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.

Introduction

Mechanosensing, including but not limited to responding to surface stiffness, is well-established to be an important cellular function in eukaryotes (1, 2). Much less is known about mechanosensing by prokaryotes (3–5). A few recent studies have shown that during biofilm formation, bacteria can sense and respond to mechanical cues, such as those arising from contacting a surface (6–12) and varying fluid flow over surface-bound bacteria (13, 14). For the biofilm-forming pathogen Pseudomonas aeruginosa, its cell-surface-exposed protein PilY1 has been proposed as a possible mechanosensor of surface adhesion (10, 13) and fluid shear (13). PilY1 is localized at the outer membrane (9, 10) and likely found at the tip of type-IV pili (TFP) as well (15). The extension and retraction of TFP power the twitching motility of P. aeruginosa on surfaces and are suggested to contribute to bacterial mechanosensing of surfaces (8, 11) and fluid shear (13).

In vivo, bacteria can experience a wide range of surface stiffnesses, from ultrasoft (dermal fillers have elastic moduli 0.02–3 kPa and living tissues 0.2–30 kPa) to hard (orthopedic implants have elastic moduli 5–300 GPa) (16, 17). In such diverse settings, biofilm formation commonly causes chronic infection, resulting in prolonged illness and high medical costs (18–20). Some research indicating that bacteria may be capable of sensing surface stiffness has recently emerged, showing that the initial accumulation of bacteria varied on surfaces of different stiffness (21–24). However, these earlier studies varied stiffness by varying characteristics such as cross-linking density or polymer concentration, which could also affect surface porosity or the density of attachment sites – in essence, changing at least two variables of the surface encountered. Furthermore, an inappropriate fabrication of surfaces with different stiffness may introduce unintended changes to other surface properties, such as adhesivity (SI discussion). Perhaps as a result, the literature on the effect of surface stiffness on bacterial accumulation on surfaces does not show consistent trends (21, 22, 24). Furthermore, there is currently very little understanding of how bacteria perceive the stiffness of surfaces to which they attach and how this input signal might allow the microbes to modulate their post-attachment accumulation accordingly. In addition, whether bacteria distinguish and respond to surface stiffness in later, post-accumulation stages of biofilm formation are also unknown. These knowledge gaps are of critical importance because they prevent the design of strategies for controlling biofilm development by manipulating surface stiffness.

To address these knowledge gaps, in the present study, we used thin and thick hydrogels coated on glass coverslips to create stiff and soft composite materials with the same surface chemistry, but different effective stiffnesses, and monitored P. aeruginosa through the early stages of biofilm formation on these surfaces. For surfaces exposed to a suspension of bacteria for one hour, we used quantitative microscopy to measure bacterial accumulation on materials of different effective stiffnesses. For the immediately-following stages of biofilm formation, characterized by bacteria reproducing on a surface rather than accumulating on the surface out of a suspended (planktonic) population, we measured the duration and growth rate of the biofilm lag phase and exponential growth phase, respectively.

For both the accumulation and reproduction stages of biofilm development, we show that bacteria actively recognize and respond to surface stiffness. When bacteria initially attach to a surface, both finite element modeling and experimental measurements of the activity of mechanosensitive ion channels show that attachment to stiff surfaces causes greater changes in the mechanical stress and strain state of the bacterial cell envelopes than does attachment to soft surfaces. PilY1 acts as a mechanosensor to transduce mechanical changes in the bacterial envelope into different intracellular levels of the second messenger cyclic-di-GMP (c-di-GMP). Using a proxy reporter for c-di-GMP levels, we measure higher levels of PilY1-dependent c-di-GMP production on stiffer surfaces than on softer. Higher levels of c-di-GMP lead to greater reduction in motility, a reduced likelihood of detachment, and, as a result, greater accumulation on the surface. Once the initial accumulation stage has passed, higher levels of cyclic-di-GMP are associated with a longer biofilm lag phase on stiffer surfaces.

Results

PilY1 allows P. aeruginosa to differentially accumulate on substrates of different stiffness.

