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. 2018 Oct 10:4:23.
doi: 10.1038/s41522-018-0067-0. eCollection 2018.

Mucins trigger dispersal of Pseudomonas aeruginosa biofilms

Julia Y Co #  1   2 , Gerardo Cárcamo-Oyarce #  3 , Nicole Billings  1 , Kelsey M Wheeler  1   2 , Scott C Grindy  4 , Niels Holten-Andersen  4 , Katharina Ribbeck  1
Affiliations

Mucins trigger dispersal of Pseudomonas aeruginosa biofilms

Julia Y Co et al. NPJ Biofilms Microbiomes. .

Abstract

Mucus is a biological gel that lines all wet epithelia in the body, including the mouth, lungs, and digestive tract, and has evolved to protect the body from pathogenic infection. However, microbial pathogenesis is often studied in mucus-free environments that lack the geometric constraints and microbial interactions in physiological three-dimensional mucus gels. We developed fluid-flow and static test systems based on purified mucin polymers, the major gel-forming constituents of the mucus barrier, to understand how the mucus barrier influences bacterial virulence, particularly the integrity of Pseudomonas aeruginosa biofilms, which can become resistant to immune clearance and antimicrobial agents. We found that mucins separate the cells in P. aeruginosa biofilms and disperse them into suspension. Other viscous polymer solutions did not match the biofilm disruption caused by mucins, suggesting that mucin-specific properties mediate the phenomenon. Cellular dispersion depended on functional flagella, indicating a role for swimming motility. Taken together, our observations support a model in which host mucins are key players in the regulation of microbial virulence. These mucins should be considered in studies of mucosal pathogenesis and during the development of novel strategies to treat biofilms.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Natively purified mucins MUC5AC and MUC2 trigger the disruption of P. aeruginosa biofilms. a PAO1-GFP biofilms (48 h) were exposed to medium with increasing concentrations of mucins. The flow rate of the medium was 0.5 μL/min. At 0.5% (w/v) and above, MUC5AC reduced biofilm mass. Scale bars = 20 μm. b Confocal images of biofilms were analyzed using COMSTAT to quantify biomass after 1 h of exposure to mucins. Dotted lines indicate average values for 48 h biofilms before exposure (t = 0 h). Error bars represent standard error (n ≥ 3). *P ≤ 0.05, unpaired Student’s t test. c Mucin-mediated biofilm erosion is affected by the flow rate. Quantification of 48 h PAO1-GFP biofilm biomass after 1 h of exposure to LB with or without 0.5% mucins at slow flow (0.5 μL/min) or fast flow (10 μL/min). Values are normalized to biofilm biomass before exposure. Error bars represent standard error (n ≥ 3). *P ≤ 0.05, unpaired Student’s t test. A comparison between mucin MUC5AC and MUC2 shows that both mucins disassemble P. aeruginosa biofilms. d Live confocal imaging and e biofilm biomass quantification of 48 h PAO1-GFP biofilms before (t = 0 h) and after (t = 1 h) exposure to medium with or without 0.5% mucins at 0.5 μL/min flow. Scale bars = 20 μm. Error bars represent standard error (n = 3). *P ≤ 0.05, unpaired Student’s t test
Fig. 2
Fig. 2
Mucins disrupt P. aeruginosa biofilms without killing the bacteria. a Prolonged mucin exposure suppresses biofilm development. 48 h PAO1-GFP biofilms were exposed to medium with or without MUC5AC (0.5% w/v) over 20 h and the remaining surface-attached biofilm biomass quantified. Error bars represent standard error (n = 3). b Biofilms eroded by exposure to mucins remain viable. 48 h PAO1-GFP biofilms were first eroded via exposure to 0.5% MUC5AC for 1 h at 0.5 μL/min flow, then incubated for an additional 19 h in medium containing MUC5AC as indicated. Biofilms resumed development after being moved to mucin-free medium, indicating that eroded biofilms are still viable. Scale bars = 20 μm. c Quantification of biofilm biomass at 20 h. Dotted lines indicate value after the initial 1 h mucin treatment. Error bars represent standard error (n ≥ 3). **P ≤ 0.005, unpaired Student’s t test. d Exposure to mucins does not significantly impair P. aeruginosa viability. PAO1-GFP cells were grown in suspension in medium with or without 0.5% mucins. Colony-forming units (CFUs) were counted to assess cell viability. Error bars represent standard error (n = 3)
Fig. 3
Fig. 3
Viscous polymer solutions do not disrupt P. aeruginosa biofilms to the same extent as mucins. a Quantification of PAO1-GFP biofilms biomass (48 h) after exposure to 0.5% (w/v) polymer solutions in LB medium at 0.5 μL/min flow for 1 h shows that only mucins induce significant biofilm disassembly. Reported values represent biofilms after 1 h of exposure normalized to biofilms before exposure. Error bars represent standard error (n = 3). *P ≤ 0.05 versus medium-only treatment, one-way ANOVA. The medium-only treatment and non-mucin polymer treatments did not significantly differ from each other. b Viscosity of polymers (0.5% (w/v) solutions, n = 3) is not associated with the ability to disrupt P. aeruginosa biofilms
Fig. 4
Fig. 4
P. aeruginosa flagellar motility is required for mucin-associated biofilm erosion. a Live confocal imaging and b biofilm biomass quantification of PAO1-GFP (WT), PAO1ΔfliD-GFP, and PAO1ΔmotABCD-GFP biofilms (48 h) exposed to LB medium with or without 0.5% mucins at 0.5 μL/min flow for 1 h. Scale bars = 20 μm. Biofilm biomass was quantified using COMSTAT analysis of confocal images. Reported values represent biofilms after 1 h of exposure normalized to WT biofilms before exposure (t = 0 h). Error bars represent standard error (n ≥ 3). *P ≤ 0.05, unpaired Student’s t test
Fig. 5
Fig. 5
Exposure to mucins induces dispersal of P. aeruginosa biofilms under static growth conditions. a Mucins prompt active biofilm dispersal in P. aeruginosa. Live confocal imaging of 48 h PAO1-GFP biofilms before (t = 0 h) and after (t = 3 h) exposure to ABTG minimal medium or ABTG medium + 0.5% (w/v) MUC5AC. Scale bars = 20 μm. b Dispersed cells display a distinctive spatial organization. 3D representation of the cellular distribution of dispersed 48 h PAO1-GFP biofilms after 3 h of exposure to ABTG medium or to ABTG medium + 0.5% mucins. c Exposure to mucins reduces biomass of P. aeruginosa biofilms. Values are normalized to biofilm biomass before exposure. Error bars represent standard error (n ≥ 3). *P ≤ 0.05, unpaired Student’s t test. d Exposure to mucins increases the number of dispersed cells from P. aeruginosa biofilms. Viable dispersed cells were quantified by counting colony-forming units (CFUs). Error bars represent standard error (n ≥ 3)
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