1. Microbiology and Infectious Disease
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Biofilms deform soft surfaces and disrupt epithelia

  1. Alice Cont
  2. Tamara Rossy
  3. Zainebe Al-Mayyah
  4. Alexandre Persat  Is a corresponding author
  1. Institute of Bioengineering and Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
Research Article
Cite this article as: eLife 2020;9:e56533 doi: 10.7554/eLife.56533
6 figures, 1 video, 2 tables and 1 additional file

Figures

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 mechanical properties of host tissues. (B) In-plane and cross-sectional confocal visualizations show that V. cholerae Rg biofilms growing on hydrogels display large gaps at their core. (C) Embedding fluorescence tracer particle in the hydrogel films allow for visualization of deformations. V. cholerae Rg biofilms formed at the surface of the films deform the substrate. (D) P. aeruginosa Rg biofilms similarly deform the soft substrates. Hydrogel elastic modulus: (B and C) E = 12 kPa, (D and E) E = 38 kPa. Scale bars: (C and D) 100 µm, (B and E) 20 µm.

Figure 2 with 2 supplements
Biofilms deform their substrate by buckling.

(A) Morphological parameters δmax (maximum deformation amplitude) and λ (half max full width) computed from resliced deformation profiles. Dashed line indicates the baseline position of the gel surface. (B) Timelapse visualization of V. cholerae Rg biofilm growth (brightfield, top) with deformation (reslice, bottom). Dashed lines indicate biofilm position and size on the corresponding hydrogel profile. (C) Superimposition of these profiles shows the rapid deformation and the emergence of a recess at biofilm edges. Each color corresponds to the same biofilm at different times. (D) Time evolution of δmax shows a rapid increase after 6 to 7 hr of growth. (E) The dependence of δmax on biofilm diameter highlights a critical biofilm diameter dc above which deformation occurs. For D and E, each line color corresponds to a different biofilm. (F) Hydrogel strain field computed by digital volume correlation between 11 hr and 12 hr of growth. We superimposed the vector strain field with a brightfield image of the biofilm. For visualization purposes we only display data for the top right quarter of the biofilm shown in inset (dashed lines). E = 38 kPa. Scale bar: 10 µm for inset t = 0 hr in (B), else 20 µm.

Figure 2—source data 1

Deformation amplitude and λ 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 δmax. (B) Biofilm diameter-dependence of λ. Both δmax 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 in LB. (C) Biofilm diameter-dependence of maximum deformation for rugose and smooth variants of V. cholerae. Data points correspond to biofilms grown in two microfluidic chambers for PAO1 Rg, PAO1 WT and Vc WT and to biofilms grown in one microfluidic chamber for Vc Rg. E = 38 kPa. Scale bars: 20 µm.

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 surface. (B) Comparison of hydrogel deformations by P. aeruginosa Rg and cdrA- biofilms. (C) Dependence of maximum deformations on P. aeruginosa Rg, cdrA-, pel- and psl- biofilm diameter. All matrix mutants tend to generate weaker deformations compared to Rg. Data points correspond to different biofilms grown in two microfluidic chambers. (D) A model for the mechanism of biofilm deformation of soft substrates. Buildup of mechanical stress in the biofilm induces buckling. Adhesion between the biofilm and the surface transmits buckling-generated stress to the hydrogel, inducing deformations. E = 38 kPa. Scale bars: 20 µm.

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 (reslice, bottom). E = 38 kPa. Scale bars: 20 µm.

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) Deformation profiles generated by V. cholerae Rg biofilms of equal diameters on three hydrogels with different stiffness. (C) Biofilm diameter-dependence of maximum deformation for four different hydrogel composition representing a typical range of tissue stiffnesses. The softest hydrogel can deform up to 80 µm for a biofilm diameter of 220 µm. Data points correspond to different biofilms grown in one microfluidic chamber. Scale bar: 20 µm.

Figure 5—source data 1

Deformation amplitude and λ 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 δmax 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) monolayers of CMT-93 cells. Yellow arrow indicates gaps in the epithelial monolayer (ii and iii), blue arrow shows deformed ECM (iv). (C) Confocal images of uninfected (i) and infected (ii-iii) monolayers of MDCK cells, also showing delamination and rupture as illustrated in (D). Scale bars: 20 µm.