To eliminate effects arising from physicochemical properties of surfaces other than stiffness, such as adhesivity or porosity, we fabricated thin and thick hydrogels atop glass coverslips. (Fig. 1A and Fig. S1 A). Different hydrogel thickness had the same chemical compositions and the fabrication methods used did not alter the surface chemistry or topography (Fig. S1 B and C, Fig. S1 F and G). We chose this geometry-based approach to modifying substrate stiffness to avoid inadvertently altering material adhesivity along with stiffness, which has been observed before (SI discussion) - for instance, poly(dimethylsiloxane) (PDMS) can have different surface adhesivities associated with different stiffnesses, shown by polymer beads found to accumulate more on soft PDMS than on stiff PDMS (25).

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.

To check that the composite materials we made had the same surface adhesivity regardless of gel thickness, we incubated both thin and thick hydrogel composites with a suspension of fluorescent polystyrene polymer beads for one hour, and imaged the number of beads attached using confocal microscopy. We verified that the numbers of polystyrene beads that attached did not significantly differ with hydrogel thickness (SI Discussion, Fig. S1 D and E). Thus, we conclude that hydrogel thickness does not impact passive physicochemical adhesion to surfaces.

However, the thickness of the hydrogel coated onto rigid glass coverslips does impact the stiffness of the resulting composite material. Linear elasticity theory was used to derive a closed-form expression for the effective elastic modulus (E_effective) of hydrogel-coverslip composites (SI Discussion and Equation. S7). For a hydrogel thickness (t_gel) comparable to the 1 micron size of a bacterium, the E_effective increases sharply with decreasing t_gel (Fig. 1B). According to this model, the composites with thin (~5 μm) hydrogels are approximately 16 times stiffer than those with thick (~150 μm) (Fig. 1B and Table S1). We also used a nanoindenter to experimentally impose loads on both types of composites that achieved indentation depth comparable to what bacteria realistically experience in the experiments (Fig. S1 H). The indentation for a given force and tip geometry was consistently less for the thin gel than for the thick gel, thereby validating that the composite made with thin gel is stiffer than the composite made with thick (SI Discussion, Fig. S1 H).

To assess the impact of surface stiffness on the accumulation of bacteria on the surface, we incubated the bacterial suspension for one hour with hydrogel-coverslip composites and measured the bacterial accumulation on these surfaces by visualizing the number of bacteria using phase contrast microscopy. Consistent with some previous reports (21–23) but not with others (24), wild type P. aeruginosa cells (WT) accumulated significantly more on stiffer composites than on softer (Fig. 1 C and D), as did mutants without TFP (ΔpilA) and mutants without the PilT retraction motor (ΔpilT) (Fig. 1 C and D), by a factor of ~3.3 for all three strains (Fig. 1E). Thus, while functional TFP can increase the “baseline” accumulation, they have no measurable impact on the greater likelihood of accumulating on stiffer surfaces. Similar effects were found for the more starkly-contrasting case of glass versus agarose gel surfaces, (SI Discussion, Fig. S2 A).

In contrast, mutants without PilY1 (ΔpilY1) accumulated equally on effectively-stiff and effectively-soft composites (Fig. 1 C and D). This indicates that P. aeruginosa requires the cell-surface-exposed protein PilY1 for distinguishing between, and responding to, different surface stiffnesses. Again, similar effects were found for the more starkly-contrasting case of glass versus agarose gel surfaces, implying a much more muted response to stiffness difference by ΔpilY1 (SI Discussion, Fig. S2 A).

