An 18 kDa Scaffold Protein Is Critical for <i>Staphylococcus epidermidis</i> Biofilm Formation

<div><p>Virulence of the nosocomial pathogen <i>Staphylococcus epidermidis</i> is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from <i>S</i>. <i>epidermidis</i> biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative <i>S</i>. <i>epidermidis</i> mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of <i>S</i>. <i>epidermidis</i> cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an <i>S</i>. <i>epidermidis</i> biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative <i>S</i>. <i>epidermidis</i> mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of <i>sbp</i> in commensal and invasive <i>S</i>. <i>epidermidis</i> populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.</p></div

Functional role of Sbp in Aap-mediated <i>S</i>. <i>epidermidis</i> biofilm formation.

<p><b>(A)</b> Biofilm phenotypes of 1457-M10, 1457-M10Δ<i>aap</i>, 1457-M10Δ<i>sbp</i>, 1457-M10Δ<i>aap</i>Δ<i>sbp</i>, 1457-M10Δ<i>aap</i>(pRBDomain-B) and 1457-M10Δ<i>aap</i>Δ<i>sbp</i>(pRBDomain-B) were tested in static biofilm assays in the absence (black columns) or presence of varying rSbp concentrations (grey columns). All columns represent mean of 12 values obtained in 3 independent experiments. Error bars represent standard deviations. Significant differences (p< 0.05; one-way ANOVA with Dunnett’s correction for multiple testing) are indicated (*, p<0.05; ***, p<0.001). n.s., not significant. <b>(B)</b> Recruitment of rSbp to the surface of 1457-M10Δ<i>aap</i>Δ<i>sbp</i> in the presence of absence of <i>in trans</i> expressed Aap domain-B. Western blot of cell surface protein extracts from identical numbers of bacteria suspended in PBS containing 50, 10, or 5 μg/ml rSbp. PBS without rSbp served as a negative control. rSbp was detected by bioluminescence using a polyclonal rabbit anti-rSbp antiserum and anti-rabbit IgG coupled to peroxidase. <b>(C)</b> Distribution of Aap domain-B and rSbp in living biofilms. 1457-M10Δ<i>aap</i>Δ<i>sbp</i>(pRBDomain-B) was grown in the presence of 1.5 μg/ml rSbp-DyLight550. Bacteria were stained with SYTO 9, Aap domain-B was detected using a polyclonal anti-rDomain-B antiserum and a Cy5-labelled anti-rabbit IgG antibody. Panels I–III represent images of each fluorescence channel, image IV is a merge depicting Aap domain-B and rSbp (IV). A zoom-in shows a representative area with Aap domain-B–rSbp co-localizations (purple; double arrow head). Arrow, Aap domain-B expressing cells without Sbp-recruitment (blue); arrow head; Sbp deposition independent from Aap domain-B (red).</p

Strains used in the study.

<p>Cm: Chloramphenicol; Ery: Erythromycin; Tet: Tetracycline; TMP: trimethoprim</p><p>*Plasmid pRBDomain-B was originally referred to pRB<i>aap</i>R_T [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004735#ppat.1004735.ref016" target="_blank">16</a>]</p><p>Strains used in the study.</p

Functional role of Sbp in primary attachment.

