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/fks1
Δ, 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
]. 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 [8
]. 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 ( 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).
Figure 1 FKS1 is required for fluconazole biofilm resistance in vitro and in vivo. (A) The FKS1/fks1Δ and FKS1-S645F biofilms are more susceptible to 48h fluconazole treatment. Assays were performed on two occasions in triplicate. (B) The TET-FKS1 mutant (more ...)
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 (). 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 (). 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) ().
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 (). 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).
Fks1p produces biofilm matrix glucan which confers resistance to non-biofilm cells by drug sequestration
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% (). 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 ().
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 [2
]. 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/fks1
Δ) (). 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 (). 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 [2
]. 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 ().
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).