Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa

Abstract

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.

Keywords: PilT; PilY1; Pseudomonas aeruginosa; bacterial mechanosensing; biofilm; biomechanics; cell envelope mechanics; cyclic-di-GMP; surface stiffness sensing.

Fig. 1.

More bacteria accumulate on stiffer surfaces during one hour’s incubation for initial attachment. (A) Schematic illustration of composites with different thicknesses of hydrogel, t_gel, on top of glass coverslips with constant thickness t_glass. (B) The effective Young’s modulus of the hydrogel-coverslip composite (E_effective), where E_{bulk gel} is the modulus of bulk hydrogel. (C and D) The accumulation of WT, ΔpilA, ΔpilT and ΔpilY1 on thin and thick hydrogel composites after incubating with surfaces for one hour. Data are means ± SD. ***P <0.001; NS, not significant (P = 0.28 for agarose; P = 0.29 for alginate); analysis of variance (ANOVA) test. (E) The ratio of accumulated bacteria on thin to that on thick hydrogel composites.

Fig. 2.

Adhesion to a stiffer surface leads to greater changes in mechanical stress/strain in the bacterial envelope and increased permeation of the bacterial cell membrane by sodium. (A) The finite element model and schematic illustration. Displacement along –X coordinate is applied on curve abc to bring the cell into contact with the surface. The heat map denotes the circumferential stress on OM (outer membrane). Note that the stress result on the substrate is not shown here. Inset: The representative elements analyzed in this study. (B) Contact area with different degree of indentation (displacement along –X coordinate). Contact area is normalized to the cellular surface area in the undeformed configuration. The dash line denotes when the cell first contacts the substrate. (C) OM stresses become less tensile whereas IM (inner membrane) strain increases at element #1 upon surface adhesion. The degree of changes is greater on a stiffer substrate. Contact pressure is greater on a stiffer substrate. Subscript c denotes the circumferential direction and subscript a denotes the axial direction. Stresses are normalized to their respective values during the free-floating state and strains are the net change with respect to their respective values during the free-floating state. (D) The histogram shows the average intracellular fluorescence intensity per cell of attached WT on thin and thick agarose gel composites after incubating with surfaces for one hour. Inset: Dot plot of the histogram, shown with median values. ***P <0.001; Mann-Whitney u test. This indicates a statistically-significant difference between fluorescence intensity distributions and between median fluorescent intensities for cells on thin and thick gel composites.

Fig. 3.

Adhered bacteria spin during the first hour of accumulation. (A-D), Phase contrast images of WT and the ΔpilY1 mutant adhered to thin and thick agarose gel composites. Insets: Tracked trajectories of bacterial centers-of-mass over 62.6 s. (E and F), Histograms showing speed distributions of WT and the ΔpilY1 mutant on thin and thick gel composites. Insets: Dot plots of the corresponding histogram. The median value is written to the right of each plot. ***P <0.001; Mann-Whitney u test. *** indicates a statistically-significant difference in the distributions of WT speeds on thin and on thick gel composites and that the median speed of WT adhered to thick gel composites was higher, with statistical significance, than that of WT to thin gel composites. In contrast, NS (not significant) indicates that there is no statistically-significant difference in the distributions of speeds or in the median speeds of the ΔpilY1 mutant on the two composite types (P = 0.66, Mann-Whitney u test).

Fig. 4.

On surfaces with different stiffnesses, PilY1 acts to mediate the duration of the lag phase in biofilm growth and the levels of the intracellular signal c-di-GMP, and PilT is required to mediate the growth rate of the exponential phase of biofilm growth. (A-C), Fluorescent reporter for changes in intracellular c-di-GMP in WT and the ΔpilY1 and ΔpilT mutants during accumulation, lag phase, and exponential phase. The initial hour of accumulation on a surface is designated by −1 to 0 h, shown by hollow color bars. For each sample, exponential phase was observed for two hours, shown by solid color bars. Squares represent mean levels of c-di-GMP at each time point, linked by lines as a guide to the eye. Shaded regions correspond to 95% confidence intervals. The inset in (E) shows c-di-GMP reporter intensity in the ΔpilY1 mutant with a smaller y-axis range. (D-F), Growth dynamics of attached WT, and the ΔpilY1 and ΔpilT mutants on thin and thick agarose gel composites. Data are means ± SD. The data at 0 time point corresponds to the end of one hour of bacterial accumulation on gel surfaces. Hatched color bars show the length of the lag phase. The doubling time, T, is calculated by the equation T = ln2/α, where α is the growth rate of bacteria on surfaces (equations of exponential regression, f(t) = A𝑒^αt , where t is the incubation time). **P <0.01; *P <0.05; NS, not significant; analysis of covariance (ANCOVA) test. ** and * indicate that the growth rate α_thin is significantly different from α_thick for WT and for the ΔpilY1 mutant, while NS means the difference in growth rates on thin and thick gel composites are not significant for ΔpilT (P > 0.1