Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans,but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase

The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP⁺-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.The alveolar macrophage can produce microbicidal amounts of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS) following phagocytosis (27, 53). Despite this, the opportunistic fungal pathogen Cryptococcus neoformans is able to inhabit and replicate within phagocytes of the mammalian host and to exit these cells unharmed (1, 2, 40). The intracellular pathogenicity of C. neoformans is most likely facilitated by stress resistance mechanisms, including a number of antioxidant proteins and pathways involved in the detoxification of ROS and RNS. Specifically, these include the synthesis of mannitol, a free radical scavenger (9, 20); the small protein flavohemoglobin denitrosylase (Fhb1), which is essential for resistance of C. neoformans to nitrosative stress (10, 14, 32); and the glutathione and thioredoxin antioxidant systems, which are both important for stress resistance and virulence (42, 43, 45).Even with different mechanisms of catalysis and/or cellular localization, one thing that these stress resistance proteins and pathways have in common is the requirement for NADPH as a cofactor. NADPH is used as an electron donor either in recycling of oxidized, inactive enzymes to reduced, active forms or directly in catalytic activity. For example, Fhb1 binds NADPH during its catalytic activity and uses it directly as an electron donor for the reduction of NO· to NO₃ (21). Catalases, which are highly conserved antioxidants that dismute H₂O₂ to molecular oxygen and water, consist of four units each with a molecule of NADPH bound in the core (18, 36, 59). The tripeptide glutathione (GSH) is oxidized to glutathione disulfide (GSSG), a homodimer held together by a disulfide bridge, during its oxidative state. GSSG can be reduced back to GSH by glutathione reductase, an enzyme that requires NADPH for electrons used in reduction. Similarly, glutathione peroxidase and thiol peroxidase ultimately depend on NADPH for recycling from an oxidized, inactive form back to a reduced, active form (57).NADPH is classically recognized as being produced by the highly conserved, cytosolic pentose phosphate pathway. This pathway has been shown to be important for reductive biochemistry during oxidative stress in many organisms. The pentose phosphate pathway is an essential factor in maintaining health of erythrocytes, cells that, due to their biological function, have considerable risk for oxidative damage. Humans deficient in the pathway have hemolytic anemia, as their erythrocytes are unable to maintain sufficient pools of reduced glutathione (68). Also, the pressure of oxidative stress can stimulate the pentose phosphate pathway. This has been shown in human lymphocytes (56); in the rat adrenal gland, liver, and pancreas (15, 16); and in bacteria (63).In fungi, the pentose phosphate pathway has been implicated in both oxidative stress resistance and adaptation to oxidative stress. In the model yeast Saccharomyces cerevisiae, NADPH-generating systems, including the pentose phosphate pathway, are critical for the ability of this organism to resist and adapt to high levels of oxidative stress (35, 47). It has also been shown that the cytosolic copper/zinc superoxide dismutase and the pentose phosphate pathway have overlapping roles in protecting S. cerevisiae from oxidative stress and that both systems are critical for maintaining the intracellular redox state (62). Furthermore, fungi may rely on the pentose phosphate pathway for more than reducing oxidative stress. Aspergillus nidulans requires a functional pentose phosphate pathway for nitrogen metabolism. Four A. nidulans mutants with independent defects in the pentose phosphate pathway were unable to grow on nitrite, nitrate, or various carbon sources, including 1% glucose, d-xylose, or d-glucoronate (28).The pathway has two phases, the oxidative phase and the nonoxidative phase. The oxidative phase consists of two successive oxidations and results in the production of NADPH. The first enzyme in the oxidative phase of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and is highly specific for NADP⁺ as a cofactor (49, 67). There is abundant evidence supporting the role of Zwf in oxidative stress resistance. In addition to Zwf deficiency causing hemolytic anemia, Zwf has been also been implicated in maintenance of DNA repair systems during oxidative stress, as some cancers and aging disorders have also been linked to Zwf deficiency (30). For instance, Chinese hamster ovarian cells that are Zwf null have enhanced radiation sensitivity and a reduced ability to repair double-strand breaks due to the