Adhesive forces will tend to increase the area of the bacterium in contact with the surface, by deforming the bacterium and the surface. The energy costs for deforming the bacterium and the surface will depend on the elasticity of each. Mechanical equilibrium will be found by minimizing the sum of elastic energy costs (from cell and substrate deformation) and the adhesive energy benefit (from contacting area). Therefore, for constant adhesive area and bacterial elasticity, we expect that the deformation of the bacterial cell envelope will depend on the elasticity of the substrate. In Escherichia coli, cell membrane proteins can sense and respond to changes in mechanical stresses in the bacterial cell envelope resulting from surface attachment (3, 26, 27). We hypothesized that, upon adhesion to surfaces of different stiffnesses, P. aeruginosa should undergo different changes in mechanical stress and strain upon surface engagement, which might be perceived by the cell-surface-exposed protein PilY1. Notably, this type of mechanical stress is distinct from biological stress, involving unfolded or misregulated proteins in the cell envelope, which has also been shown to relate to surface sensing in P. aeruginosa (28).

Stiffer substrates lead to greater changes in mechanical stress and strain in the bacterial cell envelope.

To elucidate the relationship between surface stiffness and mechanical stresses in adhering bacteria, we developed finite element models (Fig. 2A, and Fig. S3 ) to simulate bacterial attachment to gel-coverslip composites. At the molecular level, bacterial surface properties and how they impact attachment to substrates are complex and not well-known (29). Therefore, we approximated the adhesion process by displacing bacteria into contact with surfaces (SI Discussion, Fig. S3 C and D). Using our models, we compared the bacterial envelope mechanics for bacteria interacting with stiff and soft surfaces for a range of contact-increasing displacements. For any given displacement, the total contact area is greater for bacteria on a soft surface than on a stiff one (Fig. 2B), reflecting the fact that the energy cost for deforming a soft material is lower than the cost for deforming a stiff one by the same amount. The initial, free-floating cells were subjected only to a turgor pressure (biologically, this arises from the osmolarity difference between the cytoplasm and the exterior), so that bacteria were in a pre-stressed state. Contact with a surface leads to a decrease in membrane stresses on the outer membrane, an increase in circumferential strain on the inner membrane, and the development of contact pressure (Fig. 2C, Fig. S4 A to I). These changes are all more pronounced when the surface is stiffer.

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.

To validate the trends shown by our modeling results, we compared the membrane tension in bacteria attached to gel-coverslip composites of different effective stiffnesses, by comparing the activity of mechanosensitive ion channels. These channels are located on the inner, cytoplasmic membrane (30) and act as transducers of membrane tension - closed when the membrane is at low tension and open when the membrane is at high tension, allowing ions to pass through (30, 31). The two major mechanosensitive ion channels are large-conductance- and small-conductance- (MscL- and MscS-, respectively) type channels. When open under increased membrane tension, these channels provide non-selective pores of large and small diameter, respectively, through which sodium ions, Na⁺, can pass in very similar ways (31). We pre-loaded bacteria with a fluorescent indicator for Na⁺ and then allowed them to sit for one hour attached to thin and thick agarose gels, in the presence of excess external Na⁺, before measuring the indicator brightness distribution as a proxy for internal Na⁺ levels.

The brightness distribution for bacteria on stiff substrates had a peak at 100–200 arbitrary units (a.u.), whereas the brightness distribution for bacteria on soft substrates had a peak, representing more than 60% of cells, at 0 to 100 a.u. (Fig. 2D). Both the median (Fig. 2D inset) and the mean fluorescence intensity of bacteria on stiff substrates were significantly greater than that of cells on soft substrates – cells on stiff substrates had a mean fluorescence intensity of 2840.70 a.u. [2217.75 a.u., 3463.65 a.u.] (95% confidence interval) and cells on soft substrates had a mean fluorescence intensity of 677.97 a.u. [478.28 a.u., 877.67 a.u.] (95% confidence interval). These results show that bacteria on stiff substrates are more permeable to Na⁺ than are bacteria on soft substrates. Since mechanosensitive ion channels increase permeability upon increased membrane tension, we interpret this finding as indicating that bacteria have higher membrane tension when attached to stiffer materials.

Thus, these experimental results are consistent with our modeling results and therefore support the idea that the assumptions underlying our modeling are reasonable in this regime.

