Superoxide Dismutase Influences the Virulence ofCryptococcus neoformansby Affecting Growth within Macrophages

Abstract

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.Invasive fungal infections in humans are increasing in prevalence in parallel with the growing population of immunocompromised patients. There is a need for new antifungal drugs to treat these infections since the drugs currently available are either excessively toxic or lack broad fungicidal properties. Studies on the pathogenesis of fungal infections should provide insights that can help with the diagnosis and treatment of these important human diseases. Cryptococcus neoformans is a basidiomycetous yeast that has been used successfully as a model pathogenic fungus in a variety of molecular pathogenesis studies. We used C. neoformans to evaluate the contribution of superoxide dismutase (SOD) to the pathogenesis of fungal infections.SODs are metalloenzymes that detoxify oxygen radicals through the conversion of superoxide to hydrogen peroxide and oxygen (20). These enzymes are present in virtually all cells, and this very high degree of conservation is testament to their importance in cellular homeostasis. The primary role of SODs is to protect cells from endogenously generated superoxide anion, which is a by-product of normal aerobic respiration. SODs can be complexed with iron, manganese, and copper plus zinc. The iron and manganese SODs are genetically similar to each other, whereas the Cu,Zn SOD exhibits no significant homology with the other two enzymes (16,20,21,32). Eukaryotic cells generally contain an Mn SOD in the mitochondrial matrix and a Cu,Zn SOD which is located pre...

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

“…Previous studies of C. neoformans virulence factors have implied that Cu plays important, but poorly characterized, roles in fungal virulence. This hypothesis is supported by the Cu dependence of many biological processes and virulence-related proteins, including melanin biogenesis, superoxide dismutase activity and Fe uptake through Cu-dependent ferroxidase activity [12][13][14][15] . However, recent studies also demonstrated that the Cu-detoxification proteins known as MTs are essential for fungal survival during pulmonary colonization and for virulence, indicating that C. neoformans encounters elevated Cu levels in the host early in the infectious process 5 .…”

Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans

“…Third, besides the TS phenotype, the tps1 mutant at lower environmental temperatures did not display any apparent deficiencies in classic virulence factors such as melanin, capsule, or urease production, but the tps1 mutant was slightly more susceptible than the tps2 mutant to osmotic and oxidative challenges. However, the reduced protection against these conditions did not translate into a detectable intracellular growth defect, which has been linked with a lower-virulence phenotype in C. neoformans (1,11,12,24).…”

“…C. neoformans strains were wild-type H99, a serotype A clinical isolate (46), and H99R, a spontaneous uracil auxotroph derived from H99 (11). The tps1 and tps2 mutant strains were created from H99R, while the nth1 mutant originated from H99.…”

In the Absence of Reactive Oxygen Species, T Cells Default to a Th1 Phenotype and Mediate Protection against Pulmonary Cryptococcus neoformans Infection

“…This would imply that, in some instances at least, ROS may be toxic to this fungus. Melanin-deficient C. neoformans possess a reduced capacity to kill mice (17), and CuSOD lacking Cryptococcus displaying attenuated growth within macrophages (18). The improved killing of C. neoformans in the absence of ROS has been described previously in vitro, whereby incubation of peritoneal macrophages with SOD and catalase augments anticryptococcal activity owing to a concomitant increase in NO production (38).…”

“…Furthermore, this fungus displays an array of protective mechanisms (superoxide dismutase (SOD), mannitol, melanin) to confer resistance to ROS, suggesting that in some instances they may be toxic and adverse to its survival. C. neoformans deficient in melanin exhibit reduced virulence in vivo (17), and CuSOD lacking mutants are more susceptible to oxygen radicals in macrophages (18).…”