<p><b>(A)</b> Quantification of surface bound bacteria on pro-adherent polystyrene after one hour (rapid adherence). Binding of 1457 and 1457Δ<i>sbp</i> to cell culture treated polystyrene (NunclonΔ, Roskilde, Denmark) and non-adhesive polystyrene (NAP; Greiner, Frickenhausen, Germany). Each data point represents the mean A₄₀₅ value derived from 18 replicate measurements obtained in two independent experiments. Error bars indicate standard deviation. <b>(B)</b> Prolonged <i>S</i>. <i>epidermidis</i> adherence to unmodified and rSbp-coated non-adhesive polystyrene (NAP). Gfp-expressing 1457-M10(pGFP) and 1457-M10Δ<i>sbp</i>(pGFP) were grown in TSB under static conditions for 8 and 24 hours. After washing adherent cells were quantified by determining fluorescence intensity (excitation 485 nm, emission 535 nm). Columns represent normalized fluorescence values (i.e. fluorescence intensities normalized against respective bacterial cell densities). Error bars indicate standard deviation. Significant differences (p<0.05; one-way ANOVA with Bonferroni’s correction for multiple testing) are indicated by stars (**, p<0.01; ***, p<0.001). n.s., not significant. <b>(C)</b> Biofilm accumulation on non-adhesive polystyrene (NAP). 1457-M10(pTX<i>icaADBC</i>) and 1457-M10Δ<i>sbp</i>(pTX<i>icaADBC</i>) were grown under static conditions for 24 hours in the presence or absence of 3% [wt/vol] xylose. After washing, adherent cells were stained with Gentiana violet and biofilm formation was quantified at 570 nm. Biofilm formation of 1457-M10Δ<i>sbp</i>(pTX<i>icaADBC</i>) under inducing was also tested after coating of NAP surfaces with rSbp. Columns represent means of 6 values obtained in 3 independent experiments. 1457-M10 and 1457-M10Δ<i>sbp</i> complemented with plasmid pTX<i>icaADBC</i> (allowing for inducible PIA production) was used here to avoid confounding effects of <i>sbp</i> inactivation on PIA production (as demonstrated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004735#ppat.1004735.g008" target="_blank">Fig. 8A</a>). Error bars indicate standard deviation. Significant differences (p<0.05; one-way ANOVA with Bonferroni’s correction for multiple testing) are indicated by stars (*, p<0.05; ***, p<0.001). n.s., not significant. <b>(D)</b> Adhesion of <i>S</i>. <i>epidermidis</i> strain 1457, 1457Δ<i>sbp</i> and the complemented mutant to HaCaT keratinocytes. Columns represent mean of 10 values obtained in 5 independent experiments, error bars depict standard deviation. Differences between 1457 and 1457Δ<i>sbp</i> as well as 1457Δ<i>sbp</i> and the complemented mutant were significant different (p<0.01, Wilcoxon rank sum test).</p

Functional role of Sbp in <i>S</i>. <i>epidermidis</i> 1457 biofilm formation.

<p><b>(A)</b> Photometric quantification of biofilm formation after overnight growth of 1457, 1457Δ<i>sbp</i>, 1457Δ<i>aap</i>, 1457Δ<i>aap</i>Δ<i>sbp</i>, 1457Δ<i>sbp</i> complemented with pRB<i>sbp</i> and 1457Δ<i>aap</i>Δ<i>sbp</i> complemented with pRB<i>sbp</i>. 1457-M10 (PIA-negative) served as a control. Adherent cells were stained with gentiana violet before absorption at 570 nm was assessed. Columns represent means of twelve values obtained in three independent experiments. Error bars indicate standard deviations. Significant differences (Kruskall-Wallis one-way ANOVA with Dunn’s multiple comparison test) are indicated (*, p<0.05). n.s. not significant. <b>(B)</b> Analysis of biofilm formation under flow conditions. <i>S</i>. <i>epidermidis</i> 1457, 1457Δ<i>sbp</i> and the complemented mutant were grown in TSB + 0.5% glucose for 48 h at a flow rate of 1 ml/min. <b>(C)</b> Microscopic analysis of <i>S</i>. <i>epidermidis</i> 1457, 1457Δ<i>sbp</i>, 1457-M10Δ<i>aap</i>(pRBDomain-B), and 1457-M10Δ<i>aap</i>Δ<i>sbp</i>(pRBDomain-B). Cells were scraped from cell culture plates after static over night growth and appropriate dilutions were allowed to dry on glass cover slips. Bacteria were Gram-stained. Images were taken at 1000 x magification. <b>(D)</b> Induction of biofilm formation by exogenous recombinant rSbp. Biofilm formation of 1457Δ<i>sbp</i> and 1585Δ<i>sbp</i> was quantitatively assessed in the presence of varying amounts of purified rSbp. After overnight growth, adherent cells were stained using gentiana violet, and biofilms were quantified spectrophotometrically at 570 nm. Columns represent means of 6 values obtained in three independent experiments. Error bars indicate standard deviation. Significant differences compared to the control (no exogenous rSbp; One-way ANOVA with Dunnett’s correction for multiple testing) are indicated (***, p<0.001). n.s., not significant.</p

Integrated model of Sbp functions in <i>S</i>. <i>epidermidis</i> biofilm formation.

