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Medical devices provide an ecological niche for microbes to flourish as a biofilm community, protected from antimicrobials and host defenses. Biofilms formed by Candida albicans, the most common fungal pathogen, survive exposure to extraordinary high drug concentrations. Here we show β-glucan synthase Fks1p produces glucan which is deposited in the biofilm matrix. The extracellular glucan is required for biofilm resistance and acts by sequestering antifungals, rendering cells resistant to their action. These findings provide the genetic basis for how biofilm matrix production governs drug resistance by impeding drug diffusion and identify a useful biofilm drug target.
Candida spp. are the 4th most frequent cause of bloodstream infections in hospitalized patients . Their ability to grow as biofilms on medical devices is in part responsible for the escalating disease prevalence. Despite a growing antifungal armamentarium there remains no effective medical treatment of this disease due to the profound resistance associated with biofilm growth. The discovery that Candida biofilm cells assemble an extracellular matrix composed of β-glucan led us to investigate and identify a contribution of this material to biofilm resistance .
The current studies examine of the role of β-1,3 glucan synthase gene FKS1/GSC1 in triazole drug susceptibility, matrix production, and drug sequestration during C. albicans biofilm growth. Fks1p utilizes UDP-glucose to synthesize cell wall β-1,3 glucan during planktonic growth . By examining strains with modulated FKS1 expression, we found that β-1,3 glucan synthesis and deposition in the matrix is necessary for biofilm drug resistance, but is not needed for planktonic cell resistance. This carbohydrate traps the azole drug, fluconazole, preventing its intracellular action.
Animals were maintained in accordance with the University of Wisconsin animal care guidelines.
Yeast strains were stored in 15% glycerol at -80°C and maintained on yeast extract(1%)-peptone(2%)-dextrose(2%) medium with uridine 80 μg/ml prior to experiments.
Strains were constructed from BWP17 (genotype ura3λimm434/ura3λimm434, arg4hisG/arg4hisG, his1hisG/his1hisG) and reference strain DAY185 . Heterozygous deletion mutant FKS1/fks1Δ was constructed by PCR product-directed disruption using template plasmid pFA-URA3 and primers FKS1-S1 and FKS1-S2 . Deletion was confirmed by PCR with primer sets (FKS1-G1,U2 and U3,FKS1-G4). Overexpression strain TDH3-FKS1 was constructed by transforming Day185 with PCR products from template pCJN542 and primers FKS1-F-TDH3-OE and FKS1-R-TDH3-OE . Correct construct placement was confirmed with primers FKS1-OE-F-det and Nat-OE-R-det2-CJN. MY2378A strain (TET-FKS1) has been described . Strain C48 (FKS1-S645F) has homozygous mutation C1934T of FKS1, which renders the strain resistant to echinocandins .
RNA isolation and real-time PCR methods were completed as previously described . Strains were grown in RPMI/MOPS at 37°C with orbital shaking at 200RPM for 6h. The data analysis was completed using the Ct (2−Ct) method which generated data as transcript fold-change normalized to a constitutive reference gene transcript (ACT1) and relative to the reference strain.
A tetrazolium salt (XTT - 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) reduction assay was used to assess biofilm response to fluconazole (4-1000 μg/ml) . A microbroth susceptibility method was used to test the impact of matrix on fluconazole activity (0.125-128 μg/ml) against planktonic Candida . Matrix was harvested from 6-well biofilms, concentrated 10-fold by vacuum centrifugation, heat treated at 60°C (10 min), and added to the assay.
A rat central venous catheter infection model was used for in vivo biofilm studies [9,10]. To measure glucan, blood was drawn from the catheter at 24h. For drug treatment experiments, viable burden was determined following fluconazole (250 μg/ml) instillation in the catheter for 24h . Scanning and transmission electron microscopy were undertaken as described previously .
A murine disseminated candidiasis model was used as an in vivo non-biofilm comparison . Mice were injected with 105 cells/ml via tail vein and treated (3 per group) with one of three fluconazole dosing regimens (3.1, 12.5, or 50 mg/kg/12h) for 24h. Total body burden was estimated by viable burden in mouse kidneys.
Fractionation by alkali-extraction and enzymatic digestion was used to measure cell wall glucan . For extracellular glucan analysis, a Glucatell or Fungitell (1,3)-Beta-D-Glucan Detection Reagent Kit (Associates of Cape Cod, MA) was used [2,10].