inactivation of Ku, a heterodimer DNA repair protein. In this case, the inactivation of Ku is the result of overoxidation of key cysteine residues on the protein due to the lack of sufficient reduced GSH (3). In the model yeast Saccharomyces cerevisiae, deletion of ZWF1 results in sensitivity to oxidative stress. ZWF1 is also important for the adaptive response to oxidative stress in S. cerevisiae. ZWF1-null mutants and wild-type cells were pretreated with 0.2 mM H₂O₂ and then challenged with 2 mM H₂O₂. While a large increase in tolerance to the high level of H₂O₂ was observed in the wild-type cells pretreated with 0.2 mM H₂O₂, the zwf1Δ strain was unable to tolerate the higher concentration (33). In Candida albicans, another pathogenic fungus, ZWF1 is upregulated during oxidative stress (38).Another source of NADPH is NADP⁺-dependent isocitrate dehydrogenase (Idp) (55), a ubiquitous enzyme that in systems ranging from humans to yeasts to plants has been found in the cytosol, peroxisomes, or mitochondria (12, 19, 70). Although this enzyme can be targeted to mitochondria, it is distinct from the NAD⁺-dependent isocitrate dehydrogenase (Idh) that functions in the mitochondria as part of the Krebs cycle. However, similarly to Idh, Idp catalyzes the decarboxylation of isocitrate to α-ketoglutarate (29). This reaction can be performed in the mitochondria, in the cytosol, or in peroxisomes using isocitrate formed from citrate exported across the mitochondrial membrane. This allows for the production of NADPH in cellular compartments without reliance of active transport of NADPH across membranes (11). It is important to have reductive power produced directly within organelles for protection from exogenous as well as endogenous stressor. For example, NADPH is consumed in peroxisomes by enzymes such as catalase and uric acid oxidase, that counteract the ROS produced during breakdown of lipids (4, 5, 31). Mitochondria particularly require reductive capability, as these organelles are susceptible to endogenous ROS produced during cellular respiration and also to exogenous RNS (52). The proteins that make up the electron transport chain are prone to damage by nitric oxide, peroxynitrite, and S-nitrosothiols (6). Nitric oxide and peroxynitrite have been shown to cause irreversible damage to cytochrome c reductase, NADH dehydrogenase, and the succinate-ubiquinone complex; the common mechanism of damage is sequestration of iron/sulfur centers of the proteins (54, 69). Thus, without a means of detoxification, the mitochondrial membrane loses potential and the ability to continue respiration, leading to death of the stressed cell. In C. neoformans, some antioxidant enzymes that are located at the mitochondria and dependent on NADPH for function include catalases, superoxide dismutases, cytochrome c peroxidase, and flavohemoglobin denitrosylase (7, 24, 25, 26). These enzymes are important for stress resistance or virulence of C. neoformans due to their role in high-temperature growth (24, 25) or nitrosative stress resistance (10, 14, 26).In humans, there is one IDP gene that results in mitochondrial and peroxisomal products (22). In S. cerevisiae, there are three IDP genes, which encode mitochondrial (IDP1), cytosolic (IDP2), and peroxisomal (IDP3) forms of the protein. Deletion of both ZWF1 and any one of the IDP genes in S. cerevisiae results in sensitivity to oxidative stress, likely due to a substantial decrease in NADPH produced in these double deletion mutants (41). In C. neoformans there is one predicted IDP gene (IDP1). Microarray data have indicated that this gene is upregulated 2.5-fold during nitrosative stress and thus may have a role in resistance to this stressor (44).Since so many factors essential for stress resistance in C. neoformans utilize NADPH, we hypothesize that the sources of this cofactor are likewise critical for stress resistance. Although Zwf1 is important for adaptation to oxidative stress in the fungi S. cerevisiae and C. albicans, we had previously found that C. neoformans is unable to adapt to oxidative stress (S. M. Brown and J. K. Lodge, unpublished data), and thus we had reason to suspect that the role of Zwf1 in C. neoformans may be different than that in other organisms. The role of Idp1 in stress resistance, especially in resistance to nitrosative stress, is relatively unknown. In this study we used biochemical and genetic approaches to compare the roles of Zwf1 and Idp1 in resistance to oxidative and nitrosative stress in C. neoformans. We found that the Zwf1 is dispensable for viability, for resistance to oxidative and nitrosative stress, and for NADPH production. In contrast, we found that Idp1 is important for resistance to nitrosative stress, specifically for maintaining healthy mitochondria during exposure to nitrosative stress.