Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450(14DM) (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BENr was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Plant pathogenic fungi of the genus Fusarium cause agriculturally important diseases of small grain cereals and maize. Trichothecenes are a class of mycotoxins produced by different Fusarium species that inhibit eukaryotic protein biosynthesis and presumably interfere with the expression of genes induced during the defense response of the plants. One of its members, deoxynivalenol, most likely acts as a virulence factor during fungal pathogenesis and frequently accumulates in grain to levels posing a threat to human and animal health. We report the isolation and characterization of a gene from Arabidopsis thaliana encoding a UDP-glycosyltransferase that is able to detoxify deoxynivalenol. The enzyme, previously assigned the identifier UGT73C5, catalyzes the transfer of glucose from UDP-glucose to the hydroxyl group at carbon 3 of deoxynivalenol. Using a wheat germ extract-coupled transcription/translation system we have shown that this enzymatic reaction inactivates the mycotoxin. This deoxynivalenol-glucosyltransferase (DOGT1) was also found to detoxify the acetylated derivative 15-acetyl-deoxynivalenol, whereas no protective activity was observed against the structurally similar nivalenol. Expression of the glucosyltransferase is developmentally regulated and induced by deoxynivalenol as well as salicylic acid, ethylene, and jasmonic acid. Constitutive overexpression in Arabidopsis leads to enhanced tolerance against deoxynivalenol.

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP‐binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Δ pdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Δ pdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy‐dependent extrusion of water‐soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

Summary: Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real‐time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro. ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro. The reduced viability of sod5Δ/Δ and sod4Δ/Δsod5Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox^−/− mice defective in the oxidative burst activity, demonstrating a ROS‐dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans, and perhaps many other microbial pathogens, can evade host immune surveillance in vivo.

Infectious Phenotype: The pathogenic yeast Candida albicans needs to adopt a filamentous form to invade tissues. The distantly related yeast species Saccharomyces cerevisiae also takes on a filamentous form for nutrient foraging. Comparing genome-wide deletion libraries between the two species, Ryan et al. (p. 1353 ) identified genes involved in three different filamentous yeast phenotypes and found unique genes for each of these phenotypes. However, in addition, core genes, including a previously unknown conserved regulator, appear to have homologous roles in regulating filamentous growth in these distantly related yeast species.