Biofilms deform soft surfaces and disrupt epithelia
- Institute of Bioengineering and Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
Figures
Figure 1
Biofilms deform soft substrates.
(A) Illustration of experimental setup where we generate thin hydrogel films at the bottom surface of microchannels. These devices allow us to study biofilm formation on hydrogels reproducing …
Figure 2 with 2 supplements
Biofilms deform their substrate by buckling.
(A) Morphological parameters (maximum deformation amplitude) and (half max full width) computed from resliced deformation profiles. Dashed line indicates the baseline position of the gel …
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Figure 2—source data 1
Deformation amplitude and as a function of time and diameter.
- https://cdn.elifesciences.org/articles/56533/elife-56533-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Biofilm diameter-dependence of δmax and λ.
(A) Biofilm diameter-dependence of . (B) Biofilm diameter-dependence of λ. Both and λ linearly scale with the diameter d of the biofilm.
Figure 2—figure supplement 2
Hydrogel deformation field computed at different growth stages, superimposed with a brightfield image of the biofilm.
Scale bar: 20 µm. The force field at each timestep is normalized by its maximum displacement, thereby showing relative deformations.
Figure 3 with 1 supplement
Wild-type and rugose biofilms deform soft-substrates.
(A) Biofilm diameter-dependence of maximum deformation for rugose and smooth variants of P. aeruginosa. (B) Smooth variant of V. cholerae A1552 deforms hydrogels when growing in M9 medium, but not …
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Figure 3—source data 1
Deformation amplitude for WT strains.
- https://cdn.elifesciences.org/articles/56533/elife-56533-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Biofilm diameter-dependence of maximum deformation for the smooth variant of different V. cholerae strains grown in M9.
Data points correspond to different biofilms grown in two microfluidic chambers for A1552 and to biofilms grown in one microfluidic chamber for C6706 and N16961.
Figure 4 with 2 supplements
EPS composition drives biofilm and substrate deformations.
(A) Deformations of hydrogel substrates by V. cholerae Rg, rbma- and bap1- biofilms. Biofilms formed by rbma- and bap1- fail to deform the substrate. bap1- biofilms delaminate from the hydrogel …
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Figure 4—source data 1
Deformation amplitude for PAO1 matrix mutants.
- https://cdn.elifesciences.org/articles/56533/elife-56533-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Deformation behaviour for vpsL deletion mutant and complementation strains.
(A) VpsL deletion mutant can not form biofilms. Complementation of (B) V. cholerae rbmA and (C) bap1 deletion mutants (brightfield, top) restore the ability of the biofilm to deform the hydrogel …
Figure 4—figure supplement 2
P. aeruginosa biofilms on substrates with different stiffness.
Increasing hydrogel stiffness to 200 kPa induces delamination of biofilms, as observed on glass. Scale bars: 20 µm.
Figure 5 with 2 supplements
Biofilms generate large traction forces.
(A) Traction force microscopy measurements at the hydrogel-biofilm interface. The dashed line shows the edge of the biofilm. Traction force is largest at the biofilm center, reaching 100 kPa. (B) …
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Figure 5—source data 1
Deformation amplitude and for substrates of different stiffness.
- https://cdn.elifesciences.org/articles/56533/elife-56533-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Biofilm diameter-dependence of λ for substrates with different moduli.
λ scales linearly and it is not substrate-stiffness dependent.
Figure 5—figure supplement 2
Power-law relationship between deformation and substrate moduli (E).
Values for δmax/r were extrapolated from linear regression of the data points in Figure 5C for r = 50 µm (d = 100 µm).
Figure 6 with 3 supplements
Biofilms deform and disrupt epithelial cell monolayer.
(A) CMT-93 and MDCK cells grow at the surface of a soft ECM into a tight monolayer on which we seed a liquid inoculum of V. cholerae Rg. (B) Confocal images of uninfected (i) and infected (ii-v) …
Figure 6—figure supplement 1
Confocal images of uninfected (i) and infected (ii-iv) monolayers of Caco-2 cells.
Scale bars: 20 µm.
Figure 6—figure supplement 2
Biofilms perturb the viability of MDCK cell monalyers.
(i) Confocal in plane visualization of a Vc Rg biofilm growing on top of an MDCK cell monolayer stained with CellTracker, Hoechst and Calcein-AM. Cross-section visualization of the infected MDCK …
Figure 6—figure supplement 3
Biofilms increase the permeability of MDCK cell monolayers.
(i) Bright-field in plane image of an uninfected MDCK cell monolayer grown at the surface of a soft ECM. (ii) Confocal cross-section shows that uninfected monolayers are impermeable to FITC labeled …
Videos
Video 1
Timelapse visualization of V. cholerae Rg biofilm growth (brightfield) and corresponding hydrogel deformation (E = 38 kPa) in the xy and xz planes.
