Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSFriday, March 15 3:00 PM–6:00 PMFred Farr ForumOxidative Stress, ROS Signaling and Adaptation to HypoxiaCo-chairs: Geraldine Butler and Barry ScottTranscriptional regulatory networks controlling the early hypoxic response in Candida albicans. A. Nantel, M. van het Hoog, A. Sellam, C. Beaurepaire, F.Tebbji, M. Whiteway. National Research Council of Canada, Montreal, Quebec, Canada.The ability of Candida albicans to colonize or invade multiple host environments requires that it rapidly adapts to different conditions. Our group hasbeen exploiting ChIP-chip and transcription profiling technologies, together with computer modeling, to provide a better understanding of selecttranscription factor (TF) networks. We used DNA microarrays to measure the changes in transcriptional profiles that occur immediately following thetransfer of C. albicans to hypoxic growth conditions. The impressive speed of this response is not compatible with current models of fungal adaptation tohypoxia that depend on the inhibition of sterol and heme biosynthesis. Functional interpretation of these profiles was achieved using Gene Set EnrichmentAnalysis, a method that determines whether defined groups of genes exhibit a statistically significant bias in their distribution within a ranked gene list.The Sit4p phosphatase, Ccr4p mRNA deacetylase and Sko1p TF were identified as novel regulators of the early hypoxic response. While cells mutated inthese regulators exhibit a delay in their transcriptional responses to hypoxia their ability to grow in the absence of oxygen is not impeded. Promoteroccupancy data on 26 TFs was combined with the profiles of 375 significantly-modulated target genes in a Network Component Analysis (NCA) to producea model of the dynamic and highly interconnected TF network that controls this process. The NCA also allowed us to observe correlations betweentemporal changes in TF activities and the expression of their respective genes, thus allowing us to identify which TFs are potentially subjected to posttranscriptionalmodifications. The TF network is centered on Tye7p and Upc2p which are associated with many of the genes that exhibit the fastest andstrongest up regulations. While Upc2p only associates with downstream promoters, Tye7p is acting as a hub, its own promoter being bound by itself and 7additional TFs. Rap1 and Ahr1 appear to function as master regulators since they bind to a greater proportion of TF gene promoters, including those ofUpc2p and Tye7p. Finally, Cbf1p, Mrr1p and Rap1p show the greatest numbers of unique gene targets. The high connectivity of these models illustratesthe challenges that lie in determining the individual contributions of specific TFs.Proteomic analysis of the hypoxic response of the human-pathogenic fungus Aspergillus fumigatus. Olaf Kniemeyer 1,4,5 , Kristin Kroll 1,5 , Vera Pähtz 1,4,5 ,Martin Vödisch 1,5 , Falk Hillmann 1,5 , Kirstin Scherlach 2 , Martin Roth 3 , Christian Hertweck 2 , Axel A. Brakhage 1,5 . 1) Department of Molecular and AppliedMicrobiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany; 2) Department of Biomolecular Chemistry, LeibnizInstitute for Natural Product Research and Infection Biology (HKI), Jena, Germany; 3) Bio Pilot Plant, Leibniz Institute for Natural Product Research andInfection Biology (HKI), Jena, Germany; 4) Integrated Research and Treatment Center, Center for Sepsis Control and Care Jena, University Hospital (CSSC),Jena, Germany; 5) Department of Microbiology and Molecular Biology, Friedrich Schiller University Jena, Beutenbergstr. 11a, 07745 Jena, Germany.Aspergillus fumigatus is a ubiquitous, filamentous fungus which may cause a broad spectrum of disease in the human host, ranging from allergic orlocally restricted infections to invasive mycoses. The most fatal A. fumigatus disease, invasive aspergillosis occurs in patients who are severelyimmunocompromised and is characterized by a high mortality. During the course of the infection A. fumigatus has to cope with several kinds of stressconditions including low oxygen levels (hypoxia). Just recently it was shown that hypoxia adaptation is an important virulence attribute of A. fumigatus. Toidentify novel hypoxia-sensing and adapting pathways we have characterized the changes of the A. fumigatus proteome in response to short (3-24 hours)and long periods (7-10 days) of hypoxia (1% O 2). To maintain reproducible culture conditions, an oxygen-controlled fermenter was used. During long-termcultivation under hypoxia, proteins involved in glycolysis, the pentose phosphate shunt, amino acid biosynthesis, NO-detoxification and respiration showedan increased level. In contrast, proteins involved in sulfate assimilation and acetate activation were down-regulated. Strikingly, hypoxia also inducedbiosynthesis of the secondary metabolite pseurotin A. The proteomic response of A. fumigatus to short periods of hypoxia showed some similarities, butalso marked differences: The level of glycolytic, NO-detoxifying and amino acid biosynthesis enzymes increased under both hypoxic growth conditions.However, the abundance of enzymes of the pentose-phosphate pathway decreased, whereas enzymes involved in ethanol fermentation significantlyincreased. To get a deeper knowledge about the specific role of metabolic pathways in adaptation to hypoxia, we have started to characterize candidategenes for their role in hypoxia by generating deletion mutants. First data will be presented and discussed.Fgap1-mediated response to oxidative stress in trichothecene-producing Fusarium graminearum. M. Montibus, N. Ponts, E. Zehraoui, F. Richard-Forget,C. Barreau. INRA, UR1264-MycSA, BP81, F-33883 Villenave d’Ornon, France.The filamentous fungus Fusarium graminearum infects cereals and corn. It is one of the main causal agent of “Fusarium Head Blight” and “Maize EarRot”. During infection, it produces mycotoxins belonging to the trichothecenes family that accumulate in the grains. Although the biosynthetic pathwayinvolving specific Tri genes has been elucidated, the global regulation of toxin biosynthesis remains enigmatic. It is now established that oxidative stressmodulates the production of toxins by F. graminearum. H 2O 2 added in liquid cultures of this fungus enhances trichothecenes accumulation and increasesTri genes expression. Our working hypothesis is that a transcription factor regulates redox homeostasis, and is involved in Tri genes regulation. In the yeastSaccharomyces cerevisiae, the transcription factor Yap1p mediates response to oxidative stress via nuclear re-localization and activation of genes codingfor detoxification enzymes. In this study, we investigate the role of Yap1p homolog in F. graminearum, Fgap1, in response to oxidative stress and itseventual role in the regulation of trichothecene production. A deleted mutant and a strain expressing a constitutively activated form of the Fgap1 factor inF. graminearum were constructed. We cultured these mutants in GYEP liquid medium supplemented with H 2O 2 to evaluate their sensitivity to oxidativestress and analyse their toxin production. The nuclear localization of constitutively activated Fgap1p as well as wild-type Fgap1p under oxidative stress byH 2O 2 was analyzed. Expression profiles of genes encoding oxidative stress response enzymes potentially controlled by Fgap1p and of genes involved in thebiosynthesis of type B trichothecenes were analyzed by Q-RT-PCR. Trichothecene accumulation is strongly enhanced in the deleted strain, with an increasein Tri genes expression. On the other hand, Tri genes expression and toxin accumulation are drastically repressed in the mutant in which Fgap1p isconstitutively activated. Moreover, the level of expression of two genes encoding catalases is modulated in both mutants. The involvement of Fgap1 inother types of stress has also been investigated. In particular, cadmium and osmotic stress affect growth in the deleted strain.72