A 3[H]-labeled fluconazole accumulation protocol was adapted for biofilm use . Biofilms were grown in 6-well plates for 48h. To degrade the glucan-rich matrix, 24h biofilms were treated with β-1,3 glucanase (0-6 units/ml) for an additional 24h . Fluconazole was prepared by adding 30 μCi to 18 ml of RPMI/MOPS (1,686,500 dpm/ml). After addition of 0.6 ml to each washed biofilm, plates were incubated for 30 min at 37°C with 50RPM. Biofilms were washed, dislodged, and collected. After biofilm disruption by vortexing and sonication (10 min), cells were pelleted (4500g for 20 min) to collect the soluble matrix. Experiments were performed 2-4 times with liquid scintillation counting (TRI-CARB 2100TR, Packard). Data were normalized for dry cell weight.
We examined a collection of mutants with varied FKS1 expression to discern the role of FKS1 in biofilm formation. The strains produced similar biofilms in vitro in the wells of polystyrene plates and in vivo on the luminal surfaces of rat venous catheters (data not shown). We constructed a heterozygous deletion mutant, FKS1/fks1Δ, with reduced FKS1 expression (30% by RT-PCR). However, we were not successful in constructing a FKS1 null mutant, which is presumed essential in C. albicans . We therefore utilized the TET-FKS1 mutant with one FKS1 allele under control of a tetracycline repressible promoter and one allele deleted to further explore FKS1 function . The TET-FKS1 strain formed a biofilm under repressed and non-repressed conditions. Likewise, overexpression of one FKS1 allele (3.8-fold by RT-PCR) by an inserted TDH3 promoter did not impact biofilm formation. The final strain examined, FKS1-S645F, containing homozygous point mutations in FKS1 conferring reduced glucan synthase activity similarly produced biofilms . Each of the strains generated both yeast and hyphae (data not shown).
We next tested the impact of FKS1 disruption on biofilm drug resistance using an XTT reduction assay. Reference strain biofilms were resistant to fluconazole concentrations (1000 μg/ml) more than 2000× higher than the planktonic susceptibility (Figure 1A and data not shown). However, FKS1/fks1Δ biofilms were reduced by more than 80% following a 48h 250 μg/ml fluconazole treatment. The FKS1-S645F mutant with reduced glucan synthase capacity recapitulated the susceptible biofilm phenotype. For both strains, dose-dependent biofilm reduction was observed with fluconazole concentrations as low as 4 μg/ml (data not shown). Like the reference strain, the TDH3-FKS1 overexpression strain was maximally resistant to fluconazole. Heterozygous disruption of FKS1 did not alter planktonic fluconazole susceptibility. Under planktonic conditions, MICs for FKS1/fks1Δ and the reference strain were identical (0.25 μg/ml).
We utilized the TET-FKS1 strain to further gauge how the expression level of FKS1 impacted fluconazole resistance. TET-FKS1 biofilms with doxycycline-repressed FKS1 were more susceptible to fluconazole than non-repressed controls (Figure 1B). Optimum FKS1 repression for fluconazole susceptibility occurred at doxycycline concentrations of 7.5-30 ng/ml, with up to 85% biofilm reduction. These doxycycline concentrations did not impact the reference strain.
Using an in vivo vascular catheter biofilm model, we similarly tested the impact of FKS1 mutation on biofilm resistance. Treatment with fluconazole (250 μg/ml) in the catheter lumen was ineffective against reference strain biofilms, but decreased the viable burden in FKS1/fks1Δ biofilms by 100-fold (Figure 1C). This altered fluconazole susceptibility was specific to biofilm cells. Comparison of the reference strain and the FKS1/fks1Δ mutant in a non-biofilm disseminated candidiasis model found no difference in drug efficacy across a wide fluconazole range (3.1-50 mg/kg/12h) (Figure 1D).
The biochemical impact of FKS1 modulation on the cell wall and biofilm matrix was examined next. Using alkali-extraction and enzymatic digestion, we measured biofilm cell wall β-1,3 glucan. Reductions of approximately 30% were observed for the FKS1/fks1Δ and FKS1-S645F biofilm cell walls compared to the reference strain (Figure 2A). Doxycycline FKS1 repression of the TET-FKS1 strain lowered cell wall glucan by greater than 50% compared to the untreated controls. Cell wall ultrastructure was explored using transmission electron microscopy (TEM). Discernable microscopic cell wall changes were not observed among the study strains by this method (data not shown).
Production of extracellular glucan, including matrix glucan, was quantified in the FKS1 mutant biofilms using a limulus lysate assay. Both supernatant and matrix glucan production were reduced in the FKS1/fks1Δ biofilms by approximately 60% (Figure 2B). Noteworthy, the TDH3-FKS1 biofilms overexpressing FKS1 produced over 10-fold more glucan. Using a rat vascular catheter biofilm model, we confirmed a role for FKS1 in extracellular glucan production in vivo. High magnification scanning electron micrographs of the biofilms growing in rat venous catheters were consistent with these findings. Compared to the reference strain, the FKS1/fks1Δ biofilm appeared to have less adherent matrix material, while the TDH3-FKS1 biofilm had more (Figure 2C).
