<i>Pseudomonas aeruginosa</i> cells attached to a surface display a typical proteome early as 20 minutes of incubation

<div><p>Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen <i>Pseudomonas aeruginosa</i> in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.</p></div

Attachment capacity of <i>P</i>. <i>aeruginosa</i> PAO1 reference strains and isogenic mutants.

<p>The attachment capacity of reference strains, mutants and complemented mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180341#pone.0180341.s011" target="_blank">S9A Table</a>) was assayed after 20 min at 37°C in our glass wool system (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180341#sec021" target="_blank">materials and methods</a>). Only the attachment capacity of <i>P</i>. <i>aeruginosa</i> PAO1 was presented for reason of clarity, the other reference strains (PAO1-L and MPAO1) showed results similar to those obtained for PAO1. In the same manner, the attachment capacities of the different mutants were not altered by introducing the empty plasmid pUCP20 or pUCP22. For each of the mutants presented herein, the complemented strain displayed an attachment capacity ranging from 80% to 140% of the reference strain (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180341#pone.0180341.s010" target="_blank">S8 Table</a>). The results corresponded to the average of 3 independent experiments (bar = SD).</p

NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

1<p>WCE, whole cell exctract.</p>2<p>glyco, partially purified glycosome fraction.</p>3<p>Mean ± SEM of n experiments (mU/mg of protein).</p>4<p>+gluc: cells cultured in SDM79 containing 10 mM glucose.</p>5<p>−gluc: cells cultured in glucose-depleted SDM79GluFree.</p><p>NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.</p

Phenotypic analysis of Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell.

<p>(A) growth curve of WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells in glucose-rich (SDM79 with 10 mM glucose) or glucose-free (SDM79GluFree) conditions. (B) Global protein abundance in the partially purified glycosome fraction of WT (x-axis) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells (y-axis). Each protein identification is presented by a point at log₁₀ of normalized peptide count values taken from the proteome data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s004" target="_blank">S4 Figure</a>. Proteins on the dashed grey line have identical normalized peptide counts in both samples; the grey lines represent a 2-fold abundance in one condition.</p

TAG species analysis and uptake of labeled oleate.

<p>(A) Dominant TAG species in procyclic <i>T. brucei</i> cells identified by ESI/MS/MS after oleate feeding for three days (black columns) or in the control (white columns). For a complete list of TAG species detected see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s001" target="_blank">S1 Figure</a>. The nomenclature 54:X indicates the total carbon number of all three acyl chains and the sum of all unsaturated double bonds within the acyl chains. (B) Uptake kinetics upon growth in the presence of radiolabeled oleate for up to 8 h. The incorporation of ¹⁴C oleate into lipid species was quantified by HPTLC and a Storm 860 phosphorimager. PPL, phospholipids; TAG, triacylglycerol; SE, Steryl-esters; DAG, diacylglycerol.</p

Oleate feeding stimulates lipid droplet formation in procyclic <i>T. brucei</i> cells.

<p>Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628-Bird1" target="_blank">[36]</a>. An example of several experiments is shown.</p

LD and TAG turnover in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

<p>Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.</p

Quantification of the oleate-induced lipid droplet formation.

<p>(A) BODIPY 493/503 stained LDs were counted in stacks of confocal laser scanning microscopy (CLSM) images; the average number of LDs per cell is given after oleate feeding (black column) or in the control (white column). (B) Distribution of LD numbers per cells in the population after oleate feeding (black columns) or in the control (white columns). (C) Quantification of BODIPY-stained LDs by flow cytometry after oleate feeding (black column) or in the control (white column). BODIPY 493/503 preferentially stains nonpolar lipids. Error bars give the SEM (n = 3) of values normalized to the control. (D) Quantification of TAG content by HPTLC and densitometry after oleate feeding (black columns) or in the control (white columns). Values are normalized to the control.</p

Anti-tumor activity of pFasL.

<p><b><u>Panel A:</u></b> Tumor growth in mice having received subcutaneously 10⁵ A431 cells at day 0, and 0.1 mL of concentrated pFasL (white boxes) or pFasL-free control (grey boxes) locally at days 2 and 7 (n = 6 mice per group). Tumor volumes are expressed in mm³. Values are presented as median, 25^th and 75^th percentiles (horizontal line, bottom and top of boxes), and 10^th and 90^th percentiles (bottom and top range bars) (**p = 0.04, * p = 0.05). <b><u>Panel B:</u></b> Kaplan-Meier analysis of cumulative percentage of mice without detectable tumor,, xenografted with A431 cells and treated with pFasL (black circles) or pFasL-free control (black squares) (p = 0.02). n = 20 mice per group, from two experiments pooled.</p

Biochemical characterization of the FasL/gp190 chimeras.

<p><b><u>Panel A:</u></b> Supernatants from COS cells transfected with the FasL constructs were quantified by ELISA and 10 μg of FasL protein were loaded per lane. Migrations were performed under reducing (SDS-PAGE) or non-reducing (BN-PAGE) conditions. FasL was revealed by immunoblot. <b><u>Panel B:</u></b> 2 μg of FasL construct were loaded on the gel filtration column. FasL was quantified by ELISA in elution fractions, and cytotoxicity was measured using the MTT assay. <b><u>Panel C:</u></b> Affinity measurement using Biacore®. Fas-Fc was immobilized on the chip, before the indicated soluble FasL constructs were added. A range of concentrations was tested for each analyte, but only the graph obtained with the highest concentration tested is displayed. <b><u>Panel D:</u></b> The apparent molecular weights and degree of oligo/polymerization of the FasL chimeras were estimated from the non denaturing gel electrophoresis and gel filtration experiments.</p