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

P3HT-Based Solar Cells: Structural Properties and Photovoltaic Performance

Each year we are bombarded with B.Sc. and Ph.D. applications from students that want to improve the world. They have learned that their future depends on changing the type of fuel we use and that solar energy is our future. The hope and energy of these young people will transform future energy technologies, but it will not happen quickly. Organic photovoltaic devices are easy to sketch, but the materials, processing steps, and ways of measuring the properties of the materials are very complicated. It is not trivial to make a systematic measurement that will change the way other research groups think or practice. In approaching this chapter, we thought about what a new researcher would need to know about organic photovoltaic devices and materials in order to have a good start in the subject. Then, we simplified that to focus on what a new researcher would need to know about poly-3-hexylthiophene:phenyl-C61-butyric acid methyl ester blends (P3HT: PCBM) to make research progress with these materials. This chapter is by no means authoritative or a compendium of all things on P3HT:PCBM. We have selected to explain how the sample fabrication techniques lead to control of morphology and structural features and how these morphological features have specific optical and electronic consequences for organic photovoltaic device applications

Intratumor heterogeneity index of breast carcinomas based on DNA methylation profiles

Abstract Background Cancer cells evolve and constitute heterogeneous populations that fluctuate in space and time and are subjected to selection generating intratumor heterogeneity. This phenomenon is determined by the acquisition of genetic/epigenetic alterations and their selection over time which has clinical implications on drug resistance. Methods DNA extracted from different tumor cell populations (breast carcinomas, cancer cell lines and cellular clones) were analyzed by MS-MLPA. Methylation profiles were used to generate a heterogeneity index to quantify the magnitude of epigenetic heterogeneity in these populations. Cellular clones were obtained from single cells derived of MDA-MB 231 cancer cell lines applying serial limiting dilution method and morphology was analyzed by optical microscopy and flow cytometry. Clones characteristics were examined through cellular proliferation, migration capacity and apoptosis. Heterogeneity index was also calculated from beta values derived from methylation profiles of TCGA tumors. Results The study of methylation profiles of 23 fresh breast carcinomas revealed heterogeneous allele populations in these tumor pieces. With the purpose to measure the magnitude of epigenetic heterogeneity, we developed an heterogeneity index based on methylation information and observed that all tumors present their own heterogeneity level. Applying the index calculation in pure cancer cell populations such as cancer cell lines (MDA-MB 231, MCF-7, T47D, HeLa and K-562), we also observed epigenetic heterogeneity. In addition, we detected that clones obtained from the MDA-MB 231 cancer cell line generated their own new heterogeneity over time. Using TCGA tumors, we determined that the heterogeneity index correlated with prognostic and predictive factors like tumor size (p = 0.0088), number of affected axillary nodes (p = 0.007), estrogen receptor expression (p < 0.0001) and HER2 positivity (p = 0.0007). When we analyzed molecular subtypes we found that they presented different heterogeneity levels. Interestingly, we also observed that all mentioned tumor cell populations shared a similar Heterogeneity index (HI) mean. Conclusions Our results show that each tumor presents a unique epigenetic heterogeneity level, which is associated with prognostic and predictive factors. We also observe that breast tumor subtypes differ in terms of epigenetic heterogeneity, which could serve as a new contribution to understand the different prognosis of these groups

<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

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

<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

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

LC3 and Rab7 are recruited to the <i>S. marcescens</i>-containing vacuole.

<p><b>A</b>) CHO-EGFP-LC3 cells (green fluorescence) or <b>B</b>) CHO cells transfected with EGFP-Rab7 (green fluorescence), were infected with wild-type <i>S. marcescens</i> and cells were fixed at 120 min p.i (a-d) or 360 minutes p.i (e-h). 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. Inset images show higher magnification of the boxed areas in merged images. Bars: 10 µm. <b>C)</b> CHO-EGFP-LC3 cells or CHO cells transfected with pEGFP-Rab7 were infected with wild-type <i>S. marcescens</i>. Cells were fixed at the indicated time points and bacteria were detected with antibodies anti-<i>Serratia</i> coupled with a secondary antibody labeled with Cy3. The percentage of colocalization of EGFP-LC3 (•) or EGFP-Rab7 (▴) with bacteria was determined by fluorescence microscopy. At least 200 infected cells were counted for each time point. The average ± S.D. for three independent experiments is shown.</p