Glycoside hydrolase processing of the Pel polysaccharide alters biofilm biomechanics and Pseudomonas aeruginosa virulence

Abstract

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.

Fig. 1. A pelA hydrolase mutant produces increased adherent biofilm biomass.

a Catalytic domain architecture of PelA. S, signal sequence; GH166, glycoside hydrolase family 166 domain referred to as PelA_h; Deacetylase; deactylase domain. * denotes the approximate location of the glycoside hydrolase catalytic variant residue, E218A. Crystal violet microtitre plate assay to quantify adherent biofilm biomass for the b P_BAD pel and c PA14 strain backgrounds, respectively. ΔpelF serves as a negative control. Error bars represent standard error of the mean of five independent trials. Statistical significance was evaluated using an ordinary one-way analysis of variance with Tukey corrections for multiple comparisons. ns, no significant difference, P ≥ 0.05; ***P < 0.001; and ****P < 0.0001. P_BAD pel, PAO1 ∆wspF ∆psl P_BADpel.

Fig. 2. CFU counts from P_BADpel and PA14 strains show no compromise in viable cell numbers.

CFU counts from planktonic cultures of indicated a P_BAD pel and b PA14 strains, respectively. Error bars represent standard error of the mean of three independent trials. CFU counts from biofilms for c P_BAD pel and d PA14 strains, respectively. Error bars represent standard error of the mean of three independent trials. Statistical significance was evaluated using an ordinary one-way analysis of variance with Tukey corrections for multiple comparisons. ns no significant difference, P ≥ 0.05; and ***P < 0.0001. P_BAD pel, PAO1 ∆wspF ∆psl P_BADpel.

Fig. 3. PelA hydrolase activity is responsible for generating secreted Pel.

Dot blot of crude cell-associated and secreted Pel samples from the indicated strains. Pel was detected using α-Pel primary antibody with HRP-conjugated secondary antibody. P_BAD pel, PAO1 ∆wspF ∆psl P_BADpel.

Fig. 4. Cell-associated Pel is a key component of biofilm microcolonies in flow cells.

a Representative confocal flow cell images stained with SYTO62 (pink) and the Pel-specific lectin WFL-FITC (green) to detect biomass and Pel, respectively. Scale bars = 100 μm. b Dot blot of crude cell-associated and secreted Pel fractions of the indicated strains. Pel was detected using WFL-HRP. P_BAD pel, PAO1 ∆wspF ∆psl P_BADpel.

Fig. 5. Secreted Pel contributes to the physical and mechanical properties of PA14 biofilms.

a Standing pellicle assay. Top, pellicles of the indicated strains grown in borosilicate glass tubes. Bottom, corresponding pellicles poured into a petri dish. b Congo red colony morphologies. c Representative images of water droplets on colony biofilms used for the contact angle measurements. d Illustration of contact angle (θ) measurement and its implication for surface hydrophobicity. e Contact angle hydrophobicity assay. Error bars represent standard deviation of four independent trials. f Young’s modulus quantified from uniaxial indentation rheology. Error bars represent standard deviation of eight independent trials. Statistical significance was evaluated using an ordinary one-way analysis of variance with Tukey corrections for multiple comparisons. ns, no significant difference, P ≥ 0.05; ***P < 0.001, ****P < 0.0001.

Fig. 6. PelA hydrolase activity reduces P. aeruginosa PA14 virulence.

a C. elegans slow killing assay Kaplan-Meier survival curve for PA14 strains. Data are pooled from three independent experiments (n = 105–120 worms per PA14 strain per experiment). Statistical significance was evaluated using a log-rank Mantel-Cox test. b C. elegans fast killing for PA14 strains. Survival was assessed after 24 h. Error bars represent standard error of the mean of eight independent experiments (n = 35–40 worms per PA14 strain per experiment). Statistical significance was evaluated using a one-tailed, unpaired Student’s t-test with Bonferroni corrections for multiple testing. c D. melanogaster oral infection Kaplan-Meier survival curve for PA14 strains. Data are pooled from six independent experiments (n = 20 flies per PA14 strain per replicate). Survival was assessed every 24 hours. Statistical significance was evaluated using a log-rank Mantel-Cox test. ns, no significant difference, P ≥ 0.05; *P = 0.01–0.05; **P = 0.001–0.01; and ****P < 0.0001.

Fig. 7. Summary of results and implications.

Schematic of a Properties that PelA hydrolase activity imparts to P. aeruginosa PA14 Pel biofilm biomechanics. b Additional properties implicated by our study. ROS reactive oxygen species.