Adhesion-induced changes can only happen following, not preceding, bacterial contact with surfaces. Since gel thickness does not impact physiochemical surface adhesivity (Fig. S1 D and E), we expect bacteria to have equal likelihood of encountering and initially sticking to stiff and soft substrates. Therefore, this is the first report, to our best knowledge, showing that greater accumulation on stiffer composites must arise as the result of something that happens after initial surface engagement – i.e., an active bacterial response to substrate stiffness. We hypothesized that WT initially adhered to thick hydrogels will be more likely to detach than cells initially adhered to thin hydrogels and that this difference should require PilY1.

PilY1 transduces substrate stiffness to adjust flagellar spinning and the detachment rate.

Shortly after encountering a surface, many P. aeruginosa cells are reversibly tethered by their flagella, which drive in-place spinning (32); spinning facilitates detachment from surfaces (33, 34). Deficiency in spinning is associated with decreased probability of detachment (33). Bacteria can also use TFP to move laterally on surfaces, but, during the first hour after bacteria were introduced to hydrogels (i.e., what we have termed the accumulation stage), we found that the vast majority of surface motility was in the form of spinning (Fig. S5 A and B). Therefore, we tracked the center-of-mass speed of surface-adhered bacteria (Fig. 3 A–D) as for a measure of spinning motility. We expect that a population with faster-spinning bacteria will have a higher rate of detachment (35).

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).

For WT, the distribution of spinning speeds on soft substrates was much broader than stiff substrates (Fig. 3E). Both the median (Fig. 3E inset) and the mean speeds on soft substrates were significantly higher than on stiff substrates - mean speed of 20.06 μm/min [18.43 μm/min, 21.68 μm/min] (95% confidence interval) on soft composites and mean speed of 11.46 μm/min [10.95 μm/min, 11.97 μm/min] (95% confidence interval) on stiff composites. In summary, WT are more likely to spin rapidly on soft substrates than on stiff substrates. Upon tracking cells, we indeed found that WT were significantly more likely to detach from soft substrates (30 detachment events among 673 tracked cells) than from stiff substrates (10 detachment events among 1609 tracked cells) (P 0.001, χ² test) (Fig. S5 C). This is an active bacterial response to substrate stiffness.

For the ΔpilY1 mutant, the peak spinning speed was unchanged from that of WT (Fig. 3 E and F), suggesting that loss of PilY1 does not intrinsically disrupt spinning motility. However, for the ΔpilY1 mutant, neither the distributions of spinning speeds nor the median spinning speeds were significantly different on stiff and soft substrates (Fig. 3F). The mean speed was 15.08 μm/min [14.16 μm/min, 16.01 μm/min] (95% confidence interval) on thin gels and 14.86 μm/min [13.92 μm/min, 15.81 μm/min] (95% confidence interval) on thick. Furthermore, the ΔpilY1 mutant was equally likely to detach from thin and thick gels (P = 0.78, χ² test) (Fig. S5 C). These results are strikingly unlike those for WT, and imply that P. aeruginosa lacking PilY1 do not adjust their spinning motility, and therefore their likelihood of detachment, in response to surface stiffness.

From these findings, we infer that PilY1 is required for early sensing of substrate stiffness in P. aeruginosa, and PilY1 is linked to regulating flagellar activity either up or down - increasing spinning speed on soft surfaces and decreasing spinning speed on stiff surfaces (Fig. S5 D and E). Notably, we find a linear correlation between spinning speed and the probability of detachment (Fig. S5 D and E) This finding raises the question of what provides the causative linkage between PilY1 and changes in flagellar activity.

Substrate stiffness impacts c-di-GMP signaling in a PilY1-dependent manner during accumulation.

The intracellular second messenger c-di-GMP is broadly used by many bacteria to regulate many cellular processes, including the sessile-to-motile transition, biofilm formation, and flagella-mediated motility (36). Therefore, to see whether PilY1 modulates c-di-GMP dynamics in response to surface stiffness, we used a validated reporter plasmid, P_cdrA::gfp, that produces green fluorescent protein (GFP) in response to increases in c-di-GMP (37); this plasmid was previously used to study c-di-GMP signaling in bacterial mechanosensing of shear (13).

