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The Candida albicans NOT4 gene was disrupted in order to investigate the role of Not4p in growth, morphogenesis and pathogenicity. Heterozygote (NOT4/not4), null (not4/not4) and reconstructed heterozygote ([NOT4]/not4) strains of C. albicans, as well as CAF2-1, the parental strain, were grown under conditions that promote hyphal formation. When cultured in liquid medium 199 the heterozygote, reconstructed and wild-type strains began the yeast-to-hyphal transition within 3 h and continued hyphal growth for the duration of experiments. The null mutant also began hyphal growth within 3-5 h but hyphae tended to be shorter and distorted. Subsequently, hyphal growth was arrested and growth returned predominantly to the yeast form. Similar differences were observed when strains were grown on solid Spider medium and medium 199. The parental, heterozygote and reconstructed strains formed normal filamentous networks emanating from colonies. In contrast, the null mutant failed to form hyphae on all solid media tested. The ability of the NOT4 null strain to form biofilms was also investigated, and it was observed that biofilm development does not readily occur for this strain. Virulence of each strain was examined utilizing the mouse model of systemic candidiasis. Mice infected with CAF2-1 succumbed to infection within 3-7 days. All mice infected with the null strain survived for the duration of experiments, while the heterozygote and reconstructed heterozygote strains showed an intermediate level of virulence. These findings suggest that NOT4 may play a role in affecting strain pathogenicity, possibly by regulating expression of certain genes that effect cellular morphogenesis and virulence.

The basis of cerulenin resistance of Candida albicans strains 4918-2 and 4918-10 has been investigated. Parasexual genetic analyses established that cerulenin resistance to concentrations of at least 5 micrograms ml-1 is dominant in both strains. The results also showed that strain 4918-2 is heterozygous for resistance, while the change from resistance to sensitivity of strain 4918-10 is reversible. Experiments to define the mechanism(s) responsible for resistance focused on cerulenin uptake and fatty-acid synthase activity. Cerulenin uptake by strains 4918-2 and 4918-10 was 24% of that of the wild-type (strain 4918). Uptake was restored in UV-induced cerulenin-sensitive segregants of strains 4918-2 and 4918-10, and varied from 63% to 200% of parental values. Fatty-acid synthase from strains 4918-2 and 4918-10 was resistant to cerulenin as judged by differences in the inactivation of the enzyme by the agent. However, inactivation kinetics of fatty-acid synthase of cerulenin-sensitive segregants did not revert to the parental inactivation profile. Further investigation showed that nine out of ten segregants were resistant to cerulenin at concentrations between 1 and 4 micrograms ml-1 while strain 4918 was sensitive to cerulenin at all concentrations tested. Thus, the results suggest that alteration of fatty-acid synthase and changes in permeability contribute to total cerulenin resistance of each strain.