Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

Abstract

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure.

This work examined the presence and quantified the amount of eDNA in C.albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

introduction

Biofilms are structurally complex microconsortia of surface adhering cells embedded within a extracellular matrix (ECM) composed of substances derived from cell lysis or produced and secreted by cells [1]. The ECM contributes to the architectural preservation of biofilms by the maintenance of stable cell– cell and cell – surface interactions and interacts with the surrounding environment [2]. Although polysaccharides and proteins are the more extensively studied substances of biofilms ECM, other molecules such as lipids and nucleic acids play a crucial role on ECM functions [1]. In fact, extracellular DNA (eDNA) has been identified in the surrounding milieu as part of ECM of biofilms formed by both Gram-negative species such as Pseudomonas aeruginosa [3] and Gram-positive bacteria such as Bacillus cereus [4]. The proposed mechanisms implicated in eDNA release include: (i) cell lysis, (ii) quorum sensing and (iii) excretion from DNA containing vesicles [3]. Bacterial biofilms eDNA characterization revealed that it comprises fragments of high molecular weight (around 30 Kb) [5] that differ from genomic DNA, as indicated by the different profiles exhibited upon restriction endonuclease treatments or randomly amplified polymorphic DNA analysis [6, 7]. Evidence for a role of eDNA as a structural component of bacterial biofilms ECM arose from studies showing that eDNA is required at the initial stages of biofilm formation [8, 9]. Additionally, independent studies evaluating exogenous DNA and DNase effect on biofilm antimicrobial susceptibility [5, 10] showed an association between eDNA and biofilms increased antibiotic resistance.

Despite the intensive research in the bacterial field, the investigation focusing on eDNA in fungal biofilms is scarce.

Candida albicans is a polymorphic fungus that causes opportunistic infections in humans and its ability to form biofilms is well characterized [11]. C. albicans ECM abundance was recently shown to be regulated positively by the glucoamylases Gca1 and Gca2 and the alcohol dehydrogenase Adh5 and negatively by the alcohol dehydrogenases Csh1 and Ifd6 [12]. C. albicans ECM consists predominantly of carbohydrates but also includes proteins, hexosamine, uronic acid, and phosphorus [13]. Consistent with, the presence of phosporous, circumstantial evidence points to the presence of eDNA in ECM. Specifically, mature biofilms treatment with 50 μg/ml of DNase resulted in 30 % decrease of biofilm biomass, similarly to the observed for proteinase K treatments [13] More recently, eDNA was extracted from C. albicans 72 h biofilm ECM within micrograms per gram of biofilm wet weight[14]. Additionally, it has been demonstrated that the presence of eDNA is a common feature of biofilms formed by other Candida species [14].

However, further studies are required to extend the knowledge on the contribution of eDNA to C. albicans biofilms structure and matrix composition. Here we examined the presence of DNA in C. albicans biofilm ECM and the effect of DNase and the addition of exogenous C. albicans DNA on biofilm formation.

Material and Methods

Strain and culture conditions

The C. albicans wild-type strain SC5314 was used in this study.Cells were stored at -70°C in 20 % glycerol stocks and propagated by streaking a loopfull of cells onto Sabouraud dextrose agar medium (BD, Franklin Lakes, NJ, USA) supplemented with 100 mg l ^-1 ampicillin (Fisher Bioreagents, Fair Lawn, NJ, USA) and incubating at 30°C for 24 h. These stocks were stored at 4°C for no longer than two weeks. For all experiments, batches (30 ml in 125 ml flasks) of yeast extract-peptone-dextrose (YPD) medium [1 % (w/v) yeast extract, 2 %(w/v) peptone, 2 % (w/v) glucose] (US Biological, Swampscott, MA) were inoculated with freshly grown yeast cells in an orbital shaker at 30°C for 24 h. Cells were harvested by centrifugation and washed in sterile saline. Cells were counted with a haemocytometer and dilutions were made to prepare standardized cell suspensions (1 × 10⁶ cells/ml) in the appropriate pre-warmed growth medium (see below). Confirmation of the number and viability of cells was determined by plating serial dilutions on Sabouraud dextrose agar. Colony forming units were counted after 24-h incubation at 30°C.