For bacteria containing PilY1, we found a sharp rise in c-di-GMP levels during the initial hour of accumulation ( −1 to 0 h in Fig. 4 A and C) , which is consistent with previous findings that, c-di-GMP levels in P. aeruginosa increase upon surface attachment (9, 13, 38). At the end of the “accumulation” hour (i.e., the beginning of the incubation time), WT on stiff substrates had significantly higher c-di-GMP levels than did WT on soft substrates. This meshes with our finding that WT on stiff substrates had lower spinning motility than those on soft substrates (Fig. 3E), as high levels of c-di-GMP inhibit bacterial motility (36). It also suggests that the causative linkage between PilY1 and changes in flagellar activity (which, in turn, modulate the likelihood of detaching from the surface), is through PilY1-controlled c-di-GMP signaling.

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

On both soft and stiff substrates, the ΔpilY1 mutant had much lower c-di-GMP levels than did WT (Fig. 4 A and B); this is consistent with the role of PilY1 in regulating c-di-GMP production (9). The mean level of c-di-GMP at the end of the “accumulation” hour was ~2.9 times higher on stiff substrates than on soft for WT, while only ~1.4 times higher for the ΔpilY1 mutant (Fig. 4 A and B), consistent with a loss of the ability to discriminate surface stiffnesses. This finding is also consistent with the causative connection that PilY1 mechanosensing that controls c-di-GMP signaling levels is required for bacterial mechanoresponse to substrate stiffness during the initial “accumulation” phase.

This finding also raises the question of how PilY1 mechanosensing, and consequent changes in c-di-GMP signaling, impact the growth of the bacterial population on the surface.

PilY1 impacts biofilm growth in the lag phase, in response to surface stiffness, by modulating c-di-GMP signaling.

When planktonic bacteria are introduced into new liquid medium, they experience a temporary period of non-replication, termed the “lag phase” (39). After attachment to a glass surface, P. aeruginosa also undergoes a lag phase before exhibiting exponential growth (40, 41). However, unlike the planktonic lag phase, the lag phase of biofilm growth involves a combination of bacteria replication and detachment from surfaces, such that the population of surface-bound bacteria does not increase (40, 41).

After allowing bacterial accumulation on surfaces for one hour, we replaced the bacterial suspension with fresh, sterile culture medium. We designate this timepoint the beginning of the incubation time (0 h in Fig. 4). The duration of the lag phase, from the beginning of the incubation time to the onset of exponential growth, is given by the lag time, τ_lag, indicated by hatched color bars in Fig. 4. WT populations had a τ_lag of 4 h on stiff substrates and 1 h on soft substrates, but ΔpilY1 populations had the same τ_lag of 1 h on both substrate types (Fig. 4 D and E). Similar results were found for bacterial growth on bulk gels (soft) and glass slides (stiff) (Fig. S2 B and C). These results show that surface stiffness can markedly impact the growth of the sessile bacterial population, and that PilY1 is key for this process as well as for the accumulation preceding incubation. When PilY1 was complemented back on an arabinose-inducible plasmid, the ΔpilY1 mutant populations again had different τ_lag on stiff and soft substrates (SI Discussion, Fig. S6 C), confirming PilY1’s role in surface stiffness sensing.

On both stiff and soft substrates, c-di-GMP levels in WT fell during the lag phase and subsequently oscillated once populations entered the exponential growth phase (Fig. 4A). The high level of c-di-GMP induced by the initial mechanical stimulus of surface contact (0 h in Fig. 4A) allows bacteria to sense the surface and initiate a sessile lifestyle. However, it would be a metabolic burden for cells to maintain such high c-di-GMP levels in the following biofilm development. We speculate that bacteria may have to decrease the c-di-GMP level to allow the beginning of exponential biofilm growth on surfaces; this speculation is consistent with the work of others (42, 43). If so, the longer τ_lag for WT on stiff surfaces than on soft likely arises from the much higher initial c-di-GMP levels on stiff surfaces and the consequent need for more time to gradually decrease c-di-GMP levels. For ΔpilY1, low initial levels of c-di-GMP are associated with a short τ_lag on both stiff and soft substrates (Fig. 4 B and E).