<p>Free-floating <i>S</i>. <i>epidermidis</i> decorated with cell surface bound Sbp adhere to artificial surfaces. The <b>fast primary attachment phase</b> is apparently independent from Sbp. While <i>S</i>. <i>epidermidis</i> adheres to the surface, Sbp localizes to the bacterial–substrate interface. Sbp deposition is a <b>surface priming process</b> necessary for stable <i>S</i>. <i>epidermidis</i>–foreign material interactions and sustained adherence during <b>biofilm accumulation</b>. Most likely, priming and accumulation are processes running in parallel. Sbp is part of the extracellular biofilm matrix, partly co-localizing with PIA (<b>Zoom in</b>). PIA-dependent biofilm formation indirectly depends on the presence of Sbp that, via so far unknown mechanisms modulates <i>icaADBC</i> transcription and subsequent PIA synthesis. In addition, Sbp serves as a necessary factor during Aap domain-B mediated bacterial aggregation, potentially through direct molecular interactions. Here, the involvement of additional (protein) factors cannot be excluded.</p

Interconnection between Sbp production and PIA synthesis.

<p><b>(A)</b> Quantification of PIA production in <i>S</i>. <i>epidermidis</i> 1457, 1457Δ<i>sbp</i> 1457Δ<i>aap</i> and 1457Δ<i>aap</i>Δ<i>sbp</i> by dot blot analysis. Serial dilutions of cell wall extracts were spotted onto PVDF membranes which were then incubated with WGA coupled to peroxidase. Bound WGA was then visualized by chemiluminescence. PIA titers were defined as the highest dilution giving a signal above the background (as determined by parallel analysis of 1457-M10). Dot blots show results obtained at 1:8 (cell wall preparations) and 1:2 (supernatants) dilution. 1457-M10(pTX<i>icaADBC</i>) and 1457-M10Δ<i>sbp</i>(pTX<i>icaADBC</i>) were grown in the presence of 3% [wt/vol] xylose for induction of <i>icaADBC</i> expression. <b>(B)</b> Analysis of biofilm formation by 1457-M10 and 1457-M10Δ<i>sbp</i> complemented with pTX<i>icaADBC</i> allowing for xylose-inducible <i>in trans</i> expression of <i>icaADBC</i>. Bacteria were grown overnight in TSB or TSB supplemented with xylose (3% [w/v]), respectively. Following washing procedures adherent bacteria were stained with gentiana violet. Columns represent means of 12 values obtained in three independent experiments. Error bars indicate standard deviation. Significant differences (p<0.05; one-way ANOVA with Bonferroni’s correction for multiple testing) are indicated by stars (***, p<0.001). n.s., not significant.</p

Microscopic analysis of 1457Δ<i>sbp</i>.

<p><b>(A—C)</b> Three dimensional reconstruction of biofilms from Gfp-expressing <i>S</i>. <i>epidermidis</i> 1457 (A), 1457Δ<i>sbp</i> (B) and 1457Δ<i>sbp</i> grown in the presence of fluorescence-labelled rSbp-DyLight550 (1.5 μg/ml; red) (C). The arrowhead indicates localization of rSbp at the biofilm—substrate interface, the asterisk highlights localization of rSbp within the biofilm matrix. A detailed demonstration of rSbp-DyLight550 distribution is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004735#ppat.1004735.s006" target="_blank">S6A Fig.</a>. Fluorescence labelling did not alter the biofilm-inducing properties of rSbp (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004735#ppat.1004735.s006" target="_blank">S6B Fig.</a>). Grid unit = 11.62 μm <b>(D)</b> Quantification of mean biofilm volume and thickness. 18 randomly chosen biofilm CLSM images obtained in three independent experiments were analyzed for each strain. Analysis was carried out using the Volocity software package. Error bars depict standard deviations. Significant differences (p<0.05; one-way ANOVA with Bonferroni’s correction for multiple testing) are indicated (*. p<0.05; ***, p<0.001). Differences indicate between wild type 1457 and 1457Δ<i>sbp</i> complemented with rSbp indicate the potential importance of the Sbp source for <i>S</i>. <i>epidermidis</i> biofilm structure. rSbp-DyLight550 fully reconstituted biofilm formation in 1457Δ<i>sbp</i> in standard micro titer plate biofilm assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004735#ppat.1004735.s006" target="_blank">S6B Fig.</a>).</p