Isocitrate dehydrogenase is important for nitrosative stress resistance in Cryptococcus neoformans, but oxidative stress resistance is not dependent on glucose-6-phosphate dehydrogenase

Abstract

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 idp1Delta 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.

Fig. 1.

C. neoformans IDP1 is essential for growth at higher temperature. (a) The temperature-sensitive phenotype of an idp1Δ mutant is completely restored in complemented strains. Tenfold serial dilutions of cell suspensions of wild-type C. neoformans (KN99), the idp1Δ mutant, and two independent idp1 mutants reconstituted with the wild-type IDP1 gene (A8 and C4) were inoculated on YPD and incubated at the indicated temperatures. Pictures were taken at 2 and 4 days. (b) Real-time PCR confirmation of the presence of the IDP gene-specific transcript in the complemented strains.

Fig. 2.

NADPH in idp1Δ cells increases during nitrosative stress but stays consistent in wild-type and zwf1Δ cells during stress. Absolute amounts of NADPH and NADP⁺ were plotted as a ratio. Measurements were taken from 1 × 10⁷ wild-type (black bars), zwf1Δ (gray bars), and idp1Δ (white bars) cells. (A) NADPH/NADP⁺ ratio during normal growth. (B) NADPH/NADP⁺ ratio during oxidative stress (1 mM H₂O₂). (C) NADPH/NADP⁺ ratio during nitrosative stress (1 mM NaNO₂). Values are means ± standard deviations (n = 12). *, P < 0.05 compared with control (two-tailed Student t test).

Fig. 3.

The idp1Δ mutant displays greater sensitivity to nitrosative stress than to H₂O₂ stress. The stress phenotype is completely rescued in the two independent complemented strains. Serial 10-fold dilutions of the wild type (KN99), the idp1 mutant, and two reconstituted strains were spotted on YNB (pH 4.0) and YNB (pH 4.0) supplemented with H₂O₂ (1 mM) or NaNO₂ (0.75 mM). Plates were incubated at 25°C, and pictures were taken at days 2 and 4. The idp1Δ::IDP1-A8 and idp1Δ::IDP-C4 strains contain the wild-type IDP1 gene reinserted at its endogenous locus.

Fig. 4.

The zwf1Δ strain is not sensitive to oxidative or nitrosative stress. Three independent null isolates (zwf1Δ-1, zwf1Δ-2, and zwf1Δ-3) and the wild type (KN99α) were plated on minimal medium with or without a oxidative (H₂O₂) or nitrosative (NaNO₂) stressor and incubated at 30°C. Compared to the wild type, the null strains did not have a sensitive phenotype with either stressor. The picture was taken after 7 days of incubation.

Fig. 5.

Production of 6-PG is attenuated in the zwf1Δ strain compared to the wild type. Trimethylsilyl derivatives of 6-PG were measured by gas chromatography-mass spectrometry from lysates of 1 × 10⁸ wild-type (KN99α) and zwf1Δ cells. Triplicate measurements were made from six biological replicates of each strain grown in minimal medium without a stressor or after 2 h of exposure to oxidative stress (1 mM H₂O₂) or nitrosative stress (1 mM NaNO₂). Values are means ± standard deviations. *, P < 0.05 compared to control, using Student's t test.

Fig. 6.

Nitrosative stress induces mitochondrial damage that is pronounced in the idp1Δ strain compared to the wild type. Images were taken by confocal microscopy at a magnification of ×100. During exposure to nitrosative stress (0.75 mM NaNO₂), the idp1Δ cells retain MitoTracker (MT) (green images) but poorly retain rhodamine 123 (Rh) (red images) compared to the wild type (KN99α) under the same conditions. During normal conditions (control), both wild-type and idp1Δ cells retain MT and Rh. BF, bright field.

Fig. 7.

Percentage of cells that were rhodamine 123 positive after exposure to nitrosative stress. The percentage of cells that retained MitoTracker and also retained rhodamine 123 after 1 h of exposure to nitrosative stress (0.75 mM NaNO₂) is shown. Cells that retained both dyes are considered to have mitochondria with intact, potentiated membranes, while cells that did not retain rhodamine 123 do not have mitochondria with fully potentiated membranes. Three hundred cells of each strain that retained MitoTracker were counted, and the percentage of these 300 that also retained rhodamine 123 is represented graphically.

Fig. 8.

Possible cytosolic sources of NADPH in C. neoformans. NADPH is produced during the oxidative phase of the pentose phosphate pathway after glucose is converted to glucose-6-phosphate, which is then converted to 6-phosphogluconate (6-PG) by glucose-6-phosphate dehydrogenase (Zwf1). This reaction produces NADPH (arrow 1). 6-PG (star) is then converted to ribulose-5-phosphate by 6-phosphogluconae dehydrogenase (Gnd) in a reaction that also produces NADPH (arrow 2). The Gnd reaction requires 6-PG (star) to proceed. In C. neoformans, an alternative source of 6-PG may be gluconate kinase (Gnk), an enzyme that converts gluconate to 6-PG in a reaction that produces NADPH (arrow 3).