Serratia Invasion to Epithelial Cells Figure 5. Effect of autophagy inhibitors on Serratia invasion. A) Inhibition or neutralization of vacuolar acidification on Serratia invasion. CHO-EGFP-LC3 cells were maintained in a-MEM, incubated with DMSO 1% (which equals the final concentration of DMSO used as vehicle for bafilomycin A1), 100 nM bafilomycin A1 or 10 mM NH4Cl, as indicated in Materials and Methods. Cells were infected with wild-type S. marcescens following the gentamicin protection assay procedure. Invaded cells were lysed with 0.05% Triton X-100 and CFUs were determined on LB agar plates at 120, 240 or 360 min p.i. Percentages were calculated relative to CFUs in the inoculum. The average 6 S.D. is shown for three independent experiments, *p,0.001. B) Modulation of autophagy on Serratia invasion. CHO-EGFP-LC3 cells were maintained in a-MEM, or pre-treated in EBSS starvation medium (Starv). 100 nM Wortmannin (Wn) was added 2 h previous to the infection assay (a-MEM + Wn; or Starv + Wn) or at 60 min p.i. (a-MEM + Wn p.i). Cells were infected with wild-type S. marcescens following the gentamicin protection assay procedure. Invaded cells were lysed with 0.05% Triton X-100 and the CFUs were determined on LB agar plates at 120 or 360 min p.i. The average 6 S.D. is shown for three independent experiments (% intracellular CFUs are indicated above each bar in the graph; nd: not determined, *p,0.001). doi:10.1371/journal.pone.0024054.g005 reduction, estimated at 120 or 240 min p.i., respectively) when compared to the wild-type strain. Both adhesion and invasion were restored to wild-type levels when the flhD mutant strain was complemented by the expression of FlhD2C2 in trans from the pflhDC plasmid (Fig. 10A, black bars). Additionally, we determined that the wild-type strain and the otherwise isogenic phlA mutant strain showed equivalent invasion capacity (data not shown). These results demonstrate that the expression of the flagellum, but not of the co-regulated PhlA exoenzyme, is a key determinant in the early steps of Serratia interaction with epithelial cells. However, the three strains analyzed (wild-type, flhD and phlA) were equally capable of inducing the EGFP-LC3 autophagic pattern (i.e. punctate distribution) when colocalization of LC3 with the SeCV was monitored. Once again, the autophagy phenotype was PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e24054