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. 2010 Jul 1;202(1):171-5.
doi: 10.1086/651200.

Genetic basis of Candida biofilm resistance due to drug-sequestering matrix glucan

Jeniel E Nett  1 , Hiram Sanchez, Michael T Cain, David R Andes
Affiliations

Genetic basis of Candida biofilm resistance due to drug-sequestering matrix glucan

Jeniel E Nett et al. J Infect Dis. .

Abstract

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 extraordinarily high drug concentrations. Here, we show that beta-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 also identify a useful biofilm drug _target.

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Conflict of interest statement

Potential conflicts of interest: J. N. No conflict, H. S. No conflict, M.C. No conflict, D. A. No conflict

Figures

Figure 1
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 biofilm is more susceptible to a 24h fluconazole treatment during doxycycline 15 ng/ml (doxy) repression of FKS1. Data are from one of three checkerboard assays were performed on three occasions. (C) The FKS1/fks1Δ biofilm is susceptible to fluconazole in vivo. Biofilms in rat catheters were treated with fluconazole inside the catheter lumen. Data represent the mean of two replicates. (D) The FKS1/fks1Δ mutant and reference strains are equally susceptible to fluconazole in a non-biofilm disseminated candidiasis model. Four animals were used in each group. Statistical significance was determined by ANOVA with pair-wise comparisons using the Holm-Sidak method and standard deviations are shown. The symbol * indicates p<0.05.
Figure 2
Figure 2. Fks1p produces biofilm matrix glucan which confers resistance to non-biofilm cells by drug sequestration
(A) Modulation of FKS1 impacts biofilm cell wall β-1,3 glucan. Measurements were determined by the phenol sulfuric acid method following alkali-extraction and enzymatic digestion of biofilm cell walls. Assays were performed in triplicate. (B) FKS1 is required for extracellular β-1,3 glucan production in biofilms. Supernatant and matrix were collected from in vitro biofilms and β-1,3 glucan was measured using a limulus lysate based assay. A rat venous catheter model was used for in vivo experiments and serum was collected for glucan determination. Glucan assays were preformed in triplicate. (C) High magnification SEMs (×15,000) of rat catheter biofilms demonstrate the impact of FKS1 modulation on matrix production. Scale bars represent 1 μM. (D) Planktonic cells treated with reference strain matrix are significantly more resistant to fluconazole than those treated with FKS1/fks1Δ biofilm matrix. Matrix was harvested from FKS1/fks1Δ and reference strain biofilms and added to non-biofilm, planktonic cells in the presence of escalating fluconazole concentrations. Matrix was normalized per biofilm. Assays were preformed on two occasions in triplicate. (E) FKS1 is required for matrix sequestration of [H3] fluconazole. Intact biofilms grown from the glucan modified strains were exposed to [H3] fluconazole, washed, and harvested. Scintillation counting was used to determine the fluconazole concentration in the intact biofilms and the isolated matrix. Assays were performed on two occasions in triplicate. (F) Degradation of matrix β-1,3 glucan disrupts sequestration of [H3] fluconazole. Biofilms were treated with escalating concentrations of glucanase prior to [H3] fluconazole exposure. Statistical significance was determined by ANOVA with pair-wise comparisons using the Holm-Sidak method and standard deviations are shown. The symbol * indicates p<0.05.
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