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 monolayer stained with Hoechst (ii), Calcein-AM (iii), Cell Tracker (iv) and merged visualizations (v, vi). (vii, viii) Confocal in plane visualization of the biofilm on a focal plane above (i). Scale bars: 20 µm.

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 Dextran. (iii) Bright-field in plane visualization of an MDCK cell monolayer infected with Vc Rg. The dashed line shows the approximate edge of the biofilm. Confocal in plane (iv) and cross section (v) visualization of FITC-labeled Dextran permeability through the damaged epithelium. Blue arrows show Dextran permeability through the biofilm in direct contact with the ECM, yellow arrows indicate Dextran permeability through epithelial cells junctions. Scale bars: 20 µm.

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
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (V. cholerae A1552)Vc RgYildiz and Schoolnik, 1999Rugose variant
Strain, strain background (V. cholerae A1552)Vc WTYildiz and Schoolnik, 1999Smooth wild-type variant
Strain, strain background (V. cholerae A1552)Vc Rg ΔvpsLThis studyIn 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 ΔrbmAThis studyIn 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 Δbap1This studyIn 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 pBADrbmAThis studyVc Rg ΔrbmA harboring the plasmid pNUT1236
Strain, strain background (V. cholerae A1552)Vc Rg Δbap1 pBADbap1This studyVc Rg Δbap1 harboring the plasmid pBAD/Myc-His B
Strain, strain background (V. cholerae N16961)N16961Drescher et al., 2016Smooth wild-type variant
Strain, strain background (V. cholerae C6706)C6706Thelin and Taylor, 1996Smooth wild-typevariant
Strain, strain background (P. aeruginosa)PAO1 WTHickman et al., 2005
Strain, strain background (P. aeruginosa)PAO1 RgRybtke et al., 2012In frame deletions of wspF
Strain, strain background (P. aeruginosa)PAO1 Rg ΔpelRybtke et al., 2012In frame deletions of wspF, pelA genes
Strain, strain background (P. aeruginosa)PAO1 Rg ΔpslRybtke et al., 2012In frame deletions of wspF, pslBCD genes
Strain, strain background (P. aeruginosa)PAO1 Rg ΔcdrARybtke et al., 2015In frame deletions of wspF, cdrA genes
Cell line (Homo sapiens)Caco-2ATCCHTB-37
RRID:CVCL_0025
Cell line (Canis)MDCKSigma Aldrich84121903-1VL
RRID:CVCL_0422
Cell line (Mus musculus)CMT-93ATCCRRID:CCL-223
RRID:CVCL_1986
Recombinant DNA reagentpFY_113 (plasmid)Berk et al., 2012Plasmid for generation of in-frame rbmA deletion mutants
Recombinant DNA reagentpFY_330 (plasmid)Berk et al., 2012Plasmid for generation of in-frame bap1 deletion mutants
Recombinant DNA reagentpFY_922 (plasmid)Fong et al., 2010Plasmid for generation of in-frame vpsL deletion mutants
Recombinant DNA reagentpNUT1236 (plasmid)Hartmann et al., 2019Arabinose-inducible plasmid containing the coding region of rbmA
Recombinant DNA reagentpBAD/Myc-His B (plasmid)Fong and Yildiz, 2007Arabinose-inducible plasmid containing the coding region of bap1
Chemical compound, drugLithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP)Tokio Chemical Industries
Chemical compound, drugPEGDA (MW 3400, 6000, 10000)Biochempeg
Chemical compound, drugPEGDA (MW 700)Sigma-Aldrich
SoftwareFijiFiji
SoftwareOriginProOriginLab Corporation
SoftwareMATLABMathworks
SoftwareImarisBitplane
Algorithm3D TFMToyjanova et al., 2014
OtherSYTO9 stainInvitrogenS3485410 µM
OtherCellTracker Orange CMRA stainInvitrogenC3455110 µM
OtherHoechst stainThermo Fischer Scientific622495 µg/ml
OtherCalcein-AM stainSigma Aldrich177835 µM
Table 1
Molecular weight and concentrations of the precursors used for the generation of the hydrogels and resulting elastic modulus.
PrecursorConcentration wt/volModulus 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

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