The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37°C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37°C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

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Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b 5 reductase and cytochrome b 5 were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b 5 reductase and cytochrome b 5 , expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent K m values similar to those found in human liver microsomes and specific activities (V max ) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b 5 in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b 5 reductase and cytochrome b 5 significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b 5 reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b 5 reductase and cytochrome b 5 in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.Hydroxylamine and amidoxime compounds are metabolized in humans by an as yet incompletely characterized NADH-dependent reductase system. Hydroxylamine metabolites have been implicated in dose-dependent and idiosyncratic drug toxicity from sulfamethoxazole, dapsone, procainamide, and other arylamine drugs (Uetrecht, 2002). Amidoximes and other hydroxylated amines have been developed as prodrugs to enhance the absorption of a wide range of antihypertensive, antiprotozoal, and antithrombotic drugs (Weller et al

Cryptococcus neoformans is an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity of C. neoformans to subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated with C. neoformans resistance to oxidative stress. In the present study, we identify and characterize a C. neoformans homologue to SKN7, a transcription factor in Saccharomyces cerevisiae that regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution of SKN7 in the pathogenesis of fungal infections, we created skn7 mutants via targeted disruption. The skn7 mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly, skn7 mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed in skn7 mutants derived from other fungi. These findings demonstrate that SKN7 contributes to the virulence composite but is not required for pathogenicity in C. neoformans. In addition, flocculation of C. neoformans skn7 mutants suggests a potentially unique function of SKN7 not previously observed in other cryptococcal strains or skn7 mutants.Cryptococcus neoformans, the etiological agent of cryptococcosis, is an encapsulated fungal pathogen that infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis (31). Cryptococcosis occurs in approximately 5 to 25% of AIDS patients worldwide (37), and studies have shown that 2.8% of organ transplant recipients can develop C. neoformans infections, resulting in an overall death rate of 42% (16). Although highly active antiretroviral therapy has contributed to a significant decrease in the incidence of cryptococcosis in AIDS patients in developed countries (2), increases in organ transplant recipients and patients undergoing extensive corticosteroid therapy forecast a rise of cryptococcosis in other high-risk populations. In medically advanced countries the acute mortality rate is between 10 and 25% (37), and conventional antifungal agents are often excessively toxic, lack potent fungicidal properties, or are being rendered less effective by the emergence of drug-resistant strains. Therefore, continued studies are needed to identify novel targets for the development of drugs or vaccines to combat cryptococcal infections.Obligate aerobic microorganisms, such as C. neoformans, are under persistent exposure to endogenous oxidative stress caused by the incomplete reduction of oxygen to water, which yields reactive oxygen species (ROS) such as H 2 O 2 , the hydrox...