We next tested the hypothesis that biofilm matrix and specifically β-1,3 glucan were responsible for the drug resistance observed during biofilm growth. We used a microbroth susceptibility assay to determine the impact of matrix glucan on planktonic cell resistance . Addition of the reference strain matrix to planktonic cells rendered cells more resistant to fluconazole than the addition of matrix from the strain that produced less matrix β-1,3 glucan (FKS1/fks1Δ) (Figure 2D). This suggests that the matrix glucan alone is responsible for a degree of resistance observed during biofilm growth and that FKS1 is necessary for this resistance.
A radio-labeled fluconazole sequestration assay was then designed to measure a biofilm component-antifungal interaction by fluconazole within the intact biofilm and individual biofilm components. Approximately 50% less fluconazole associated with the intact FKS1/fks1Δ and FKS1-S645F mutant biofilms compared to the reference strain biofilm (Figure 2E). Over 70% more fluconazole accumulated in the TDH3-FKS1 overexpression strain biofilm. Nearly all the radioactivity localized to the matrix, suggesting sequestration of the fluconazole by the extracellular matrix. Unfortunately, radioactivity levels in the intracellular component were below the level of detection.
Using the same assay, we tested the impact of matrix β-1,3 glucan modification on fluconazole sequestration. Reference strain biofilms were treated with β-1,3 glucanase at concentrations known to enhance the activity of fluconazole . Treatment with glucanase significantly reduced the amount of radioactive fluconazole sequestered by the biofilm matrix in a dose-dependent manner, further supporting a glucan-antifungal interaction (Figure 2F).
While the current observations strongly suggested that the impact of FKS1 modulation was due to β-1,3 glucan sequestration of antifungal, we also considered the possibility that FKS1 disruption may lead to a breach of cell wall integrity, rendering cells more susceptible to stress-inducing agents, such as antifungal drugs. We measured the planktonic and biofilm susceptibility of the reference strain and the FKS1/fks1Δ mutant to a variety of cell stressors, including hydrogen peroxide, congo red, ethanol, sodium dodecyl sulfate, and hyperosmotic stress. Differences in susceptibility were not detected between the two stains (data not shown).
Candida biofilm cells are embedded in a protective extracellular matrix and exhibit resistance to commonly used anti-infectives [13-15]. The mechanism for this resistance in Candida biofilms appears to be multi-factorial [13-15]. However, the contributions of previously identified mechanisms are quantitatively modest and a large gap in our understanding of drug resistance in this important setting remains.
Our findings show that glucan synthesis by Fks1p is critical for biofilm-specific drug resistance in C. albicans. As with planktonic cells, glucan synthase Fks1p is responsible for production of cell wall β-1,3 glucan during biofilm growth. However, we found this protein to have a unique function in Candida biofilms. It also manufactures glucan which is incorporated into an adhesive, carbohydrate-rich matrix. By producing matrix β-glucan capable of sequestering antifungal, a drug sponge prevents the therapy from reaching its target, allowing biofilm cells to survive astonishingly high drug exposures. In addition, this matrix glucan has shown to be a potential diagnostic marker for Candida biofilm infection in an animal model .
Previous investigations have postulated and explored the contribution of the biofilm matrix to antifungal diffusion through Candida biofilms . Pivotal studies by the Douglas group identified a relationship between drug susceptibility and the matrix quantity visualized by microscopy. Further studies have found some slowing of antifungal transit using a filter disk assay. However, previous studies had not addressed the biochemical or genetic basis for this phenomenon.
Defining the chemical composition and genetic regulation of Candida biofilm matrix is a promising step for development of diagnostic testing and treatment for these devastating infections. Our findings suggest azoles and echinocandins, which target Fks1p, may be synergist in biofilms. We envision future studies to address the role of this mechanism for other antifungal drug classes and determine the precise chemical nature of the drug-matrix interaction. Agents designed to disrupt this interaction should prevent or eradicate recalcitrant device-associated infections. In addition, information regarding the matrix composition may be used to design diagnostic tests specific to Candida biofilm infections.
We thank C. Douglas, A. Mitchell, C. Nobile, and D. Perlin for strains and plasmids. We thank B. Klein for critical reading of the manuscript.
This work was supported by the National Institutes of Health (RO1 AI073289-01).
Potential conflicts of interest: J. N. No conflict, H. S. No conflict, M.C. No conflict, D. A. No conflict