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 glr1Delta 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.

FIG. 1.

Survival of H99 in the presence of sodium nitrite at pH 4. Cells were grown for 16 h in YNB pH 4 and treated with various concentrations of sodium nitrite for 2 and 6 h.

FIG. 2.

Protein expression in response to nitrosative stress. Two-dimensional electrophoretic analysis of H99 untreated (a) and treated with 500 μM sodium nitrite for 6 h (b). Arrows indicate proteins with altered expression which were identified by mass spectrometry, and numbers refer to Table 3. Numbered arrows indicate the gel from which the protein spot was analyzed. Molecular masses are listed in kilodaltons. The asterisk (*) marks the Tsa1 protein spot analyzed in reference .

FIG. 3.

Gene expression in response to nitric oxide stress. Gene expression was determined by real-time PCR.

FIG. 4.

glr1Δ mutant phenotypes. Phenotypes of two independent glr1Δ isolates in the presence of oxidative or nitrosative stress (a), in the presence of RAW 264.7 macrophages (b), and in vivo using an inhalation model of murine infection (c).