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. 2013 Mar;12(3):420-9.
doi: 10.1128/EC.00287-12. Epub 2013 Jan 11.

Extracellular DNA release acts as an antifungal resistance mechanism in mature Aspergillus fumigatus biofilms

Ranjith Rajendran  1 , Craig Williams, David F Lappin, Owain Millington, Margarida Martins, Gordon Ramage
Affiliations

Extracellular DNA release acts as an antifungal resistance mechanism in mature Aspergillus fumigatus biofilms

Ranjith Rajendran et al. Eukaryot Cell. 2013 Mar.

Abstract

Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.

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Figures

Fig 1
Fig 1
Aspergillus fumigatus biofilms contain extracellular DNA. (a and b) Fluorescence micrographs of A. fumigatus (Af-GFP strain) biofilms grown for 24 and 48 h on Thermanox coverslips in a 24-well plate at 37°C. Biofilms were stained with the DNA-binding dye propidium iodide (PI). Color-saturated images of live Af-GFP-expressing cells (green) and PI-stained eDNA (diffuse red) or dead cells (punctate red) are shown. Yellow indicates the presence of both cells and eDNA. Magnification, ×200. (c) CLSM images of 24-h A. fumigatus biofilms on Thermanox coverslips at 37°C. The different planar images show PI-stained eDNA (diffuse red), which diffusely covers and intersperses with calcofluor white (blue)-stained hyphae. (d) High-magnification SEM image of 24-h A. fumigatus biofilm showing the presence of hyphae covered and interspersed with ECM. Magnification, ×2,000. The images shown are representative of three independent experiments. (e and f) Af293 biofilms grown in RPMI medium for 8, 12, 24, 48, or 72 h (e) or in RPMI medium, YPD, or YNB for 48 h (f) were gently washed with EDTA to remove the ECM, an MFA was used to produce a standard curve, and eDNA within the ECM was quantified. Experiments were performed in triplicate on 3 separate occasions. Data were analyzed by ANOVA, and Bonferroni's correction for multiple comparisons was applied (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (g) RAPD PCR was carried out on extracted genomic (G) and extracellular (E) DNA samples, _targeting seven different genes (G1, ANXC4; G2, BGT1; G3, CAT1; G4, LIP; G5, MAT1-2; G6, SODB; and G7, ZRF2), and the PCR products were electrophoresed in an agarose gel. Positive bands for seven genes indicate that the eDNA is similar in sequence to genomic DNA.
Fig 2
Fig 2
Extracellular DNA plays a role in biofilm structural integrity. (a and b) CLSM analysis. Mature 24-h biofilms were treated with vehicle control (a) or 0.25 mg/ml of DNase (b) for 24 h, and the resultant biofilms were assessed by CLSM imaging of the x-y, x-z, and y-z dimensions. Biofilms were stained with calcofluor white and propidium iodide. Artificially colored images of cells (white) and PI-stained eDNA (red) or dead cells (punctate red) are shown. (c) Biofilm depths with and without DNase were analyzed using Volocity software. The images shown are representative of three independent experiments. (d to f) Biomass analysis. Mature biofilms (d) and conidia (e) were treated with DNase (0, 0.25, 1, and 4 mg/ml), and the resultant biomasses were assessed using a crystal violet assay. (f) Salmon sperm (SS), C. albicans (Ca), and A. fumigatus (Af) genomic DNAs were added to conidia (0, 4, 8, and 20 mg/liter), and biofilms formed over 24 h at 37°C. The resultant biomasses were assessed using a crystal violet assay. Data were analyzed by ANOVA with Bonferroni's correction for multiple comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Fig 3
Fig 3
Autolysis is associated with extracellular DNA release. (a) Chitinase activity was assessed using a commercially available fluorometric assay for 8-, 12-, and 24-h biofilms, and also in the presence of azetazolamide (24 h + Aze). (b) eDNA release was assessed in the presence of azetazolamide for 8, 12, and 24 h, using the SYBR green MFA. RFU, relative fluorescence units. (c) Conidia were incubated with azetazolamide for 24 h, and the resultant biomass was assessed using a crystal violet assay. Data were analyzed by ANOVA with Bonferroni's correction for multiple comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (d to g) Fluorescence micrographs of A. fumigatus biofilm (24 h) controls (d and f) or biofilms grown in the presence of azetazolamide (e and g) on Thermanox coverslips. These biofilms were stained with calcofluor white and propidium iodide and imaged using Colorview software. Magnification, ×200 (d and e) or ×400 (f and g).
Fig 4
Fig 4
Extracellular DNA contributes to antifungal resistance. A checkerboard assay was designed to evaluate the combined effects of DNase and antifungal agents on the sMIC50. A. fumigatus Af293 biofilms were grown for 24 h at 37°C prior to treatment. DNase was serially double diluted in RPMI medium down each column (0 to 256 mg/liter), and amphotericin B (0 to 16 mg/liter) (a) or caspofungin (0 to 512 mg/liter) (b) was then serially double diluted across each row of a 96-well microtiter plate to create a concentration gradient. The biofilms were treated for 24 h prior to their viability being assessed using an XTT reduction assay. The green areas indicate metabolically active growth of A. fumigatus, and the black areas indicate the inhibition of metabolism (<50% of the control).
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