The Aspergillus fumigatus Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model

Abstract

Macrophages and neutrophils kill the airborne fungal pathogen Aspergillus fumigatus. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of A. fumigatus challenged by H 2 O 2 . Since many of the identified proteins and genes are apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified and designated Afyap1. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Afyap1 gene led to drastically increased sensitivity of the deletion mutant against H 2 O 2 and menadione, but not against diamide and NO radicals. Proteome analysis of the ⌬Afyap1 mutant strain challenged with 2 mM H 2 O 2 indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Afyap1 deletion mutant did not show attenuated virulence in a murine model of Aspergillus infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of A. fumigatus in an immunocompromised host. This conclusion was further supported by the finding that killing of A. fumigatus wild-type and ⌬Afyap1 mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.In the last few decades Aspergillus fumigatus has become the most important airborne fungal pathogen of humans. Diseases caused by A. fumigatus can be divided into three categories: allergic reactions and colonization with restricted invasiveness are observed in immunocompetent individuals, while systemic infections with high mortality rates occur in immunocompromised patients. Due to the improvement in transplant medicine and the therapy of hematological malignancies, the number of cases of invasive aspergillosis has increased. Specific diagnostics are still limited, as are the possibilities of therapeutic intervention, leading to a high mortality rate of 30 to 98% for invasive aspergillosis (8). The genome of the A. fumigatus isolate Af293 was fully sequenced. It consists of a haploid set of eight chromosomes with a total size of 29.4 Mb, of which 9,926 protein-encoding sequences were identified (44). With the genome data available the regulation of genes and the expression profile of proteins of A. fumigatus can be analyzed on a global scale, including the conditions that are related to infection.The infectious agent of A. fumigatus are conidia, which are inhaled during routine daily activities (8). Therefore, in immunocompromised patients, the lung is the site of infection of A. fumigatus. In immunocompetent individuals, mucociliary clearance and phagocytic defense normally prevent the disease. Alveolar macrophages are the major resident cells of the lung alveoli; they, along with neutrophils ...

Fungal antioxidant pathways promote survival against neutrophils during infection

“…The transcription factor Yap1 is activated in A. fumigatus exposed to oxidative conditions and regulates production of intracellular antioxidants and enzymes that convert ROS into less reactive products (36). For example, A. fumigatus superoxide dismutases (SOD1/2/3) convert superoxide to the less-reactive hydrogen peroxide (37), whereas A. fumigatus catalases (CatA/1/2) convert hydrogen peroxide to H 2 O (38).…”

“…Exposure of A. fumigatus to oxidative stress activates the transcription factor Yap1, which upregulates genes involved in anti-oxidative processes (36,42). During oxidative stress, the Yap1-dependent proteins allergen aspf3 and peroxiredoxin Prx1 are highly upregulated.…”

“…During oxidative stress, the Yap1-dependent proteins allergen aspf3 and peroxiredoxin Prx1 are highly upregulated. These are putative thioredoxin peroxidases (peroxiredoxins) in the thioredoxin antioxidant pathway (36) (44). Thioredoxin is then further reduced by the enzyme thioredoxin reductase using H + equivalents from NADPH + (44).…”

bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans

“…Similar to the reported roles in yeast, these proteins typically are associated with resistance to ROS or antifungals. The Yap1 orthologues Afyap1, Aoyap1, Apyap1 and MoAP1 in the human pathogen Aspergillus fumigatus, the mycotoxic pathogens Aspergillus ochraceus and Aspergillus parasiticus, and the rice pathogen Magnaporthe oryzae, respectively, all mediate the oxidative stress response, and in the latter pathogen, MoAP1 is critical for pathogenicity (Guo et al, 2011;Lessing et al, 2007;Qiao et al, 2008;Reverberi et al, 2007Reverberi et al, , 2012. Yap-like proteins in Aspergillus nidulans and Neurospora crassa have also been characterized in ROS and heavy metal resistance Tian et al, 2011).…”

Proteomics Methodology Applied to the Analysis of Filamentous Fungi - New Trends for an Impressive Diverse Group of Organisms

“…The differentially expressed proteins belonged to carbohydrate metabolism, cell stress, amino acid metabolism, translation, ubiquitin-dependent protein degradation, transcription, cytoskeletal proteins, cell wall and associated proteins and others including hypothetical proteins. Proteome analysis of the enzymatic reactive oxygen intermediates detoxifying system was studied in A. fumigatus challenged by H 2 O 2 together with the comparative proteome analysis of a mutant deleted in the transcription factor Afyap1, which is involved in the oxidative stress response (Lessing et al, 2007). Differential gel electrophoresis (DIGE) analysis, followed by mass spectrometry using an Ultraflex I MALDI-TOF/TOF (Bruker Daltonics) mass spectrometer, identified 27 protein spots that displayed an increase and 17 protein spots that displayed a decrease in intensity following A. fumigatus exposure to H 2 O 2 .…”