Surface Physicochemical Properties At The Micro And Nano Length Scales: Role On Bacterial Adhesion And Xylella Fastidiosa Biofilm Development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.8e7524

Surface morphology and potential alterations of silicon and cellulose surfaces induced by culture media.

<p>AFM topography (<b>A</b>-<b>F</b>) and surface potential (SP; <b>G-I</b>) images of the substrate surfaces. (<b>A</b>-<b>C</b>) show topography of pristine silicon (Si), ethyl cellulose (EC) and cellulose acetate (CA) surfaces, respectively; (<b>D</b>-<b>F</b>) illustrates topography of Si, EC and CA surfaces after incubation in PW medium for 24h, respectively; (<b>G</b>-<b>I</b>) present SP images of the surfaces shown in (<b>D</b>-<b>F</b>). Scale bars correspond to 1µm.</p

Interaction forces of bacterial adhesin XadA1 on silicon and cellulose surfaces.

<p>Schematics of the force spectroscopy (<b>A</b>) measurements at five different positions on the surface where force curves were acquired. The force histograms (<b>B</b>-<b>D</b>) for bare silicon (Si, B) ethyl cellulose (EC, <b>C</b>) and cellulose acetate (CA, <b>D</b>), show the total number of events (please notice the different scales for the number of events); multiple rupture events were observed for EC and CA. Typical retraction force-distance curves for AFM tips coated with XadA1 adhesion protein (<b>E</b>) for Si (i), EC (ii) and CA (iii). The inset shows the fluorescence image of the functionalized AFM tip and cantilever visualized using fluorophore labeled second antibodies (scale bar = 40µm). Measured interaction forces (<b>F</b>-<b>H</b>) between XadA1 coated AFM tip and bare Si (<b>F</b>), EC (<b>G</b>) and CA (<b>H</b>) substrates in PBS buffer shown in sequential acquisition order. The colors indicate a different region of the sample probed in each set of force curves. The bin sizes for (<b>B</b>), (<b>C</b>) were adjusted to 22pN and (<b>D</b>) to 5pN to allow accurate visualization of the molecular interaction characteristics.</p

Surface potential of culture media conditioning film formed on pristine silicon.

<p>AFM topography (<b>A</b>) and surface potential (<b>B</b>) images showing the edge of the thin conditioning film caused by periwinkle wilt (PW) medium on silicon (Si) substrate (scale bar 2 µm). The corresponding profiles of height (1) and surface potential differences (2) are shown below.</p

Comparison of biofilm growth on pristine silicon and cellulose acetate surfaces.

<p>AFM topography images of <i>Xylella fastidiosa</i> biofilms grown on bare silicon (Si) and cellulose acetate (CA) surfaces side-by-side in periwinkle wilt (PW) medium (scale bar 5 µm). Images are shown with different height contrasts to illustrate individually the CA topography (<b>A</b>) and biofilm (<b>B</b>) more accurately. As a control image, please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075247#pone.0075247.s002" target="_blank">Figure <b>S2A</b></a> in supplemental information.</p

Biofilm architecture and quantitative biofilm size distribution.

<p>Size distribution histograms (<b>A</b>-<b>C</b>) and color enhanced SEM images of biofilms architecture (<b>D</b>-<b>I</b>) grown on bare silicon (Si; <b>A, D, E</b>), ethyl cellulose (EC; <b>B, F</b>, <b>G</b>) and cellulose acetate (CA; <b>C, H, I</b>) substrates in PW medium. Images E, G and I show details of the biofilm edges on each substrate at higher magnification. Scale bars correspond to 20µm for D, F, H and 5µm for <b>E</b>, <b>G</b>, <b>I</b>. The insets in <b>A-C</b> show re-scaled histograms to more precisely visualize the presence of larger biofilms.</p

Endoglucanase gene expression dependence on surface composition.

<p>Gene expression fold change for the three endoglucanases genes analyzed in the present study for (<b>A</b>) 7, (<b>B</b>) 14 and (<b>C</b>) 21 days of biofilm growth on bare silicon (Si), ethyl cellulose (EC) and cellulose acetate (CA) surfaces. One-way ANOVA with Tukey’s post hoc test was applied in (<b>A</b>), (<b>B</b>), (<b>C</b>). Asterisks denote significance level of α=0.05. Bars represent ± standard deviation.</p