Superoxide dismutases (SOD) convert superoxide radicals into less damaging hydrogen peroxide. The opportunistic human pathogen Candida albicans is known to express CuZnSOD (SOD1) and MnSOD (SOD3) in the cytosol and MnSOD (SOD2) in the mitochondria. We identified three additional CuZn-containing superoxide dismutases, SOD4, SOD5, and SOD6, within the sequence of the C. albicans genome. The transcription of SOD5 was up-regulated during the yeast to hyphal transition of C. albicans, and SOD5 was induced when C. albicans cells were challenged with osmotic or with oxidative stresses. SOD5 transcription was also increased when cells were grown on nonfermentable substrates as the only carbon source. The Rim101p transcription factor was required for all inductions observed, whereas the Efg1p transcription factor was specifically needed for serum-modulated expression. Deletion of SOD5 produced a viable mutant strain that showed sensitivity to hydrogen peroxide when cells were grown in nutrient-limited conditions. Sod5p was found to be necessary for the virulence of C. albicans in a mouse model of infection. However, the sod5 mutant strain showed the same resistance to macrophage attack as its parental strain, suggesting that the loss of virulence in not due to an increased sensitivity to macrophage attack. INTRODUCTIONAerobic eukaryotic pathogens can encounter superoxide radicals (O 2 Ϫ ) generated from several sources. These sources can be internal or external. An important internal source is the mitochondrial respiratory chain (Boveris, 1978;Casteilla et al., 2001;Lenaz, 2001), and thus the rate of respiration can have a significant impact on reactive oxygen species (ROS) production. A key external source of ROS encountered by pathogens is from phagocytes. The superoxide radical is the first intermediate in the oxidative burst generated in the phagosome, and this burst is thought to be involved in pathogen killing (Reeves et al., 2002). The superoxide radicals are known to inactivate [4Fe-4S] cluster-containing enzymes by oxidizing one iron and releasing it from the cluster (Liochev and Fridovich, 1994;Fridovich, 1995). Free iron can react with hydrogen peroxide to generate toxic hydroxyl radicals (OH Ϫ ) by Fenton chemistry (Fridovich, 1978;Meneghini, 1997). The hydroxyl and superoxide radicals react with cellular components, resulting in oxidation of proteins and nucleic acids as well as lipid peroxidation. These effects can lead to inactivation of enzymes, to disruption of membranes, to mutations, and ultimately to cell death Gutteridge, 1990, 1999).To reduce the harmful effects of superoxide radicals, cells express detoxifying enzymes. Superoxide dismutase (SOD) is an antioxidant enzyme involved in elimination of superoxide anions; it catalyzes the reaction: O 2 Ϫ ϩ O 2 Ϫ ϩ 2H ϩ 3 H 2 O 2 ϩ O 2 . Normally, H 2 O 2 is still toxic to the cell; therefore, another enzyme, catalase, converts it to water. Superoxide dismutases can be classified according to metal cofactor(s) bound to them: there are iron (FeSOD), mangan...

SummaryThe gene encoding acetyl CoA:deacetylvindoline 4-Oacetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) crossreacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of µ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol Oacetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.

We have isolated a Candida albicans gene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinoline N-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicans AP-1. Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1 mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressing CAP1. Overexpression of either CAP1 or YAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved in YAP1-and CAP1-mediated multidrug resistance. YBR008c has been renamed FLR1, for fluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 or YAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.

SummaryCandida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type ( MAT ) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae . We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTL a and MTL a a a a strains, whereas disruptions in STE20 had no effect. A disruption in KEX2 , a gene involved in processing the S. cerevisiae pheromone Mf a a a a , prevented mating in MTL a a a a but not MTL a cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTL a but not in MTL a a a a cells. Disruption of two cell wall genes, ALS1 and INT1 , had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1 . The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.

We explored the host-pathogen interactions of the human opportunistic fungus Candida albicans using Drosophila melanogaster. We established that a Drosophila strain devoid of functional Toll receptor is highly susceptible to the human pathogen C. albicans. Using this sensitive strain, we have been able to show that a set of specific C. albicans mutants of different virulence in mammalian infection models are also impaired in virulence in Drosophila and remarkably display the same rank order of virulence. This immunodeficient insect model also revealed virulence properties undetected in an immunocompetent murine model of infection. The genetic systems available in both host and pathogen will enable the identification of host-specific components and C. albicans genes involved in the host-fungal interplay.