A multicopper oxidase gene from the human pathogenic yeast Candida albicans was isolated and characterized. An open reading frame of 1872 bp, designated CaFET3, was identified, encoding a predicted protein of 624 amino acids and a molecular mass of 70 5 kDa. The identity between the deduced amino acid sequences of CaFET3 and the Saccharomyces cerevisiae FET3 gene is 55 %. CaFET3 was localized on chromosome 6. A null mutant (fet3∆/fet3∆) was constructed by sequential gene disruption. Unlike the C. albicans SC5314 wildtype strain the fet3∆ mutant was unable to grow in low-iron medium. The lack of growth of a S. cerevisiae fet3∆ mutant in iron-limited medium was compensated by transformation with CaFET3. The null mutant strain showed no change in pathogenicity compared with the wild-type strain in the mouse model of systemic candidiasis.

The vacuolar H + -ATPase (V-ATPase) component Vma7p of the human-pathogenic yeast Candida albicans regulates hyphal growth induced by serum and Spider medium and is essential for virulence. In order to characterize the functions of the putative V-ATPase subunit Vma7p of C. albicans, null mutants were generated. The resulting mutants showed reduced vacuole acidification, which correlated with defective growth at alkaline pH. In addition, defects in degradation of intravacuolar putative endosomal structures were observed. vma7 null mutants were sensitive towards the presence of metal ions. It is concluded that the sequestration of toxic ions in the vacuole via a H + gradient generated by the V-ATPase is affected. The vma7 null mutant strains were avirulent in a mouse model of systemic candidiasis. In addition, C. albicans vma7 null mutants and the null mutant strain of the Vma7p-interacting phosphatidylinositol 3-kinase Vps34p showed similar phenotypes. In summary, the V-ATPase subunit Vma7p is involved in vacuolar ion transport and this transport is required for hyphal growth and virulence of C. albicans.

Candida albicans is able to generate significant amounts of reactive oxygen species (ROS). In this study, ROS generation by yeast and hyphal forms of the strain 3153 A was analyzed to determine whether ROS generation could be a major factor in the invasive behavior of germinative cells. Furthermore, the virulent strain CA6 and its avirulent and agerminative mutant VIR3 were compared. ROS were measured by lucigenin-enhanced chemiluminescence and a cytochrome c assay. During the blastoconidial phase of all strains moderate amounts of ROS were found at cell concentrations > 1 x 10(5)/ml. However, ROS generation appeared to be specifically inhibited at cell concentrations > 1 x 10(8)/ml, and this was found in both assays. As shown in comparative experiments, the medium used for measurement markedly affected the total amount of ROS. Hyphae of strain 3153 A generated a significantly higher amount of ROS than yeast cells and cells with germ tubes (P < 0.001). The strain CA6 showed significantly higher ROS generation than the VIR3 strain for both blastoconidiae and after 30 min of induction of hypha formation (P < 0.05). In conclusion, hypha formation, usually acknowledged as a major factor in Candida pathogenicity, was associated with markedly increased ROS formation. ROS generation was not closely linked to the ability to form hyphae, but was highest in germinative cells.

To determine if cellular functions of the phosphatidylinositol 3-kinase CaVps34p are related to processes governing Candida albicans pathogenicity, both copies of the gene were sequentially disrupted. Homozygous deletion of C. albicans VPS34 resulted in a mutant strain which exhibited defects not only in intracellular vesicle transport processes but also in morphogenesis. The CaVPS34 null mutant was unable to form hyphae on different solid media whilst showing a significantly delayed yeast-to-hyphae transition in liquid media. In addition, the mutant was rendered hypersensitive to temperature and osmotic stresses and had a strongly decreased ability to adhere to mouse fibroblast cells compared to the wild-type strain SC5314. Finally, evidence was obtained that CaVPS34 is essential for pathogenicity of C. albicans as the CaVPS34 null mutant was shown to be avirulent in a mouse model of systemic infection. C. albicans pathogenicity was restored to a near wild-type degree upon reintroduction of CaVPS34 into the chromosome of the null mutant, demonstrating that the observed avirulence corresponded to the loss of CaVPS34. Thus, the results suggest that CaVPS34 may serve as a potential target for antifungal drugs.

Helioferins A and B were detected as novel aminolipopeptides in cultures of Mycogonerosea DSM 8822 in the course of a screening for mediators of helianthate anion transfer from aqueous to toluene phases. Their structures as novel antibiotics and cytotoxic agents were elucidated by mass spectrometry and NMRspectroscopic methods. Antimicrobial activity was estimated against Candida albicans and Gram-positive bacteria including Mycobacterium spp. are capable of conveying water-soluble dye anions such as helianthate (methylorange) from aqueous to toluene phases5). In screening for similar helianthate-conveying metabolites we used mycelium extracts of more than 300Streptomyces strains, but amongthem we detected only two new producers of pamamycins. Our subsequent efforts employing fungal extracts disclosed a strain of M. rosea as the producer of novel aminolipopeptide antibiotics which strongly promoted the transfer of watersoluble anions to organic solvents.Here we report on screening, isolation, structure elucidation and biological properties of the helioferins A and B (Fig. 1, 1 and 2, respectively) as new representatives of the leucinostatin-trichopolyn group of aminolipopeptide antibiotics6~10). Results and DiscussionScreening for Anion-conveying MetabolitesThe search for production of helianthate-conveying metabolites by more than 300 strains of Fungi Imperfecti was carried out by use of a modified two-phase assay as was described previously3) (see Experimental).