eDNA estimation in C. albicans biofilm ECM

Biofilm formation under conditions of flow

Three different influent media were used (i) RPMI-1640 supplemented with L-glutamine (Mediatech Inc., Herdon, VA, USA), sodium bicarbonate [0.2 %(w/v)] and buffered with 0.165 M MOPS (Research Products International Corp., Mount Prospect, IL, USA), final pH 7, (ii) YPD and (iii) yeast nitrogen base (YNB) (Fluka, St.Louis, MO, USA) supplemented with glucose [0.9 % (w/v)] at final pH 7.The culture media used in the flow system were diluted in order to promote biofilm formation. Specifically, the chemically defined media RPMI and YNB were diluted 1:1 whereas the rich medium YPD was diluted 1:10. The media were supplemented with ampicillin 100 mg/l. Culturing conditions and apparatus were established as previously described [15]. Briefly, silicone elastomer (SE) strips (1 × 9cm ²) (Cardiovascular Instrument Corp. Silicone sheets, Wakefield, MA) were washed twice with ultrapure water, sterilized by autoclaving and pretreated overnight at 30°C with foetal calf serum (Sigma, St. Louis, MO, USA). C. albicans standardized cell suspensions (13 ml) were prepared using the same growth medium as the influent (1 × concentrated) and incubated with the SE at 37°C, 130 rpm for 90 min. After the adhesion period, the SE were transferred to the flow system and influent medium was set up at an initial flow rate of 0.75 ml/min [15]. Biofilms were formed at 37°C for 48 h. For subsequent analysis, two SE with biofilm were pooled together. Two independent experiments were performed for each growth medium condition.

Planktonic cultures (50 ml of standardized cell suspension per 125 ml flask; 1 9 concentrated YPD, RPMI, or YNB) were grown at 37C and 130 rpm for 48 h to validate the isolation of biofilm ECM (see the next paragraph).

Isolation of biofilm ECM

ECM was isolated as previously described [16] with slight modifications. Briefly, after formation the biofilms were transferred to polypropylene conical tubes (BD), resuspended in 10 ml of ultrapure sterile water and vortexed for 1 min. Next, biofilm cells were sonicated in an ultrasonic bath for 45 min, followed by a vortexing step of 2 min and centrifuged (2000 rpm; 20 min). The supernatant fraction was recovered and filtered through 0.2 μm acrodisc syringe filters with Supor Membrane (Pall Life Sciences, Ann Arbor, MI, USA) that are low protein and nucleic acids binding membranes. Age- and medium-matched planktonic cultures were run and processed in parallel with biofilms during ECM extraction to recover the liquid supernatants. Aliquots were taken before and after the sonication step and the confirmation of the cell viability was performed by CFU counts in Sabouraud dextrose agar medium.

ECM analyses

The DNA of the ECM fraction was extracted with an equal volume of phenol:chlorophorm:isoamyl alcohol (25:24:1, v/v) (Invitrogen, Carlsbad, CA, USA) and then precipitated with isopropanol (1:1). After centrifugation, the pellet was rinsed with 70% ethanol and air dried. The pellet was dissolved in 20 μl of TE buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA]. The same procedure was repeated for 5 ml of filtered supernatants of planktonic cultures.

DNA was quantified using the Quant-iT™ ds BR Assay kit (Molecular Probes, Invitrogen) according to the suppliers' instructions. This assay allows an accurate determination of DNA between 100 ng/ml and 1000 ng/ml. A λ DNA standard 45.7μg/ml was prepared in TE buffer from a commercially available standard solution (Promega, Madison, WI, USA) and run in parallel with samples in each assay. Additionally, a blank (TE only) was also included in each assay. Samples were read in the Quibit™ fluorometer (Molecular Probes, Invitrogen) and a new calibration was run for each assay. A minimum of one DNA measurement was performed for both biofilm and planktonic samples.

The protein content of the ECM fraction was estimated using the EZQ Protein Quantification kit (Molecular Probes, Invitrogen). Standards for the assay were prepared in nanopure water (Biorad, Hercules, CA, USA) with ovalbumin supplied with the kit. Standard concentrations range was between 0 and 0.25 mg/ml in order to include sample concentrations. Fotodyne UV 300 transiluminator (Fisher) was used for UV measurements. Standard curves were generated for mean UV intensity versus albumin concentration. Age- and medium-matched planktonic cultures supernatants were run in parallel. All measurements were made with three replicates.

