Osamu Shimokawa

Accumulation of 14-methyl sterols and defective hyphal growth in Candida albicans

Polyene-resistant mutants of Candida albicans accumulating 14-methyl sterols in place of ergosterol and defective in hyphal growth were isolated after ultraviolet mutagenesis. Analysis of revertants showed that the alteration in sterol composition, the inability to grow as hyphae, and the antibiotic resistance had returned to normal simultaneously. In addition, clotrimazole, which caused accumulation of 14-methyl sterols at a subfungistatic concentration, also inhibited hyphal formation but had little effect on yeast propagation. It is concluded that there is an intrinsic rather than a fortuitous relationship between sterol composition and morphogenesis.

Accumulation of 14α-methylergosta-8,24(28)-dien-3β,6α-diol in 14α-demethylation mutants of Candida albicans: genetic evidence for the involvement of 5-desaturase

Abstract 14α-Demethylation mutants of the fungus Candida albicans have been shown to accumulate 14α-methylergosta-8,24(28)-dien-3β,6α-diol. A derivative from one of these mutants (KD4900) that does not form this 6α-hydroxylated sterol but is still defective in 14α-demethylation (KD4950) was obtained. Mutational restitution of 14α-demethylation capacity of this derivative resulted in the formation of the 5,6-saturated sterol ergosta-7,22-dien-3β-ol as the major product, clearly indicating that 5-desaturase deficiency exists in this demethylation-proficient revertant (KD4952). This implies that its parent, KD4950, which has lost the ability to form the hydroxylated sterol, also is deficient in 5-desaturation. We infer from the results that 5-desaturase is responsible for the formation of the hydroxylated sterol. However, it is unclear whether the hydroxylation represents a genuine step of the normal 5-desaturation reaction.

Isolation of a Candida albicans mutant with reduced content of cell wall mannan and deficient mannan phosphorylation

A rough-colony mutant of Candida albicans was isolated after ultraviolet mutagenesis. The mutant contained approximately half the normal amount of the cell wall mannan, the acetolysis pattern of which was indistinguishable from that of the wild-type counterpart. However, the extent of phosphorylation in the mutant mannan was about 12% of the value for wild type.

Increased drug sensitivity in Candida albicans cells accumulating 14-methylated sterols

Multiple increases in drug sensitivity were observed in Candida albicans cells that were accumulating 14-methylated sterols as a result of a mutation or clotrimazole treatment. The possibility was suggested that a change in membrane permeability caused by the alteration in sterol composition was responsible for the increased susceptibility.

A Candida albicans rough-type mutant with increased cell surface hydrophobicity and a structural defect in the cell wall mannan

A Candida albicans rough-type mutant showing increased hydrophobicity of the cell surface was isolated. The mutant cells contained about one-quarter as much mannan as the wild type, and were defective in antigenic factors 1, 4, 5, and 6. Acetolysis fingerprinting demonstrated that the mutant mannan had a structural defect in its carbohydrate skeleton.

A Candida albicans mutant conditionally defective in sterol 14α-demethylation

A conditional sterol 14α-demethylation mutant of Candida albicans was isolated whose defect was dependent both on the growth temperature and on the culture medium used. Under restrictive conditions, this mutant showed a deficiency in hyphal growth and an increased susceptibility to diverse chemicals, in accordance with the phenotypes of the already known non-conditional mutants of 14α-demethylation. In addition, the mutant was shown to have a conditional defect in respiration. The impaired respiration was also seen in the non-conditional mutants.

Relationship between MIC and Minimum Sterol 14α-Demethylation-Inhibitory Concentration as a Factor in Evaluating Activities of Azoles against Various Fungal Species

ABSTRACT The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14α-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous Candida yeasts tested were clearly divided into two groups: group I, consisting of C. albicans, C. tropicalis, and C. lusitaniae, had MICs that were much higher than the MDICs, whereas group II, comprising C. glabrata, C. parapsilosis, C. guilliermondii, and C. krusei, had MICs that were approximately equal to the MDICs. In the ascomycetous fungi Aspergillus fumigatus and Sporothrix schenckii, the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi Cryptococcus (Filobasidiella) neoformans, C. curvatus, and Trichosporon asahii, the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14α-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.

Phenotypes of Candida albicans sterol mutants deficient in Δ8,7-isomerization or 5-desaturation

Two Candida albicans sterol mutants deficient in Δ8,7-isomerization and 5-desaturation, respectively, were studied in relation to those phenotypes associated with the 14α-demethylation deficiency, i.e. repression of hypha formation, inability to grow with non-fermentable substrates, and increased sensitivity to nonpolyene-nonazole chemicals. It was found that the Δ8,7-isomerization deficiency, resulting in the accumulation of Δ8 sterols (mostly Δ24(28) or Δ5,22), was associated with severe reduction in hyphal growth and utilization of non-fermentable substrates. These cellular activities were reduced only slightly when 5-desaturation was deficient, causing the accumulation of Δ7 sterols. On the other hand, both the isomerization and desaturation deficiencies were accompanied by multiple increases in drug sensitivity which were small in magnitude compared with those associated with the 14α-demethylation deficiency.

Acetate-mediated production of orotic acid by ura3 mutants of Candida albicans.

Candida albicans ura3 mutants were found to produce large amounts of orotic acid when the growth medium was supplemented with sodium acetate. Experiments with 13C‐labeled acetate showed that the acetate served as a precursor of orotic acid. This system of acetate‐mediated production of orotic acid is similar to other documented microbial producers in yield but unique for its acetate requirement.

Glucose metabolism in resting cells ofCandida albicans as studied by13C NMR

Abstract The metabolism of glucose in resting cells of Candida albicans was studied by 13 C NMR spectrometry using 13 C-labeled glucose. Under aeration, the formation of ethanol, glycerol, arabitol, mannitol, and trehalose was observed. The addition of inhibitors of the respiratory chain or the omission of aeration resulted in a total loss of formation of those polyols and trehalose, with ethanol being the only detectable product. Thus, respiration seems to favor the production of polyols including glycerol, as well as that of trehalose. With regard to glycerol, this finding is in contrast with the previous observation in Saccharomyces cerevisiae that oxygenation represses its production.