Fatty acid synthase impacts the pathobiology of Candida parapsilosis in vitro and during mammalian infection

Abstract

Cytosolic fungal fatty acid synthase is composed of two subunits alpha and beta, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14ratio0), palmitic acid (16ratio0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.

Figure 1. Disruption of Fas2 genes in C. parapsilosis.

Schematic representation of disruption construct (A), genotype of wild type with Fas2 loci (B), disrupted locus with SAT1 cassette (C), and disrupted locus without SAT1 cassette (D). Southern blot analysis of wild type strain (lane1, genotype of B/B), heterozygous resistant strains (lane 2, genotype B/C), heterozygous non-resistant strain (lane 3, genotype B/D), homozygous resistant strain (lane 4, genotype C/D), homozygous non-resistant strain (lane 5, genotype D/D), and reconstituted strain (lane 6, genotype B/D). Southern blot probe was PCR amplified from the upstream fragment of plasmid pSFS2Fas2.

Figure 2. Growth rates of wild type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains.

Yeast cell growth was compared in YPD (A), YPDT40 (YPD plus fatty acids) (B), YNB (C), and YNBT40 (YNB plus fatty acids) (D) broth. Growth of the Candida cells was measured by cell density at OD₆₀₀. Illustration of the distribution of yeast strains on plates (E). Growth of WT, HET, KO, and RE in solid YPD without fatty acids (F), YPD with 0.5% (w/v) oleic acid (G), and YPD with 1% (v/v) Tween 40 and 0.01% (w/v) myristic and stearic aicds (H). Pictures were taken after 2 days of incubation at 30°C. Experiments were repeated twice with four replicates for each strain with the same results.

Figure 3. Growth dependence of Fas2 KO strain on different exogenous fatty acids in YPD.

Different amounts of myristic (14∶0), palmitic (16∶0), stearic (18∶0), oleic (18∶1) acids, and T80 (Tween 80) were added to YPD broth. Yeast growth was determined by cell density after 48 hours at 30°C. The results were the mean of two independent experiments.

Figure 4. Relative expression levels of Fas1 and Fas2.

Gene expression levels were determined by qRT-PCR in wild type C. parapsilosis in YP (1% yeast extract, 2% peptone), YPD with 1% (w/v) glucose (YPD), YP with 0.1% (w/v) myristic acid (14∶0), stearic acid (18∶0), or oleic acid (18∶1), and YP with 0.5% (v/v) Tween 40 or Tween 80. The cells were grown at 30°C for 20 hours. The results are the mean values from two experiments performed with triplicates.

Figure 5. Growth of the wild type (WT) and knockout (KO) in PBS (0) or 10, 20, 30, 40, or 50% of human serum diluted in PBS

(A). Susceptibility test of WT, HET, and RE strains with indicated concentrations of cerulenin (µg/ml) in YPD agar incubated at 30°C for 2 days (B). Inhibition of WT growth by indicated concentration of cerulenin in 50% human serum (C). The number of CFU was expressed as log10. Experiments were repeated twice with triplicates. Error bars indicate standard deviation. *P<0.01; **P<0.05 (ANOVA).

Figure 6. Comparison of biofilm formation of wild type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains on polysterene and silicone surfaces.

Metabolic activity of the cells was measured by XTT assay on polysterene plates (A), and plates containing silicone disk (B). Microscopic analysis of biofilm structures of the WT (C) and KO (D) strains formed after 48 hours on polysterene plates. XTT assay was measured at 492 nm. Experiments were performed twice with triplicates that reproduced similar results. Error bars indicate standard deviation. *P<0.01 (ANOVA).

Figure 7. Phagocytosis and killing assays of the wild type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains with murine-like macrophages J774.16.

Phagocytosis of C. parapsilosis strains by J774.16 (A). Determination of CFUs after 2 hour co-culture of yeast cells with J774.16 (B). Intracellular viability of yeast as determined by acridine and crystal violet staining of the WT (C) and the Fas2 KO strains (D) in the macrophages. The green yeast cells (arrow heads) are alive whereas the orange-red cells (arrows) are dead. Pictures are the merge of the red, green, and phase channels. Experiments were repeated at least twice and similar results were obtained. Error bars indicate standard deviation. *P<0.01 (ANOVA).

Figure 8. Lactate dehydrogenase (LDH) activity of Fas2 KO under stress conditions and growth of yeast cells in YPDT40 at different pH and temperature.

LDH activity of Fas2 KO released after exposure of the yeast cells to 0, 0.5 and 1 mM of hydrogen peroxide at 37°C for 1 hour (A). LDH activity after incubation of yeast cells at 30°C, 37°C and 40°C for 1 hour (B). LDH activity of Fas2 KO was expressed as % increase compared to WT. The results are the average from two independent experiments performed in triplicate. Spot test of Fas2 mutant and wild type grown in YPDT40 at the indicated pH (C). *P<0.05 (ANOVA).

Figure 9. Intraperitoneal infection of A/J mice with wild type (WT), heterozygous (HET), homozygous (KO), or reconstituted (RE) yeasts.

(A) CFUs in the kidney, spleen, and liver 3 days after intraperitoneal infection and (B) 5 days after infection. Each symbol represents 1 mouse. *P≤0.001, ^# P≤0.01 (Newman-Keuls). ^‡, no detectable CFU of KO mutants.