Effect of DNase and extracellular DNA treatment on C. albicans biofilm formation

DNase I from bovine pancreas (Sigma) stock solutions were prepared before each experiment in 0.15 M NaCl supplemented with 5 mM of MgCl₂ (Merck, VWR, West Chester, PA, USA). The range of concentrations chosen was based on previously described titration data for bacterial species [17]. Working solutions of DNase in two-fold increments ranging from 0.02 to 2 mg/ml were prepared in RPMI medium.

C. albicans genomic DNA, to be added as exogenous DNA for C. albicans cells treatment, was extracted with the MasterPure™ Yeast DNA Purification kit (Epicentre Biotechnologies, Madison, WI, USA) according to the suppliers' instructions. C. albicans DNA purity and concentration were estimated by measuring the A₂₆₀ and A₂₈₀. It has been previously described that in C. albicans stationary phase planktonic cultures eDNA levels are higher than 0.1 ng/ml [18]. In this sense, physiological and supraphysiological concentrations ranging from 10 to 2560 to ng/ml, with two-fold increments, were chosen for this assay. Dilutions of the genomic DNA stock solution were prepared in RPMI medium. For control experiments, DNase was heat inactivated at 95°C for 10 min and RNase (Sigma) stock solutions were prepared in TE buffer.

DNase treatment and exogenous DNA addition at different stages of biofilm formation

Standardized cell suspensions (100μl) were inoculated into polystyrene, flat-bottomed, 96-well microtitre plates (Corning Inc., Corning, NY, USA) and incubated for 0, 1, 2 and 24 h at 37°C under static conditions. At 0-h (preincubation), DNase was added to the standardized suspension. At 1-, 2- and 24-h of incubation the medium was removed and adhered cells washed two times with sterile PBS (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4) (Sigma). DNase working solutions were then added to C. albicans cells. The plates were incubated at 37°C for additional 24 h. The same procedure was performed for the addition of exogenous DNA to C.albicans cells. Positive controls for biofilm formation included RPMI only and other RPMI plus 5 mM of MgCl₂ for DNase experiments. Negative background control for subsequent analysis consisted of RPMI medium only. Four independent assays with three to eight replicates for each condition were performed. The effect was examined in terms of biofilm biomass changes using the crystal violet assay (see below) As controls for DNase enzymatic activity, RNase and heat inactivated DNase were added at 24 h as described above. This experiment was performed once with eight replicates.

The effect of DNase and/ or exogenous DNA on C. albicans viability and filamentation was examined in planktonic cultures and on preformed biofilms. For planktonic cultures, 5 ml of the standardized cell suspension plus (i) 5 mM of MgCl₂, (ii) 2 mg/ml DNase or (iii) 2560 ng/ml of exogenous DNA were incubated at37°C, 130 rpm for 2 h. Viability was examined by CFU counts in Sabouraud dextrose agar medium. Filamentation was monitored by light microscopy using an inverted microscope (Westover Scientific, Mill Creek, WA, USA). This experiment was performed twice. The highest concentrations of DNase and exogenous DNA used in these experiments did not have an effect on C. albicans planktonic cells viability and filamentation.

For preformed biofilms, DNase ranging from 2 to 0.02 mg/ml was added to 24-h mature biofilms as described above and incubated for an additional 24 h. Next, cells were removed with a micropipette tip, re-suspended in saline solution and CFU analysis carried out. Light microscopy examination of biofilms was performed in parallel. All the treatments were compared with the untreated (RPMI and RPMI plus 5 mM MgCl₂) controls..

Examination of the effect of DNase treatment and exogenous DNA on biofilm formation

The effect of DNase and eDNA on C. albicans biofilm biomass was estimated using the crystal violet assay. Briefly, the medium was removed and biofilms washed twice with PBS. Then, biofilms were stained with 50 μl of crystal violet 3 g/l [0.3 % (w/v) crystal violet (Sigma), 5 % (v/v) isopropanol, 5% (v/v) methanol, 90 % (v/v) water] for 5 min. Afterwards, each well was washed twice with PBS, air dried and destained with 100 μl of ethanol 100%. Next, 75 μl of the destaining solution was transferred to a new microtiterplate, and the A₅₅₀ was measured (Benchmark Microplate Reader, Bio-Rad, Hercules, CA, USA). Samples exhibiting very intense color, yielding “offscale”absorbance values, were diluted 1:2 before performing a second absorbance reading.

