The Role of Isocitrate Lyase (ICL1) in the Metabolic Adaptation of Candida albicans Biofilms

Abstract

Background: A major characteristic of Candida biofilm cells that differentiates them from free-floating cells is their high tolerance to antifungal drugs. This high resistance is attributed to particular biofilm properties, including the accumulation of extrapolymeric substances, morphogenetic switching, and metabolic flexibility.

Objectives: This study evaluated the roles of metabolic processes (in particular the glyoxylate cycle) on biofilm formation, antifungal drug resistance, morphology, and cell wall components.

Methods: Growth, adhesion, biofilm formation, and cell wall carbohydrate composition were quantified for isogenic Candida albicans ICL1/ICL1, ICL1/icl1, and icl1/icl1 strains. The morphology and topography of these strains were compared by light microscopy and scanning electron microscopy. FKS1 (glucan synthase), ERG11 (14-α-demethylase), and CDR2 (efflux pump) mRNA levels were quantified using qRT-PCR.

Results: The ICL1/icl1 and icl1/icl1 strains formed similar biofilms and exhibited analogous drug-tolerance levels to the control ICL1/ICL1 strains. Furthermore, the drug sequestration ability of β-1, 3-glucan, a major carbohydrate component of the extracellular matrix, was not impaired. However, the inactivation of ICL1 did impair morphogenesis. ICL1 deletion also had a considerable effect on the expression of the FKS1, ERG11, and CDR2 genes. FKS1 and ERG11 were upregulated in ICL1/icl1 and icl1/icl1 cells throughout the biofilm developmental stages, and CDR2 was upregulated at the early phase. However, their expression was downregulated compared to the control ICL1/ICL1 strain.

Conclusions: We conclude that the glyoxylate cycle is not a specific determinant of biofilm drug resistance.

Keywords: Biofilms; Candida albicans; Isocitrate Lyase (ICL1); Resistance.

Figure 1.. Inverted Microscopy Images of C. albicans Biofilms at Different Growth Stages on RPMI 1640 Medium

These images depict biofilm formation on a polystyrene surface under static conditions through the three developmental phases. A, Growing predominantly as yeast cells, attachment of the cells to the potential growth material and to each other was observed; B, metabolic activity increased with cellular density in correlation with the duration of cultivation. This image shows cells at the maturation phase; C, Microcommunities became heterogeneous and clustered. Apparent production and visible accumulation of slime extracellular matrix (ECM) enclosing the sessile yeast and filamentous cells, which increased with maturity, was observed.

Figure 2.. A, The chart represents the average metabolic activity OD of C. albicans wildtype biofilms on YPL, YPD, and RPMI 1640 media, measured with the MTT assay at each developmental stage (early, intermediate, and maturation). The results are representative of three independent experiments. Absorbance is plotted against growth intervals (time); B, Heterozygous CaICL1/icl1 mutant biofilm on YPL, YPD, and RPMI 1640 media. Formation at early, intermediate, and maturation stages. The results are representative of three independent experiments. Absorbance is plotted against growth intervals (time). Independent experiments were performed in triplicate; C, Homozygous CaIcl1/icl1 mutant biofilm on YPL, YPD, and RPMI 1640 media. Formation is shown at the early, intermediate, and maturation stages. The results are representative of three independent experiments. Absorbance is plotted against growth intervals (time). Independent experiments were performed in triplicate.

Figure 3.. A, C. albicans wildtype susceptibility testing with fluconazole 128 µg/mL and amphotericin B 16 µg/mL on lactate-containing medium. ANOVA was carried out for the statistical analysis to calculate the significant difference at each developmental level; B, Heterozygous CaICL1/icl1 mutant susceptibility testing with fluconazole 128 µg/mL and amphotericin B 16 µg/mL on lactate-containing medium. ANOVA was carried out for the statistical analysis to calculate the significant difference at each developmental level. Independent experiments were performed in triplicate.

Figure 4.. Developmental Stages of Biofilms

Figure 5.. SEM images of reference and knockout strain biofilms at maturity, before and after treatment. Biofilms were cultured for 48 hours at 37°C under static conditions in YPL medium and visualized at 500× magnification. A, Treated wildtype ICL1/ICL1; B, untreated wildtype ICL1/ICL1; C, treated ICL1/icl1; D, untreated ICL1/icl1; E, treated icl1/icl1, and F, untreated icl1/icl1. Images have been reconstructed to permit clear viewing.

Figure 6.. A, Quantification of β-1, 3-glucan present in the ECM of wildtype and mutant biofilms not treated with fluconazole at the early, intermediate, and mature phases, respectively; B, Quantification of β-1, 3-glucan present in the ECM of the wildtype and mutant biofilms treated with 128 µg/mL fluconazole at the early, intermediate, and mature phases, respectively.

Figure 7.. A, Comparative Relative Transcriptional Expression of FKS1; Results were normalized to the internal control CaACT1; B, Comparative relative transcriptional expression of ERG11. Results were normalized to the housekeeping gene CaACT1; C, Comparative relative transcriptional expression of CDR2. Results were normalized to the internal control CaACT1.