Posttranslational, Translational, and Transcriptional Responses to Nitric Oxide Stress in Cryptococcus neoformans : Implications for Virulence

Abstract

The ability of the fungal pathogen Cryptococcus neoformans to evade the mammalian innate immune response and cause disease is partially due to its ability to respond to and survive nitrosative stress. In this study, we use proteomic and genomic approaches to elucidate the response of C. neoformans to nitric oxide stress. This nitrosative stress response involves both transcriptional, translational, and posttranslational regulation. Proteomic and genomic analyses reveal changes in expression of stress response genes. In addition, genes involved in cell wall organization, respiration, signal transduction, transport, transcriptional control, and metabolism show altered expression under nitrosative conditions. Posttranslational modifications of transaldolase (Tal1), aconitase (Aco1), and the thiol peroxidase, Tsa1, are regulated during nitrosative stress. One stress-related protein up-regulated in the presence of nitric oxide stress is glutathione reductase (Glr1). To further investigate its functional role during nitrosative stress, a deletion mutant was generated. We show that this glr1⌬ mutant is sensitive to nitrosative stress and macrophage killing in addition to being avirulent in mice. These studies define the response to nitrosative stress in this important fungal pathogen.To survive the oxidative and nitrosative attack initiated by phagocytic cells of the host, pathogens must respond appropriately (reviewed in reference 35). This antimicrobial attack is established by two main systems including the inducible nitric oxide synthase pathway and the NADPH oxidase pathway (14). These two pathways generate either reactive nitrogen species (RNS) or reactive oxygen species. In the absence of either of these two pathways, mammalian hosts are more susceptible to both bacterial and fungal infections (18, 41). To cause infection, pathogens must evade the immune system by initiating a response to the stresses encountered.Previously, transcriptional responses to temperature, osmotic, and hydrogen peroxide stress as well as the stresses encountered in macrophages have been studied in fungi, including Saccharomyces cerevisiae and Candida albicans (13,25,29). A proteomic response to stress has only been determined in S. cerevisiae during hydrogen peroxide exposure (17). Proteomic analysis of the nitrosative stress response has not been studied in fungi, though transcriptional responses to RNS have been recently described in S. cerevisiae, C. albicans, and Histoplasma capsulatum (21,39,46). Though the response to nitrosative stress has not been studied in Cryptococcus neoformans, it has been implicated in both stress resistance and virulence of this fungal pathogen (10,33,36). It has been shown that macrophages produce nitric oxide in response to cryptococcal cells (20) and that the anticryptococcal activity of macrophages is mostly dependent on RNS (48). Recently, it was determined that during experimental cryptococcosis, the inducible form of nitric oxide synthase (iNOS) is expressed at increasing levels during infection (...

The effect of L-DOPA onCryptococcus neoformansgrowth and gene expression

“…The toxicity hypothesis is supported by published data showing that two of the genes, CNB04110 and CNB04240, are also induced by nitric oxide stress. 32 Furthermore, homology searches also provided information consistent with the toxicity hypothesis. CNG04630 has homology to permeases of the major facilitator family, proteins involved in removal of toxic substances from the cell, whereas CNK02300 has homology to glutathione transferases, proteins involved in metabolizing toxins inside cells.…”

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

“…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).…”

Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

“…Thus, the hypersensitivity of the pkh2-02⌬ mutant to oxidative and nitrosative stress suggested that its temperature-independent pathogenesis defect may be related to decreased replication or survival in the phagolysosome (47). To test this possibility, we compared the ability of the pkh2-02⌬ mutant to survive in the murine macrophage-like cell line J774, a well-studied model for the interaction of C. neoformans with macrophages.…”

“…A key characteristic of C. neoformans is its ability to survive and replicate within the phagolysosomes of macrophages (46). This ability is, in turn, dependent on the organism's tolerance of oxidative and nitrosative stresses (47). Since Pkc1 activity is required for oxidative and nitrosative stress tolerance in C. neoformans (37) and is reduced in pkh2-02⌬ mutants (Fig.…”

“…C. neoformans cells were opsonized with anticapsular antibody prior to addition to otherwise unactivated J774 cells (51). To control for the decreased growth rate of the pkh2-02⌬ mutant at 37°C, identical wells lacking J774 cells were processed in parallel, and growth of the C. neoformans cells in J774 cells was normalized to growth in medium alone (47). Under these conditions, WT cells replicate within J774 cells more efficiently than in medium alone while pkh2-02⌬ cells show a statistically significant decrease (2-fold decrease; P Ͻ 0.05 by Student's t test) in growth in the presence of J774 cells (Fig.…”