Statistical analyses

The association between the eDNA/ mg of protein in the different experimental conditions was analysed using the chi - square test with the Yate's correction[19]. A statistical confidence interval of 95% was established. The comparison between (i) log CFU/ml before and after sonication and (ii) each biofilm treatment and control condition was performed by two-tailed unpaired t-test (confidence interval 95 %), using GraphPad Prism, version 5.00 software for Windows (GraphPad Software, San Diego, CA, USA). Differences were considered significant at P< 0.05. Statistical analyses were performed.

Results

Presence of eDNA on C. albicans biofilm ECM

Three different media were tested for their ability to promote the accumulation of eDNA in the ECM of 48-h C. albicans biofilms developed under flow conditions.

In the extraction of ECM it was mandatory to ensure that there was no leak of cellular DNA during sample processing. To validate the ECM extraction process age- matched planktonic cultures were handled in parallel with biofilm samples. Before and after samples processing, aliquots were collected and CFU analysis carried out. Difference between means log CFU/ml before and after sonication were of: 0.00 (P= 1.00) for RPMI, -0.07 (P=0.39) for YPD and 0.00 (P= 1.00) for YNB.

As shown in Table 1, irrespective of which medium was used, eDNA was detected at measurable levels in ECM of C. albicans biofilms. Under the conditions used in this assay, the highest levels of eDNA in ECM were observed for biofilms formed using RPMI medium (3045.4 ± 227 ng eDNA/mg of protein). These levels were 9-fold higher than those observed in the ECM of biofilms grown in YPD (P< 0.01). Both the ratio of eDNA/protein and absolute quantities of eDNA were lowest in the ECM from YNB-grown biofilms when compared to biofilms grown in RPMI and YPD (P< 0.01) (Table 1). The amount of free DNA determined in the supernatant of 48-h grown C. albicans planktonic cultures revealed the following hierarchy of eDNA accumulation relative to the amount of protein: YPD > RPMI > YNB (P< 0.01) (Table 1).

Table 1.

C. albicans 48-h biofilm and planktonic cultures eDNA and protein content. DNA was extracted and further quantified. Protein content was directly assayed from the samples. Data presented represents mean ± mean deviation.

Culture	Medium	eDNA(ng)	Protein(mg)	eDNA/ protein(ng/mg)
Biofilm ECMa
	RPMIc	1518 ± 822	0.48 ± 0.2	3045.4 ± 227.3
	YPDd	180 ± 160	0.42 ± 0.1	339.6 ± 265.6
	YNBc	1.8 ± 1.6	0.10 ±0.1	13.0 ± 8.8
Planktonicb
	RPMI	4.4 ± 2.1	2.4 × 10-2 ± 1.7 × 10-2	231 ± 71.4
	YPD	85 ± 1.4	3.4 × 10-2 ± 7.1× 10-3	2661.3 ± 607.7
	YNB	1.8 ± 0.7	2.4 × 10-2 ± 5.4 × 10-4	73.3 ± 31.7

^aobtained from biofilms developed under flow conditions, corresponding to 18cm² of area

^bper ml of culture

^cused at 1:1 dilution

^dused at YPD 1:10 dilution

This data shows that eDNA is a component of C. albicans biofilm matrix and suggests that its accumulation in the ECM is affected by culture conditions.

Effect of DNase treatment on C. albicans biofilm formation

In order to examine the possible role of eDNA on C. albicans biofilm development, in a first set of experiments DNase was added to cells at different times (0, 1, 2 and 24 h) post-inoculation in the wells of microtiter plates and biofilms formation allowed to proceed for an additional 24 h (Fig. 1).The results of these experiments, assessed by a colorimetric assay for biofilm biomass showed that DNase did not exhibit major effects on C. albicans adherent cells at early time points during biofilm development. DNase was also used to treat preformed biofilms (24 h) to determine whether different concentrations of this enzyme could affect the formed biofilms (Fig. 1). As shown in Fig. 1, DNase showed a general inhibitory effect on C.albicans preformed biofilms. This was reflected by lower crystal violet readings at DNase concentrations higher than 0.03 mg/ml. For example, biofilms treated for 24 h with 0.13mg/ml DNase showed a reduction of 40 % in A₅₅₀ readings when compared with the control (P= 0.03). Furthermore, preformed biofilms treatment with RNase and heat inactivated DNase at the same concentrations used for DNase did not display a decrease in biofilm biomass (data not shown), demonstrating that DNase activity is required for the observed effect.

