Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell

The autophagic process triggered by <i>Serratia</i>.

<p><b>A) Autophagy triggered by </b><b><i>Serratia</i></b><b> requires Atg5</b>. MEF <i>Atg5</i>^+/+ and MEF <i>Atg5</i>^−/− cells were infected for 60 min with wild-type <i>S. marcescenes</i>. Thereafter, 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 on LB agar plates were determined. Units were calculated relative to CFU recovered from MEF <i>Atg5</i>^+/+ cells at 120 min p.i. The media ± S.D. is shown for three independent experiments. <b>B) </b><b><i>S. marcescens</i></b><b> actively induces autophagy from the extracellular media.</b> Overnight <i>S. marcescens</i> cultures were incubated 60 min in LB without antibiotic or supplemented with 20 or 100 µg/ml chloramphenicol (bacteriostatic condition). Subsequently, CHO-EGFP-LC3 cells were infected with bacteria and then extracellular bacteria were eliminated with gentamicin (bacteriostatic treatment was maintained throughout the invasion assay). At 180 min p.i, cells were fixed and bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Cy3. Finally, the percentage of colocalization of vesicles containing <i>Serratia</i> with EGFP-LC3, relative to total infected cells, was determined by fluorescence microscopy. The lower panel shows the percentage of cells, infected and non-infected, with an autophagic phenotype. At least 300 infected cells were counted for each condition. The average ± S.D. is shown for three independent experiments, * and ** p<0.001. <b>C)</b> CHO-EGFP-LC3 cells (green fluorescence) infected with <i>S. marcescens</i> (red fluorescence) untreated (upper panel), treated with 20 µg/ml chloramphenicol (middle panel) or treated with 100 µg/ml chloramphenicol (lower panel) are shown. Bars: 10 µm.</p

<i>S. marcescens</i> flagellum expression is involved in adhesion to CHO cells.

<p><b>A)</b> CHO cells were infected for 60 min with <i>S. marcescens</i> wild-type, <i>flhD</i> and <i>flhD/</i>p<i>flhD</i> strains (dark gray, light gray or black bars, respectively). Cells were washed with PBS and lysed with 0.05% Triton X-100 (left graphic); or incubated 120 o 240 min p.i with medium supplemented with gentamicin to eliminate extracellular bacteria. Finally, cells were lysed with 0.05% Triton X-100 (right graphic) and the CFUs were determined on LB agar plates. Adherence and intracellular CFUs percentages were calculated relative to initial inoculums. The average ± S.D. is shown for four independent experiments, * p<0.001. <b>B)</b> CHO-EGFP-LC3 cells (green fluorescence) were infected with <i>S. marcescens</i> wild-type (b and f), <i>flhD</i> (c and g) and <i>phlA</i> (d and h) strains. Non infected cells are shown as control (a and e). Then, cells were not treated (a-d) or treated with gentamicin (e-h) for 120 min. Cells were fixed and bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Cy3 (red fluorescence). Representative confocal Z-slices are shown. Bars: 10 µm.</p

The <i>S. marcescens</i>-containing vacuoles are predominantly non acidic and non degradative compartments.

<p><b>A</b>) CHO-EGFP-LC3 cells were infected with wild-type <i>S. marcescens.</i> After 60 min p.i, cells were incubated with 3 µM of LysoTracker to label acidic compartments. Cells were fixed at 240 or 360 min p.i. and intracellular bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Alexa Fluor 647. The percentages of colocalization of bacteria with EGFP-LC3 and/or with Lysotracker were determined by fluorescence microscopy. At least 300 infected cells were counted for each condition. The average ± S.D. is shown for two independent experiments, *and **p<0.001. <b>B</b>) Representative confocal laser captured images of CHO-EGFP-LC3 cells (green fluorescence, d) infected with <i>S. marcescens</i> (blue fluorescence, b) and incubated with Lysotracker (red fluorescence, c) at 360 min p.i. are shown. Inset image (f) shows higher magnification of the boxed area in the merged image (e), and highlights a non-acidic, autophagic SeCV. Arrows point at acidic vesicles. Bars: 10 µm. <b>C)</b> CHO-EGFP-LC3 cells were pre-incubated with 10 µg/ml of DQ-BSA for 4 hours to label degradative compartments. Subsequently, cells were infected with wild-type <i>S. marcescens</i> and fixed at 240 or 360 min p.i. Intracellular bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Alexa Fluor 647. The percentages of colocalization of bacteria with EGFP-LC3 and/or with DQ-BSA were determined by fluorescence microscopy. At least 300 infected cells were counted for each condition. The average ± S.D. is shown for two independent experiments, *and **p<0.001. <b>D)</b> Representative confocal laser captured images of CHO-EGFP-LC3 cells (green fluorescence, d) pre-incubated with DQ-BSA (red fluorescence,c) and infected with <i>S. marcescens</i> (blue fluorescence, b) at 360 min p.i. are shown. Inset image (f) shows higher magnification of the boxed area in the merged image (e) and highlights a non-degradative, autophagic SeCV. Arrows point at degradative vesicles. Bars: 10 µm.</p

