marcescens flhD, pBBR1-MCS2 KnR was digested with XhoI and the 59overhanging end was filled by treatment with Klenow. Finally, it was digested with HindIII restriction enzyme. flhDC was amplified by PCR using primers FlhD-serratia-Nter and FlhC-CTR (59-TCGCCCGGGATGGG- GAATATGGGTAC-39 and 59-TCCAAGCTTCAGGCTCTT- TTCTTCG-39, respectively) and the purified PCR product was digested with the SmaI and HindIII restriction enzymes. The fragment was ligated into pBBR1-MCS2 KnR and the resulting plasmid was mobilized into S. marcescens flhD by conjugation. Strains S. marcescens wild-type flhD phlAB Plasmids pBBR1-MCS2 pflhDC pphlAB pGFP pRFP-LC3 Relevant phenotype S. marcescens RM66262, clinical isolate S. marcescens RM66262 flhD::pKNOCK, non flagellated, CmR S. marcescens RM66262 phlAB::CmR KnR, broad range host pBBR1-MCS2::flhDC, KnR pBBR1-MCS2::phlAB, KnR pBBR1-MCS2::gfpmut3.1, KnR lc3 sub-cloned in the pRFP vector, as described doi:10.1371/journal.pone.0024054.t001 Source or Reference [31] This work [31] [41] [31] [33] This work This work PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e24054