Fig. 1.

Effect of DNase treatment on C. albicans biofilm formation. DifferentDNase concentrations (0, 0.02, 0.03, 0.06, 0.13, 0.25, 0.50, 1.00 and 2.00 mg/ml) were added to C. albicans cells at different times (0, 1, 2 and 24 h) post-inoculation in the wells of microtiter plates and incubated at 37°C under static conditions. The extent of biofilm formation was estimated by the crystal violet assay. Presented values are mean A₅₅₀ ± standard error of mean of four independent experiments with three to eight replicates. Statistically significant differences (compared to biofilms formed in the absence of DNase) are indicated with an asterisk. (*, P < 0.05).

Additionally, it should be noted that microscopic examination of biofilms suggested that the DNase treated biofilms resembled the control biofilms before washing in terms of density and filamentation (Fig. 2-I). However, during the biofilm processing for the determination of biofilm biomass, the cells did detach from the microtiter plate surface (Fig. 2-II), resulting indecreased A₅₅₀. Accordingly, the assessment of cell viability of pre-washed biofilms did not reveal a detrimental effect of DNase on biofilm cells viability (compared with the control). For example, for 24 h treatment with 0.13 mg/ml DNase log CFU/well was 6.1 vs.6.5 for control biofilms.

Fig. 2.

Example of microscopy evaluation of the effect of DNase treatment on 24 h C.albicans biofilm formation. Microphotographs of cells in wells before (I) and after (II) aspiration of medium and subsequent washings with PBS and crystal violet staining for biofilms treated with 0 mg/ml (a and c) and 0.13 mg/ml of DNase (b and d). The bar in the picture represents 200 μm.

This suggests that the observed effect induced by DNase treatment is not due to changes in biofilm cells growth or filamentation, but rather due to a direct effect on the biofilm ECM which is important for biofilm integrity and maintenance.

Effect of addition exogenous DNA on C. albicans biofilm formation

To further substantiate the effect of eDNA as a structural component of biofilms, exogenous DNA was added to C. albicans cells. As shown in Fig. 3, the addition of exogenous DNA to C. albicans adhered cell populations did not affect further biofilm development. In contrast, addition of exogenous DNA, at concentrations higher than 160 ng/ml to mature biofilms led to increases in biofilm biomass , as indicated by the differences in A₅₅₀ readings compared with control biofilms (grown in the absence of exogenous DNA) (Fig. 3). As an example, under the exposure to 320 ng/ml of exogenous DNA, biofilm biomass increased up to 63 % in comparison with untreated biofilms (P= 0.03).

Fig. 3.

Effect of addition of exogenous DNA on C. albicans biofilm formation. Different eDNA concentrations (0, 20, 40, 80, 160, 320, 640, 1280 and 2560 ng/ml) were added to C. albicans cells at different times (0, 1, 2 and 24 h) post -inoculation in the wells of microtiter plates and incubated at 37°C under static conditions. The extent of biofilm formation was estimated by the crystal violet assay. Presented values are mean A₅₅₀ ± standard error of mean of four independent experiments with three to eight replicates. Statistically significant differences (compared to biofilms formed in the absence of DNA) are indicated with an asterisk. (*, P < 0.05).

These experiments indicate that eDNA may contribute to the maintenance and stability of C. albicans mature biofilms, but is not required for the early stages during establishment of biofilms.

Discussion

In recent years there has been an increasing interest in the natural sources and target effects of the nucleic acids found outside the cells in several biological systems[20]. However, the study of eDNA in C. albicans biofilms has not been the subject of extensive research. Here, a series of experiments were designed to specifically address the contribution of eDNA to C. albicans biofilms matrix.

