FIGURE 6.
GPI2 mutation affects growth and ergosterol biosynthesis in BWP17 strain of C. albicans. A, BWP17, GPI2 heterozygote (BWP17-gpi2/GPI2), and conditional null GPI2 mutant (BWP17-gpi2/PMET3-GPI2) in BWP17 strain background were analyzed for growth patterns on SD-minimal plates. BWP17 and the heterozygous mutant showed similar growth. However, conditional null GPI2 mutant showed a growth defect dependent on Met/Cys concentration. The experiment was done twice in duplicate, and a representative image is shown. B, BWP17 and GPI2 heterozygote were analyzed for growth pattern in the presence of ketoconazole. The heterozygous mutant exhibits resistance to the presence of ketoconazole as compared with BWP17. The experiment was done twice, and a representative image is shown. C, expression levels of GPI2, ERG11, ERG1, and ERG3 genes were quantified in BWP17 and GPI2 heterozygote. The strains were grown in YEPD, and total RNA was extracted as under “Experimental Procedures.” GPI2 levels were decreased in the mutant to about 0.59-fold (±0.011) (****, p value = 0.0000011). ERG11 transcript levels were enhanced in the heterozygote by almost 2.1-fold (±0.29) as compared with BWP17 (***, p value = 0.00014). However, the ERG1 transcript levels were decreased to 0.64-fold (±0.017) in the mutant as compared with BWP17 (****, p value = 0.0000044). No significant difference was observed for the transcription of ERG3 (p value = 0.14). The experiment was done twice in duplicate. D, steady state anisotropy was determined in BWP17 and GPI2 heterozygote strain using DPH. The higher anisotropy in the mutant (0.171 ± 0.019) as compared with BWP17 (0.121 ± 0.021) suggests a rigid membrane in the mutant (**, p value = 0.0041), similar to GPI2 heterozygote in CAI4 background. The experiment was done twice in duplicate. E, BWP17, GPI2 heterozygote, and conditional null GPI2 mutants were grown on YEPD plate and incubated at 37 °C for 7 days. The mutants displayed defective filamentous morphology as compared with the wild type strain. The experiment was done twice for confirmation and a representative image is shown. F, BWP17, GPI2 heterozygote and conditional null GPI2 mutant were also compared for filamentation pattern on Spider medium plate at 37 °C for 7 days. The mutants showed defective filamentation as compared with the wild type strain. The experiment was done twice, and a representative image is shown.