Serratia Invasion to Epithelial Cells Figure 3. Serratia is able to induce autophagy in different epithelial cell lines. HeLa, MEFs Atg5+/+, MEF Atg52/2 and T24 cells were transiently transfected with pRFP-LC3 (left panels, red fluorescence) and invaded with wild-type S. marcescens transformed with pGFP (right panels, green fluorescence). Cells were fixed at 180 min p.i. and visualized by confocal laser microscopy. Images are representative of at least 300 infected cells monitored in two independent assays. Bars: 10 mm. doi:10.1371/journal.pone.0024054.g003 As shown in Figs. 4A and 4C, a similar distribution of vesicles was also determined at 360 min p.i. Collectively, these results clearly indicate that at late times post-infection (i.e. after 4 h p.i.), a predominant population of the internalized Serratia is contained in autophagic-like non-degradative and non-acidic compartments, while a minor proportion of total internalized bacteria is delivered to degradative, acidic vesicles (i.e. lysosomal compartments). Next, the invasion assay was carried out in the presence of bafilomycin A1 or NH4Cl to block the vacuolar type-H+-ATPase action or to neutralize vacuolar acidification, respectively. The induction of autophagy by Serratia was examined by the conversion of LC3-I to the lipidated LC3-II form (Fig. 6, compare the LC3- II/LC3-I ratio, 0.06 from control cells and 0.44 from invaded cells with no inhibitor added, which is the result from autophagy induction by Serratia and LC3 turn-over). In the presence of bafilomycin A1, the accumulation of LC3-II due to the blockage in protein turn-over was evident (Fig. 6, LC3-II/LC3-I ratio = 0.71 for NH4Cl and = 0.52 for bafilomycin compared to 0.06 in non- treated control cells). However, in the invasion assay, the LC3-II/ LC3-I ratios were similar to the values obtained by non-invaded cells (Fig. 6, compare NH4Cl or Baf in control and invasion assays). Besides, the effect of the inhibitors on the intracellular survival of Serratia contrasted to the expected outcome that would predict an increase in intracellular CFUs due to the blockage in the delivery of vacuolar compartments to the lysosomal ones. Intracellular recovered CFUs were highly diminished as early as 120 min p.i. (0.91% and 2.71% intracellular CFUs in bafilomycin A1 or NH4Cl treated cells, versus 8.26% for control cells) and remained without significant changes at 240 or 360 min p.i., when compared to non-treated cells in which Serratia proliferated intracellularly (Fig. 5A). As shown in Fig. 7, the punctate LC3 pattern microscopically observed at 360 min p.i. in either invaded or non-invaded cells treated with the inhibitors was also indicative of an effective blockage in LC3 protein turn-over. Scarce SeCVs containing only few bacteria could be detected in treated invaded cells when compared with non-treated cells, in accordance to the low % of intracellular CFUs recovered (Fig.5A, and see Fig.7). This result explains the similarity between the LC3 ratio values obtained for non-invaded and invaded cells treated with the inhibitors: because both compounds inhibit early invasion events, at 360 min p.i. gentamicin action has already eliminated extracellular bacteria giving no time for the extracellular induction of autophagy to develop. Neither bafilomycin A1 or NH4Cl, added to the culture media at the final concentrations used in the assay, affected the growth or showed toxic effects on Serratia (see Materials and Methods, data not shown). Taking into account that the two different procedures used to prevent intraluminal vacuolar acidification rendered a severe reduction in intracellular CFUs as early as 120 min p.i., it is tempting to speculate that a low pH ambient might be required for early invasion steps. Serratia promotes a non-canonical autophagic pathway In light of the previous results, we examined whether induction of autophagy by cell starvation would enhance Serratia invasion PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e24054