For this, we first determined the eDNA content from the ECM of 48-h biofilms using three different culture media, to ensure that eDNA is a component of ECM under different environmental growth conditions. Biofilms were developed in a flow model due to the predicted higher amounts of exopolymeric material that could be obtained when compared to biofilms developed under static conditions [15, 21]. Regardless of the growth medium used, eDNA was found to be a component of C. albicans ECM in all conditions used (Table 1). In this study, it was noticed that the amount of eDNA varied considerably with the growth medium used, with RPMI favouring the accumulation of eDNA in the ECM of C. albicans biofilms (Table 1). As the DNA content in the effluent medium was not determined it can not be discarded that biofilm cells grown in YPD released more eDNA compared with RPMI but the ECMamount/ composition of RPMI biofilms promotes the accumulation of DNA. It should be noted that the association between ECM eDNA content and growth medium was observed for biofilms formed in flow model.

The release of eDNA into the extracellular medium is not biofilm cells specific. It has been previously reported the release of DNA into the supernatant of C. albicans planktonic cells grown in RPMI and Hank's balanced salt solution [18]. The highest levels of eDNA attained in the stationary phase of growth have been suggested to result from cell death due to the nutrient depletion and toxic metabolites accumulation [18]. We also determined the amount of free DNA in the supernatant of 48-h grown C. albicans planktonic cultures and observed the following hierarchy of eDNA accumulation rela-tive to the amount of protein: YPD [RPMI [YNB (unpublished data). DNA was also present in C. albicans supernatants of 48-h planktonic controls (Table 1).

In bacteria, it was proposed that quorum sensing is one of the mechanisms that contributes to increased release of eDNA into the environment [22]. Recently, the eDNA content of C. albicans chk1/chk1 ECM was determined [14], which is a mutant strain known to be nonresponsive to the C. albicans quorum sensing molecule farnesol [23]. The ECM of the mutant strain contained only slightly lower eDNA and protein levels/biofilm wet weight in comparison with the wild-type strain [14], suggesting that DNA release by C. albicans biofilm cells may not be a controlled step linked to cell-cell communication through farnesol.

Regardless the factors that promote eDNA accumulation it was important to address the role of this ECM component in C. albicans biofilms. Circumstantial evidences have shown that DNA may play a structural role in C. albicans 48-h biofilms [13]. In this sense, we evaluated the impact of eDNA removal by DNase (Fig. 1) and by the addition of exogenous DNA (Fig. 3) at different stages of C. albicans biofilm formation. For the determination of the effect of DNase and C. albicans exogenous DNA on biofilms, a rapid and robust method, such as the 96-well plate model [24] that allows testing of multiple parameters in the same experiment was preferred. Furthermore, RPMI medium was the choice medium for the following experiments because it provides an optimal pH for DNase activity. Results from these series of experiments demonstrate that eDNA is an important component of C. albicans mature biofilms contributing to its maintenance and stability but is not required for the establishment of biofilms or during the very early phases of biofilm development.

Although it has not been determined how biofilms grown in 96-well plates compare to those developed under flow conditions the conjugation of the data obtained from the two models This is consistent with a predominant role for eDNA as a component of the biofilm ECM in providing structural integrity required for biofilm maintenance of mature biofilms. However, we note that eDNA role in Candida biofilms may be species/strain dependent since a previous study reported that C. tropicalis biofilm biomass was not affected by 50 μg/ml DNase treatment [13]. Data presented in this work also points to a differential role of eDNA among C. albicans and bacterial biofilms. In fact, for several bacterial species eDNA is required for the initial attachment as well as for subsequent early phases of biofilm formation, but only for some species DNA was found to be an important structural component of mature biofilms [8, 9].

In the last years several lines of evidence suggested that in vivo eDNA has a functional activity in the regulation of host immune response. On one hand, it was demonstrated that the administration of C. albicans DNA to mice at specific time points prior to infection promoted mice survival [25, 26]. On the other hand, it was demonstrated that after C. albicans infection of nonneutropenic rabbits fungal eDNA was detected in the plasma within the first 24 h and increased over the course of disease [18]. This eDNA may be available for interaction with Toll-like receptor 9 (TLR9), an intracellular fungal DNA receptor, as evidenced by studies showing C. albicans eDNA activation of bone marrow-derived myeloid dendritic cells through the TLR9/ Myeloid differentiation factor 88 signalling pathway [27] and the increased survival of TLR9 deficient mice upon challenge with C. albicans hyphae [28]. We can hypothesize that eDNA present on C. albicans biofilms may contribute to the modulation of host immune response. Overall, this work reveals novel aspects of the role of eDNA on C. albicans biofilms and properties of biofilm ECM that contribute to a better understanding of C. albicans biofilm lifestyle.