Research ArticleBACTERIAL BIOFILMS

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

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Science Advances  20 May 2016:
Vol. 2, no. 5, e1501632
DOI: 10.1126/sciadv.1501632
  • Fig. 1 The glycoside hydrolases PslGh and PelAh hydrolyze the exopolysaccharides Pel and Psl in a biofilm.

    Representative confocal images of Psl biofilms grown statically for 24 hours (top) and Pel biofilms cultivated for 48 hours (bottom) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.

  • Fig. 2 The glycoside hydrolases PelAh and PslGh catalyze the inhibition and disruption of P. aeruginosa biofilms.

    (A) Crystal violet staining of biofilms following the exogenous addition of glycoside hydrolases or catalytic variants. (B) Dose-response curves to examine the disruption of biofilm biomass by the exogenous treatment of each glycoside hydrolase and variant. (C) Dose-response curves to examine the prevention of biofilm biomass in the presence of various glycoside hydrolases. Each data point represents the mean from three independent experiments of n = 3 crystal violet microtiter plate wells. EC50 values were calculated using nonlinear least-squares fitting to a dose-response model. Error bars indicate SEM. ***P ≤ 0.001. NS, no significant difference.

  • Fig. 3 The glycoside hydrolases PelAh and PslGh are noncytotoxic.

    (A) IMR-90 cellomics assay to measure the length-to-width ratio (LWR) of the cells using CellTracker Orange CMRA. (B) IMR-90 fibroblast cell viability assay using PrestoBlue reagent. All data were normalized to a no-treatment control (100%). The C. difficile toxin TcdB was used as a positive control in cell morphology assays, and the detergent digitonin was utilized as a negative control in cell viability assays. Each data point represents the mean from three independent experiments of n = 3 cellomic and PrestoBlue measurements in microtiter plate wells. Error bars indicate SEM. ***P ≤ 0.001. NS, no significant difference.

  • Fig. 4 Glycoside hydrolases potentiate antibiotics and increase human neutrophil killing.

    (A) CFUs for PAO1 ΔwspF Δpsl PBADpel (left) and PAO1 ΔpelF PBADpsl (right) following growth in the presence or absence of glycoside hydrolases before treatment with colistin. The mean was calculated from LB agar plate counts from three independent experiments. (B) CFUs for biofilm cultures in (A) after treatment with glycoside hydrolases and colistin for 5 hours. The mean was calculated from LB agar plate counts from three independent experiments. (C) HL-60 neutrophil killing of strain PAO1 ΔwspF Δpsl PBADpel and PAO1 ΔpelF PBADpsl following biofilm formation and treatment with PelAh and PslGh and their catalytic variants, respectively. Percent killing was normalized to a no-treatment control. Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. NS, no significant difference.

  • Fig. 5 PelAh and PslGh catalyze the biofilm disruption from clinical and environmental isolates of P. aeruginosa.

    Isolates were grouped into categories as previously described, and the glycoside hydrolases PelAh and PslGh were exogenously added either individually or together and allowed to incubate for 2 hours. All strains were treated with 300 nM of each enzyme, with the exception of PA14 and CF127, which were treated with 1000 nM. Each data point represents the mean from three independent experiments of n = 3 crystal violet microtiter plate wells. Error bars indicate SEM. ***P ≤ 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/5/e1501632/DC1

    table S1. Strains and plasmids used in this study.

    fig. S1. Time course disruption of P. aeruginosa biofilms.

    fig. S2. P. aeruginosa biofilm disruption by glycoside hydrolases in the presence of serum.

    fig. S3. Biofilm prevention in standing culture pellicle assay.

    fig. S4. Protein stability of PelAh and PslGh in P. aeruginosa culture.

    fig. S5. The growth of P. aeruginosa in the presence of glycoside hydrolases.

    fig. S6. Protein stability of PelAh and PslGh in mammalian cell culture.

    References (7678)

  • Supplementary Materials

    This PDF file includes:

    • table S1. Strains and plasmids used in this study.
    • fig. S1. Time course disruption of P. aeruginosa biofilms.
    • fig. S2. P. aeruginosa biofilm disruption by glycoside hydrolases in the presence
      of serum.
    • fig. S3. Biofilm prevention in standing culture pellicle assay.
    • fig. S4. Protein stability of PelAh and PslGh in P. aeruginosa culture.
    • fig. S5. The growth of P. aeruginosa in the presence of glycoside hydrolases.
    • fig. S6. Protein stability of PelAh and PslGh in mammalian cell culture.
    • References (76–78)

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