ascorbic-acid and Cryptococcosis

We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36-66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth-defective at 37 degrees C in 19-20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37 degrees C as well at 30 degrees C in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O(2) (.-)) in the mitochondria. The sod2 was also highly susceptible to redox-cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.

Topics: Animals; Antifungal Agents; Antimycin A; Antioxidants; Ascorbic Acid; Cryptococcosis; Cryptococcus neoformans; Disease Models, Animal; DNA, Fungal; Fungal Proteins; Gene Deletion; Genes, Fungal; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oxygen; Phagocytosis; Reactive Oxygen Species; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Superoxide Dismutase; Superoxide Dismutase-1; Superoxides; Survival Analysis; Virulence

Amphotericin B methyl ester (AME) has been reported to possess in vitro antifungal activity similar to that of amphotericin B and to have less intrinsic toxicity in mice and dogs. For these reasons AME has been porposed as an alternative to amphotericin B in the therapy of deep mycoses. For comparison of the therapeutic efficacy of the two polyenes in laboratory animals before initiation of studies in humans, groups of mice were infected with Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis. Treatment consisted of two or more doses of each drug given by the intravenous route. Concurrently, studies of subacute toxicity were conducted in the same species to permit calculation of therapeutic indices. These studies have shown that AME, as the ascorbate salt, is substantially less efficacious than amphotericin B (in colloidal dispersion with sodium deoxycholate) for treatment of the fungal infections and that amphotericin B had a higher therapeutic ratio for all infections studied than did AME.

Topics: Amphotericin B; Animals; Ascorbic Acid; Blastomyces; Blastomycosis; Candida albicans; Candidiasis; Cryptococcosis; Cryptococcus; Cryptococcus neoformans; Dose-Response Relationship, Drug; Drug Evaluation; Lethal Dose 50; Male; Mice