Cryptococcus neoformansMitochondrial Superoxide Dismutase: an Essential Link between Antioxidant Function and High-Temperature Growth

Abstract

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2⌬ mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2⌬ mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 M) at 30°C, but not at 37°C. Furthermore, sod2⌬ mutant cells that were incubated for 24 h at 37°C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25°C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.Eukaryotic cells possess several specialized enzymes that provide protection against reactive oxygen species produced as a by-product of physiologic metabolic processes (46). The mitochondria are an especially important source of endogenous reactive oxygen species in the cell. Under physiological conditions, a fraction of the oxygen consumed by the mitochondria is incompletely reduced, resulting in the formation of superoxide radical (9). Although superoxide radical can undergo spontaneous degradation, mitochondria depend on a manganese-containing superoxide dismutase (Sod2) to catalyze the rapid conversion of superoxide radical to hydrogen peroxide and oxygen (15). Loss of this enzyme activity has profound consequences for the viability of many organisms. For example, mice with a homozygous SOD2 disruption exhibited heart and liver defects, metabolic acidosis, and early neonatal death (23,24). Drosophila melanogaster SOD2 mutants exhibited increased sensitivity to oxidative stress and early onset death in young adults (21), and Saccharomyces cerevisiae SOD2 mutants exhibited a rapid loss of viability in the early stationary phase (25). These studies demonstrate a conserved and essential function for Sod2 as a component of the mitochondrial antioxidant defense system of many phylogenetically diverse organisms.Cryptococcus neoformans is an opportunistic human pathogen. Most cases of C. neoformans disease involve immunocompromised patients, such as those with human immunodeficiency virus infection or receiving corticosteroid therapy. In these patients, C. neoformans ca...

Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential

“…To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different types of drugs in the same cellular or animal model or both are needed. Thus far, only limited data have been provided (77,131,225,255). Further, few studies have shown that protective effects in vivo parallel the in vitro magnitude of the catalytic rate constant for O 2 ·À dismutation or peroxynitrite reduction or both (241,255).…”

“…Bioavailability is dependent on the size, charge, shape, (conformational flexibility and overall geometry), and lipophilicity and greatly affects the in vivo efficacy; data already indicate that whereas some drugs may be good in one model, they may fail or be less efficient in another one, as a consequence of differences in the extent of accumulation in subcellular compartments targeted (88,131,255). Detailed pharmacokinetic and toxicology data are still scarce.…”

“…Driven by the mitochondrial membrane potential, potent pentacationic Mn porphyrins with no particular targeting moiety also were found to be directly taken up by mitochondria (305). An MnSOD knockout yeast study suggested that Mn salen can also enter mitochondria (at least those of yeast) (131). In addition to synthetic antioxidants that act catalytically, natural antioxidants have been used in numerous studies and clinical trials with partially satisfactory results (270,291,292,311).…”

“…With aerobic growth of SODdeficient E. coli and S. cerevisiae lacking cytosolic SOD (225) besides mM Mn, only Mn porphyrins, MnTE-2-PyP 5þ and MnTM-2-PyP 5þ (and not Mn salen EUK-8 and Mn cyclic polyamine M40403 at the mM concentrations studied) were efficacious. With MnSOD knockout yeast C. neoformans (131), only the Mn salen, EUK-8, was protective, but neither of several Mn porphyrins (cationic and anionic), nor Tempol and MnCl 2 ; the data suggest that Mn salen transports Mn into mitochondria (131). Doctrow et al (94) compared anionic porphyrin MnTBAP 3À with EUK compounds, M40403, and a combination of acetyl-l-carnitine þ lipoic acid in the survival of MnSOD À=À mice; only EUK-207 and EUK-189 were efficacious.…”

Preferential transcription of Paracoccidioides brasiliensis genes: host niche and time-dependent expression

“…During the 22-h transition from mycelium to yeast the fungus preferentially expressed transcripts encoding catalase P and Cu/Zn superoxide dismutase. It has been suggested that superoxide dismutases, which promote the rapid degradation of superoxide into hydrogen peroxide and O 2 , augment adaptation of C. neoformans to human host body temperature (Giles et al 2005). These observations suggest a nichespecific activation of the oxidative stress response by P. brasiliensis, as described for C. albicans (Enjalbert et al 2007).…”