We conclude that PilY1 is a required element for controlling P. aeruginosa’s initial c-di-GMP response to surface stiffness and consequent lag time in early biofilm growth.

Discussion

The mechanical equilibrium of a system consisting of a bacterium adhering to a surface will be found when the net mechanical energy is minimized. Adhesion energy, which is energetically favorable and negative in sign, will increase in magnitude as the adhering area increases. The elastic energy costs for deforming the bacterium and the surface, to increase the adhering area, are energetically unfavorable and positive in sign. Since the elastic energy cost for a given deformation of a soft surface is less than the elastic energy cost for the same deformation of a stiff surface, we expect more of the elastic energy cost to be borne by the bacterium when the surface is stiff than when it is soft. Therefore, for surfaces that have the same adhesive properties, as for the thick and thin gel composites we use here, equilibrium mechanics leads us to expect bacteria adhering to soft surfaces will deform less than will bacteria adhering to stiffer surfaces; this has been confirmed by finite element modeling (Fig. S3 A and B). Thus, adhesion will result in greater changes in envelope stress for bacteria attached to stiff surfaces than for bacteria attached to soft surfaces. This expectation is supported by both finite element modeling and experiments measuring differences in the membrane tension as reflected by the opening of mechanosensitive ion channels in the inner membrane.

Our experimental results show that PilY1 is a key sensor that transduces mechanical changes upon surface engagement into c-di-GMP signaling. PilY1 is a surface-exposed protein found associated with the TFP tip (9), so PilY1 may be responding to the compressive loading incurred due to surface adhesion, a stress state identified in the modeling. A recent study suggested that the conformational changes of PilY1 lead to stimulation of bacterial c-di-GMP production and biofilm formation (44). The compressive loading may hence engender the required conformational changes on PilY1 for biofilm initiation. Modeling shows that bacteria adhered to stiffer surfaces will have a greater decrease in the tension in their outer membrane than will bacteria adhered to softer surfaces. Our experimental results show that the bacteria adhered to stiffer surfaces have higher c-di-GMP levels, resulting in lower spinning motility, less detachment, and greater accumulation in the first hour after exposure to the surface. Later, bacteria attached to stiffer surfaces need a longer lag phase in which to reduce their c-di-GMP level and begin exponential growth.

At this exponential-growth phase of biofilm development, our data suggests that the pilus retraction motor PilT may also be involved in responding to surface stiffness in a way that modulates c-di-GMP level and growth rate (Fig. 4 C and F); see SI discussion. The differential response of mechanosensitive ion channels to surface stiffness (Fig. 2 D) opens the possibility that mechanosensitive ion channels may play a role in the initial development in biofilms on surfaces, although we have not investigated that specifically.

From equilibrium mechanics, changes in bacterial stiffness would be expected to change the deformation in bacterial cells upon surface attachment, as stiffer bacteria would deform less and softer bacteria would deform more. This, in turn, would alter the mechanosensing response to surface attachment. It has recently been shown that P. aeruginosa maintain tight genomic control of their stiffness (45). This clearly has benefits for protecting the bacteria against mechanical stress, such as osmotic pressure; our work suggests that this may also benefit bacteria by safeguarding the surface-sensing response, which is essential to this biofilm-former.

