The stearoyl-coenzyme A desaturase 1 is essential for virulence and membrane stress in Candida parapsilosis through unsaturated fatty acid production

Abstract

Unsaturated fatty acids (UFA) are essential components of cells. In Saccharomyces cerevisiae, stearoyl-coenzyme A (CoA) desaturase 1 (OLE1) affects cell viability through the regulation of oleic (18:1) or palmitoleic (16:1) acid production. In this study, we used a targeted gene deletion approach to determine the impact of OLE1 on the emerging human pathogenic fungus Candida parapsilosis. We found that the deletion of OLE1 resulted in an auxotrophic yeast strain (designated OLE1 KO) that required unsaturated fatty acids for growth but not saturated fatty acids. Additionally, the production of UFA by OLE1 KO yeast cells was markedly reduced, suggesting that Ole1 is essential for UFA production. In contrast to wild-type C. parapsilosis, which produced pseudohyphal growth on UFA-supplemented medium agar, pseudohyphal formation in the OLE1 KO cells was severely impaired, suggesting that Ole1 regulates morphology. Furthermore, the OLE1 KO cells were hypersensitive to various stress-inducing factors, such as salts, SDS, and H(2)O(2), especially at the physiological temperature. The results indicate that OLE1 is essential for the stress response, perhaps through the production of UFA for cell membrane biosynthesis. The OLE1 KO cells also were hypersensitive to human and fetal bovine serum, suggesting that targeting Ole1 could suppress the dissemination of yeast cells in the bloodstream. Murine-like macrophage J774.16 more efficiently killed the OLE1 KO yeasts, and significantly larger amounts of nitric oxide were detected in cocultures of macrophages and OLE1 KO cells than with wild-type or heterozygous strains. Moreover, the disruption of OLE1 significantly reduced fungal virulence in systemic murine infection. Taken together, these results demonstrate that Ole1 regulates the pathobiology of C. parapsilosis via UFA and that the OLE1 pathway is a promising antifungal target.

FIG. 1.

Disruption of OLE1 genes in C. parapsilosis. (A) Schematic representation of the disruption construct (1) and the genotype of the WT (2) with the OLE1 locus, disrupted OLE1 locus with the SAT1 cassette (3), and disrupted locus without the SAT1 cassette (4). (B) Southern blot analysis of the WT strain (lane 1), heterozygous resistant strain (lane 2), homozygous resistant strain (lane 3), homozygous nonresistant strain (lane 4), and reconstituted nonresistant strain (lane 5). The Southern blot probe was PCR amplified from the upstream fragment of plasmid pSFS2Ole1.

FIG. 2.

Growth dependence of OLE1 KO on unsaturated fatty acids. (A) Growth rates of wild-type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains. Yeast cell growth was compared in YPD, YPDPO (YPD plus fatty acids), YNB, and YNBPO (YNB plus fatty acids) broth. The growth of the Candida cells was measured by cell density at OD₆₀₀. Experiments were repeated twice with four replicates for each medium, with similar results. (B) Spot growth assays of OLE1 KO and Fas2 KO on YPD supplemented with fatty acids or Tween 20, 40, or 80. A series of 10× dilutions of yeast cells were spotted onto agar. Plates were incubated at 30°C for 3 days, and images were digitally captured. Experiments were repeated twice with similar results. (C) The growth of the mutant yeast cells was dependent upon the amount of unsaturated fatty acids. Yeast cell strains were grown in YPD supplemented with 0 to 0.01% (wt/vol) palmitoleic acid (16:1), oleic (18:1) acid, or a mixture of these fatty acids. Yeast growth was determined by cell density after 24 h at 30°C with shaking. The results are the means from two independent experiments with triplicates. (D) A model of fatty acid biosynthesis generated from the assays. Fas is the Fas1 and Fas2 complex, which is required to synthesize SFA from malonyl-CoA. Ole1 is essential for UFA production from SFA precursors.

FIG. 3.

Hypersensitivity of OLE1 KO yeast cells to stress-inducing factors. A series of 10× dilutions of yeast cells were spotted on YPDPO in the presence of the indicated stress-inducing factor. The OLE1 KO yeast cells were more susceptible to SDS, NaCl, KCl, and H₂O₂ than the wild type. The enhanced susceptibility of the OLE1 KO cells was more profound at 37°C. The experiments were repeated with similar results.

FIG. 4.

Reduced invasive growth of OLE1 KO yeast cells. Yeast cells were plated on YPDPO agar and incubated at room temperature for 10 days. Filaments were present around the colonies of WT, HET, and RE strains but not the OLE1 KO strain (10× objective). Sections from the edges of the colonies observed on glass slides showed the pseudohyphae of the yeast cells (insets, 20× objective). The experiment was repeated with similar results.

FIG. 5.

Hypersensitivity of OLE1 KO yeast cells to serum. The growth of the wild-type (WT), heterozygous (HET), reconstituted (RE), and homozygous (KO) mutant strains in 20% human (A) or fetal bovine (B) serum diluted in PBS. Error bars indicate standard deviations. *, P < 0.001 (ANOVA). (D) Effect of serum on the colony morphology of the OLE1 KO cells. The WT and OLE1 KO yeast cells were precultured in YPDPO liquid medium overnight, and aliquots of 5 × 10⁶ yeast cells were transferred to 20% FBS at 30°C. Yeast cells were plated on YPDPO agar either prior to or after 24 h of incubation in FBS medium. OLE1 KO yeast cells exhibited heterogeneous colony sizes after 3 days of incubation on agar at 30°C. (C) WT and OLE1 KO strains were subjected to SytoxGreen and PI to characterize live and dead cells after 24 h of exposure to 20% human serum. Dead cells exhibited bright red fluorescence. All experiments were repeated twice with triplicates, and all results were similar to the data shown.

FIG. 6.

Reduced virulence of OLE1 KO. Intraperitoneal infection of A/J mice with wild-type (WT), homozygous (KO), and reconstituted (RE) yeasts (A and B). Intravenous infection of A/J mice with WT and KO yeasts (C and D). (A and C) CFU from kidneys, spleens, and livers 3 days after intraperitoneal and intravenous infection, respectively. (B and D) CFU 5 days after infection. Each symbol represents 1 mouse. *, P ≤ 0.01, (Newman-Keuls). 127, no detectable CFU of KO mutants. (E and F) Representative histological sections of kidney 5 days after intravenous infection with WT and KO cells, respectively. Arrows indicate aggregates of yeast cells in WT-infected tissues.

FIG. 7.

Reduced survival of OLE1 KO yeast cells exposed to macrophages. (A) Intracellular viability of yeast cells as assessed by the acridine and crystal violet staining of the yeast cells in the macrophages. The green yeast cells (arrows) are alive, whereas the orange-red cells (arrowheads) are dead. Pictures are the merge of the red, green, and phase channels at 20×. (B) Phagocytosis of the wild-type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains with murine-like macrophage (J774.16). The phagocytosis of each C. parapsilosis strain was assessed by counting the number of phagocytosed yeast cells in more than 500 macrophages. The phagocytosis index was the ratio of the yeast cells to macrophages. Experiments were repeated twice with triplicates, and similar results were documented. (C and D) Survival of yeast cells as determined by CFU after 2 and 4 h of coculture of yeast cells with J774.16, respectively. Experiments were repeated twice with four replicates. (E) Nitric oxide production from macrophages challenged with OLE1 KO yeast cells after 2 h. Experiments were repeated twice with four to five replicates with similar results. The results from the experiments were averaged, and error bars indicate standard deviations. For panels B to E, P < 0.05 by ANOVA (*).