Scale bar 20 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (V. cholerae A1552) | Vc Rg | Yildiz and Schoolnik, 1999 | Rugose variant | |
| Strain, strain background (V. cholerae A1552) | Vc WT | Yildiz and Schoolnik, 1999 | Smooth wild-type variant | |
| Strain, strain background (V. cholerae A1552) | Vc Rg ΔvpsL | This study | In frame deletion of vpsL in rugose background obtained by matings of Vc Rg with S17 harboring deletion plasmid pFY_922 | |
| Strain, strain background (V. cholerae A1552) | Vc Rg ΔrbmA | This study | In frame deletion of rbmA in rugose background obtained by matings of Vc Rg with S17 harboring deletion plasmid pFY_113 | |
| Strain, strain background (V. cholerae A1552) | Vc Rg Δbap1 | This study | In frame deletion of bap1 in rugose background obtained by matings of Vc Rg with S17 harboring deletion plasmid pFY_330 | |
| Strain, strain background (V. cholerae A1552) | Vc Rg ΔrbmA pBADrbmA | This study | Vc Rg ΔrbmA harboring the plasmid pNUT1236 | |
| Strain, strain background (V. cholerae A1552) | Vc Rg Δbap1 pBADbap1 | This study | Vc Rg Δbap1 harboring the plasmid pBAD/Myc-His B | |
| Strain, strain background (V. cholerae N16961) | N16961 | Drescher et al., 2016 | Smooth wild-type variant | |
| Strain, strain background (V. cholerae C6706) | C6706 | Thelin and Taylor, 1996 | Smooth wild-typevariant | |
| Strain, strain background (P. aeruginosa) | PAO1 WT | Hickman et al., 2005 | ||
| Strain, strain background (P. aeruginosa) | PAO1 Rg | Rybtke et al., 2012 | In frame deletions of wspF | |
| Strain, strain background (P. aeruginosa) | PAO1 Rg Δpel | Rybtke et al., 2012 | In frame deletions of wspF, pelA genes | |
| Strain, strain background (P. aeruginosa) | PAO1 Rg Δpsl | Rybtke et al., 2012 | In frame deletions of wspF, pslBCD genes | |
| Strain, strain background (P. aeruginosa) | PAO1 Rg ΔcdrA | Rybtke et al., 2015 | In frame deletions of wspF, cdrA genes | |
| Cell line (Homo sapiens) | Caco-2 | ATCC | HTB-37 RRID:CVCL_0025 | |
| Cell line (Canis) | MDCK | Sigma Aldrich | 84121903-1VL RRID:CVCL_0422 | |
| Cell line (Mus musculus) | CMT-93 | ATCC | RRID:CCL-223 RRID:CVCL_1986 | |
| Recombinant DNA reagent | pFY_113 (plasmid) | Berk et al., 2012 | Plasmid for generation of in-frame rbmA deletion mutants | |
| Recombinant DNA reagent | pFY_330 (plasmid) | Berk et al., 2012 | Plasmid for generation of in-frame bap1 deletion mutants | |
| Recombinant DNA reagent | pFY_922 (plasmid) | Fong et al., 2010 | Plasmid for generation of in-frame vpsL deletion mutants | |
| Recombinant DNA reagent | pNUT1236 (plasmid) | Hartmann et al., 2019 | Arabinose-inducible plasmid containing the coding region of rbmA | |
| Recombinant DNA reagent | pBAD/Myc-His B (plasmid) | Fong and Yildiz, 2007 | Arabinose-inducible plasmid containing the coding region of bap1 | |
| Chemical compound, drug | Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) | Tokio Chemical Industries | ||
| Chemical compound, drug | PEGDA (MW 3400, 6000, 10000) | Biochempeg | ||
| Chemical compound, drug | PEGDA (MW 700) | Sigma-Aldrich | ||
| Software | Fiji | Fiji | ||
| Software | OriginPro | OriginLab Corporation | ||
| Software | MATLAB | Mathworks | ||
| Software | Imaris | Bitplane | ||
| Algorithm | 3D TFM | Toyjanova et al., 2014 | ||
| Other | SYTO9 stain | Invitrogen | S34854 | 10 µM |
| Other | CellTracker Orange CMRA stain | Invitrogen | C34551 | 10 µM |
| Other | Hoechst stain | Thermo Fischer Scientific | 62249 | 5 µg/ml |
| Other | Calcein-AM stain | Sigma Aldrich | 17783 | 5 µM |
Table 1
Molecular weight and concentrations of the precursors used for the generation of the hydrogels and resulting elastic modulus.
| Precursor | Concentration wt/vol | Modulus kPa |
|---|---|---|
| PEGDA MW 10000 (Biochempeg) | 10% | 12.1 ± 0.8 |
| PEGDA MW 6000 (Biochempeg) | 10% | 38.3 ± 1.0 |
| PEGDA MW 3400 (Biochempeg) | 10% | 30.9 ± 2.0 |
| PEGDA MW 700 (Sigma-Aldrich) | 15% | 203.3 ± 13.7 |