<i>Serratia</i> is able to induce autophagy in different epithelial cell lines.

<p>HeLa, MEFs <i>Atg5</i>^+/+, MEF <i>Atg5</i>^−/− and T24 cells were transiently transfected with pRFP-LC3 (left panels, red fluorescence) and invaded with wild-type <i>S. marcescens</i> 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 µm.</p

Confocal microscopy analysis of the effect of altering vacuolar acidification on <i>Serratia</i> invasion.

<p>CHO-EGFP-LC3 cells subjected to the invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024054#pone-0024054-g005" target="_blank">Fig. 5A</a> were visualized by confocal laser microscopy. a and b panels show representative images of control, non-invaded cells and c-f panels depict invaded cells incubated with α-MEM, α-MEM +100 nM Baf or α-MEM +10 mM NH₄Cl, as indicated. Bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Cy3. Representative DIC (c), EGFP-LC3 green fluorescence (d), red fluorescence-labeled bacteria (e) and merged images (f) are shown. At least 300 cells were inspected for each condition in two independent experiments. Bars: 10 µm.</p

Analysis of LC3-I/LC3-II conversion.

<p>CHO-EGFP-LC3 cell monolayers subjected to the invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024054#pone-0024054-g005" target="_blank">Figure 5</a> were harvested and resuspended in SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE (30 µg of total protein was loaded per lane), transferred to nitrocellulose and detected with anti-GFP polyclonal antibodies (“invasion”, lower panel blots). Control, non-invaded CHO-EGFP-LC3 cells, that were subjected to the same treatments were also analyzed (“control”, upper panel blots). The positions of LC3-I and LC3-II bands are indicated. Protein loading was controlled according to the α-tubulin content (the blots detected with antibodies anti-α-tubulin are shown). Blots are representative of three independent experiments. The bands intensity was quantified with the ImageJ Programe, and the LC3II/LC3I (II/I) was calculated for the immunodetection experiment shown in the figure. The histogram represents the average ± S.D. of the relative intensities of LC3-I or LC3-II <i>versus</i> α-tubulin of three independent experiments.</p

Strains and plasmid used in this study.

<p>Strains and plasmid used in this study.</p

<i>S. marcescens</i> invades and replicates inside non-phagocytic cells.

<p><b>A)</b> CHO cells or <b>B)</b> CHO, HeLa, MEF <i>Atg5</i>^+/+ and T24 cells were infected for 60 min with wild-type <i>S. marcescens</i> 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>(B)</b> fold change in CFUs were calculated relative to the values obtained at 120 min p.i. The average ± S.D. for two independent experiments is shown (* p<0.001).</p

Effect of autophagy inhibitors on <i>Serratia</i> invasion.

<p><b>A) Inhibition or neutralization of vacuolar acidification on </b><b><i>Serratia</i></b><b> invasion.</b> CHO-EGFP-LC3 cells were maintained in α-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024054#s2" target="_blank">Materials and Methods</a>. Cells were infected with wild-type <i>S. marcescens</i> 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 ± S.D. is shown for three independent experiments, *p<0.001. <b>B) Modulation of autophagy on <i>Serratia</i> invasion.</b> CHO-EGFP-LC3 cells were maintained in α-MEM, or pre-treated in EBSS starvation medium (Starv). 100 nM Wortmannin (Wn) was added 2 h previous to the infection assay (α-MEM + Wn; or Starv + Wn) or at 60 min p.i. (α-MEM + Wn p.i). Cells were infected with wild-type <i>S. marcescens</i> 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 ± S.D. is shown for three independent experiments (% intracellular CFUs are indicated above each bar in the graph; nd: not determined, *p<0.001).</p