Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1

Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger. A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen. One C. neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and we describe a homologous gene, LAC2, that is found 8 kb away from LAC1 in the genome. In this study we report a role for both laccases, in addition to the thiol peroxidase, Tsa1, in oxidative and nitrosative stress resistance mechanisms of C. neoformans. With use of real-time PCR, similar changes in expression of the two laccase genes occur in response to oxidative and nitrosative stresses, but only the regulation of the LAC2 gene during stress is influenced by Tsa1. Both laccases contribute to melanin production using L-dopa as a substrate and are differentially localized in the cell based on green fluorescent protein fusions. A single deletion of either LAC1 or LAC2 alone had no effect on sensitivity to H2O2 or nitric oxide. However, deletion of either LAC1 or LAC2 in combination with a TSA1 deletion resulted in a slight peroxide sensitivity, and a lac2Delta tsa1Delta deletion strain was sensitive to nitric oxide stress. In addition, the deletion of both laccases reduces survival of C. neoformans in primary macrophages. Based on our expression and functional analysis, we propose a novel model for the interaction of these two systems, which are both important for virulence.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

Cryptococcus neoformans, a fungus under stress

Cryptococcus neoformans is a human fungal pathogen that survives exposure to stresses during growth in the human host, including oxidative and nitrosative stress, high temperature, hypoxia, and nutrient deprivation. There have been many genes implicated in resistance to individual stresses. Notably, the catalases do not have the expected role in resistance to external oxidative stress, but specific peroxidases appear to be critical for resistance to both oxidative and nitrosative stresses. Signal transduction through the HOG1 and calcineurin/calmodulin pathways has been implicated in the stress response. Microarray and proteomic analyses have indicated that the common responses to stress are induction of metabolic and oxidative stress genes, and repression of genes encoding translational machinery.     

Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation,Oxidative and Nitrosative Stress

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO^•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.