Finally, our study has implications for what types of bacteria are likely to mechanosense surface contact through an envelope protein. The effective modulus of our composites with thin gel was roughly 1 MPa and the effective modulus of composites with thick gel was slightly less than 100 kPa (Table S1). These values bracket the stiffnesses reported for P. aeruginosa and other Gram-negative bacteria (46–48). Bacteria themselves are a composite material, comprising the softer cytoplasmic interior and the stiffer envelope. The Young’s modulus for the envelope material per se of Gram-negative bacteria is roughly several tens of MPa, and the envelope material of Gram-positive bacteria probably has a similar modulus (49, 50). Our finite element modeling identifies bending as the major envelope deformation modality in the contact zone as bacteria attach to surface. According to the Kirchhoff-Love plate theory (51), the flexural rigidity of a thin plate (effectively the modulus that measures the energy cost for bending a plate) is characterized by Et³ /12(1−v²) ∝t³ , where E is the Young’s modulus of the plate, v is the Poisson’s ratio, and t is the plate thickness. Gram-negative bacteria have a much thinner peptidoglycan cell wall than do Gram-positive bacteria (the cell wall of P. aeruginosa (Gram-negative) is ~3 nm thick (52) and that of B. subtilis (Gram-positive) is ~30 nm thick (53)). Therefore, it is unsurprising that whole cells of Gram-positive bacteria appear to be stiffer (54) than Gram-negative bacteria (49, 55–57). This suggests that Gram-positive bacteria will deform less than will Gram-negative bacteria upon adhesion to the same surface, because the energetic cost for deforming Gram-positive bacteria will be higher, and therefore that Gram-positive bacteria may be less well-adapted to using surface proteins to sense and respond to surface stiffness. This inference is in agreement with previous reports that Gram-positive bacteria do not respond to surface stiffness in the same way as Gram-negative bacteria (58, 59).

In conclusion, the work presented here provides a new understanding of bacterial response to surface mechanics in early biofilm development, which may point the way to new approaches to control biofilm infection on devices by manipulating the mechanical properties of materials. In the current state of the art, antimicrobial coatings have been developed that kill bacteria (60, 61) and antifouling surfaces have been developed that resist the attachment of bacteria (60–64). Manipulating mechanical properties to hinder biofilm development has not been explored, and materials with biofilm-hindering mechanics could also incorporate antimicrobial or antifouling properties, potentially to synergistic effects.

Our study may also suggest how different environmental mechanics in vivo could affect the course of infection. Examples of important sites of biofilm infection where mechanics can vary include airway mucus in Cystic Fibrosis patients, which is stiffer and more amenable to biofilm formation than that of healthy people (65). Wounds that are kept moist are less susceptible to infection and have better healing than wounds that are allowed to dry (66); drying, by reducing water content, will act to increase stiffness.

Materials and Methods

We used P. aeruginosa PAO1 WT and ΔpilA, ΔpilT, ΔpilY1 mutants (67). Studies of bacterial accumulation, motility, growth, and c-di-GMP production were done with bacteria that contained the plasmid P_CdrA::gfp. This plasmid is a verified reporter for cyclic-di-GMP (c-di-GMP); the cdrA gene, and thus green fluorescent protein (GFP), is upregulated by c-di-GMP level (37). Strains containing a promotorless control plasmid pMH487 were used to measure background GFP expression independent of c-di-GMP levels (13). Strains with the P_CdrA::gfp and pMH487 plasmids were grown with 60 μg/mL Gentamycin (Sigma-Aldrich, G1914) for plasmid selection. Fluorescence measurements using the sodium ion indicator were performed using WT that did not contain any plasmid. Details of fabrication of hydrogel composites, microscopy measurement of bacterial accumulation, biofilm growth on surfaces and c-di-GMP signaling, finite element modeling of the cell-surface interaction, intracellular level of sodium ions in surface-adhered bacteria, tracking motility of surface-adhered bacteria, and image and data analysis are described in SI Materials and Methods.

Importance.

Bacteria colonize many types of biological and medical surfaces with a large range of stiffnesses. Colonization leads to the formation of biofilms, which cause costly and life-impairing chronic infections. However, whether and how bacteria can sense and respond to the mechanical cue provided by surface stiffness has remained unknown. We find that bacteria do indeed respond to surface stiffness in a way that is both consistent with expectations based on equilibrium continuum mechanics and that quantitatively impacts multiple aspects of early biofilm formation. This is a new understanding for the nascent field of bacterial mechanobiology. Furthermore, this finding suggests the possibility of a new category of approaches to hindering biofilm development by tuning the mechanical properties of biomedical surfaces.