Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa

Abstract

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia. The viscoelasticity of mucus is governed in part by low energy interactions that are difficult to assess. The CYS domain is a good candidate to support low energy interactions between GFMs and/or mucus constituents. Our aim was to stiffen the mucus from HT29-MTX cell cocultures and the colon of mice through the delivery of a recombinant protein made of hydrophobic CYS domains and found in multiple copies in polymeric mucins. The ability of the delivery of a poly-CYS molecule to stiffen mucus gels was assessed by probing cellular motility and particle diffusion. We demonstrated that poly-CYS enrichment decreases mucus permeability and hinders displacement of pathogenic flagellated bacteria and spermatozoa. Particle tracking microrheology showed a decrease of mucus diffusivity. The empirical obstruction scaling model evidenced a decrease of mesh size for mouse mucus enriched with poly-CYS molecules. Our data bring evidence that enrichment with a protein made of CYS domains stiffens the mucin network to provide a more impermeable and protective mucus barrier than mucus without such enrichment.

Figure 1

Mucus permeability in MTX:MTX-rCYSx12 cocultures. (a) Schematic representation of a monomer of the intestinal mucin Muc2 and the recombinant protein rCYSx12 (not to scale). The mucin CYS domain is depicted in blue and the S/T/P repeats carrying the numerous O-glycans are in grey. The recombinant protein rCYSx12 is made of 12 consecutive CYS domains. (b) Study design to obtain and to characterize mucus enriched in rCYSx12 from cell culture. MTX cells were transfected with a 20.2-kb vector carrying the first two exons, first intron, and 5 part of intron No. 2 of mouse Tff3. Intron No. 2 is followed by a third exon coding rCYSx12. The transgene is driven by the Tff3 promoter. A floxed Neo cassette (orange) under the control of a hybrid promoter LacUV5-SV40 was inserted to select recombinant clones. The white rectangles represent the vector backbone. After stable transfection, the native MTX cell line and recombinant MTX-rCYSx12 clones were amplified and cocultured at five ratios to obtain a range of rCYSx12 production. Production and secretion of rCYSx12 in a dose-dependent manner was observed by immunofluorescence of cell cocultures and by immunohistochemistry. (c) Distribution of beads in the mucus layer from MTX and MTX-rCYSx12 cells after 45 min sedimentation. (d) Beads distribution as a function of the mucus depth in MTX and MTX-rCYSx12 cultures. The width of each blob is proportional to the number of beads in each mucus section.

Figure 2

Decreased motility of P. aeruginosa in MTX mucus coculture enriched in rCYSx12. (a) Study design for the analysis of motile cell displacements in mucus overlaying MTX:MTX-rCYSx12 cocultures. Tracking and analysis were performed with Fiji and Icy, respectively. (b) Representative trajectories of P. aeruginosa in the mucus overlaying MTX and MTX-rCYSx12 cells. (c) Analysis of P. aeruginosa linearity in the mucus of the 5 MTX:MTX-rCYSx12 cocultures in situ using bean plots. (d) Analysis of P. aeruginosa swimming speed in the mucus of the 5 MTX:MTX-rCYSx12 cocultures in situ using bean plots. For (c,d), average and median are indicated by a horizontal bold line and a cross, respectively. The number (n) of tracked bacteria is given at the bottom of the figure and the statistical analyses were conducted using a two-sided Wilcoxon-Mann-Whitney test. Results are representative of three independent experiments.

Figure 3

Decreased cell motility in rCYSx12-enriched mucus scraped from mouse colons. (a) Study design for the analysis of motile cell displacements in mucus scraped from the colons of mice. Tracking and analysis were conducted with Fiji and Icy, respectively. (b) Representative trajectories of fluorescent S. enterica in the mucus from wild-type and transgenic mice. (c) Analysis of S. enterica linearity in mucus from wild-type and transgenic mice ex vivo using bean plots. (d) Analysis of S. enterica swimming speed in mucus from wild-type and transgenic mice ex vivo using bean plots. (e) Representative trajectories of spermatozoa in the mucus from wild-type and transgenic mice. (f) Analysis of spermatozoa linearity in mucus from wild-type and transgenic mice using bean plots. (g) Analysis of spermatozoa swimming speed in mucus from wild-type and transgenic mice using bean plots. For (c,d,f,g), and g, average and median are indicated by a horizontal bold line and a cross, respectively. The number of tracked cells (n) is indicated at the bottom of the figure and the statistics were conducted using a two-sided Wilcoxon-Mann-Whitney test. Results are representative of three independent experiments.

Figure 4

Microrheological analysis of CYS domain-enriched mucus scraped from mouse colons. (a) Study design for the analysis of PEG-bead diffusion. After checking their PEG coating, fluorescent beads were loaded in mucus scraped from the colons of wild-type and transgenic mice. Tracking and analysis of bead diffusion were conducted using Fiji and Icy, respectively. (b) Coating of 200 nm diameter YG beads (green fluorescence) with low-molecular-weight PEG. Effective coating was checked by incubation with avidin–rhodamine (red fluorescence). The avidin adsorbs onto uncoated beads (yellow fluorescence on merged pictures), but not beads coated with PEG (see the x3 enlargements). Scale bar = 50 µm. (c) MSD ± sem as a function of time for 200 nm PEG-coated beads embedded in mucus from wild-type (in blue) or transgenic (in purple) mice. (d) Diffusion coefficients distribution (t = 1 s) of 200 nm fluorescent beads embedded in colonic mucus from wild-type (in blue) or transgenic (in purple) mice. The average diffusion coefficient is represented by the vertical dashed line. (c,d) Data are averaged from 5 independent experiments. (e) Mucus mesh-size distribution calculated with the obstruction-scaling model in mucus from wild-type and transgenic mice. Data were pooled from 5 independent experiments. (f) MSD ± sem as a function of time for 1 µm PEG-coated beads embedded in mucus from wild-type (in blue) or transgenic (in purple) mice. (g) Diffusion coefficients distribution (t = 1 s) of 1 µm fluorescent beads embedded in colonic mucus from wild-type (in blue) or transgenic (in purple) mice. The average diffusion coefficient is represented by the vertical dashed line. (f,g) Data are averaged from 3 independent experiments. (h) MSD of 200 nm (filled circle) and 1 µm (empty square) particles in mucus of wild-type (WT, blue) and transgenic (Tg, purple) mice scaled by their radius R. (d,g) Diffusion coefficients lower than the uncertainty were excluded from the analysis.