Function of the thioredoxin proteins in Cryptococcus neoformans during stress or virulence and regulation by putative transcriptional modulators

Abstract

SummaryThe thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, is known to protect cells against oxidative stress. This disulphide reducing system is present in Cryptococcus neoformans and consists of two small, dithiol thioredoxin proteins and one thioredoxin reductase. In this study, we describe the thioredoxin proteins, Trx1 and Trx2, and present their importance not only to stress resistance, but also to the virulence of C. neoformans . Using real-time polymerase chain reaction, we show the induction of both thioredoxin genes during oxidative and nitrosative stress. We describe through deletion studies that the trx1 D D D D mutant has a severe growth defect and is sensitive to multiple stresses, while the trx2 D D D D mutant is only sensitive to nitric oxide stress. Using gene replacement studies, we demonstrate that the thioredoxin protein products are partially redundant in function, but there is differential gene regulation which is especially important to nitrosative stress resistance. We have also identified two putative transcription factors, Atf1 and Yap4, which appear to differentially regulate the thioredoxin system under different conditions. Atf1 is necessary for oxidative stress induction, and Yap4 is necessary for nitrosative stress induction of the thioredoxin genes in C. neoformans. While these two putative transcription factors each appear to be dispensable for survival in macrophages and virulence in mice, we show the more highly expressed thioredoxin, TRX1 , is necessary for survival of C. neoformans in the oxidative environment of macrophages and important for virulence of this fungal pathogen.

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

“…Since 750 M sodium nitrite was toxic to the cryptococcal cells and 500 M appeared to be cytostatic, changes in protein expression were examined with 250 M and 500 M NaNO 2 for 6 h to ensure that the protein changes observed were in response to nitric oxide stress and not due to severe nitric oxide-mediated toxicity. For transcriptional expression changes, a stress of 2 h with 250 M NaNO 2 was used because previous work had shown that changes in the transcriptional profile were apparent at this shorter time point (33,34).…”

“…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).…”

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

“…Most of these are limited by their cytotoxic effects on the host, limited efficacy, or development of resistance in the pathogen upon long-term usage (4)(5)(6)(7). In addition, sessile biofilms act as a store house for antibiotic tolerant fungal cells that can lead to persistent systemic infections (8,9). The lack of antifungal agents active against biofilm-forming varieties increases the urgent need for novel agents in the drug pipeline (5,10).…”

Eca1, a Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase, Is Involved in Stress Tolerance and Virulence inCryptococcus neoformans

“…Tolerance to thermal stress (caused by body temperature) and oxidative stresses (from mammalian host defenses) is critical for successful infection. C. neoformans mutants with increased sensitivity to oxidative or nitrosative challenge, including those lacking superoxide dismutase Sod1 or Sod2 (18,35), alternative oxidase Aox1 (2), flavohemoglobin denitrosylase Fhb1 (23), thiol peroxidase Tsa1 (48), or thioredoxin Trx1 (47), exhibit attenuated virulence. Temperature-sensitive mutants have been isolated in C. neoformans, including cna1 (calcineurin catalytic subunit), cnb1 (calcineurin regulatory subunit), cpa1 (cyclophilin A), and ras1 strains, and all of these have reduced virulence in mammalian host models (3,29,55,73).…”