Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSHendrickson 1 , Nora Grahl 2 , Dawoon Chung 2 , Bridget Barker 1 , Robert A. Cramer 2 . 1) Immunology & Infectious Disease, Montana State University, Bozeman,MT; 2) Microbiology & Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH.The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host based on evolutionary mechanisms derived from itsecological niche as a composter or saprobe. SrbA, a member of the helix-loop-helix family of transcription factors, plays a significant role in A. fumigatushypoxia adaptation, antifungal drug responses, and virulence. SrbA is a direct transcriptional regulator of several key enzymes in the ergosterolbiosynthesis pathway, which has been verified by in vivo ChIP-SEQ analyses. The sterol intermediate profile of DsrbA revealed a significant accumulation ofC4-methyl sterols, which correlates with the loss of erg25 (C4-sterol methyl oxidase) mRNA abundance in the SrbA-null mutant. We hypothesized that thisC4-methyl sterol accumulation may contribute to the significant phenotypes observed in DsrbA. We have characterized the two genes predicted to encodeC4-methyl sterol oxidases (Erg25) in A. fumigatus. Genetic deletion of both erg25 genes, A and B, is lethal in A. fumigatus, while single genetic deletions ofthe respective genes are viable. Although loss of both erg25A and erg25B resulted in accumulation of C4-methyl sterols, Derg25A accumulated far moreC4-methyl sterol intermediates than Derg25B, suggesting that Erg25A is the predominant C4-sterol methyl oxidase in A. fumigatus. No dramatic in vitro orin vivo phenotypes under various stress conditions were observed in Derg25A or Derg25B mutants; however, a moderate reduction in hypoxia growth wasobserved in Derg25A. Generation of a strain that constitutively expresses erg25A in the DsrbA background biochemically relieved a majority of the C4-methyl sterol buildup in DsrbA. Significantly, restoration of erg25A mRNA levels in DsrbA with a promoter replacement fully restored the in vitro hypoxiagrowth defect of DsrbA. These results indicate that erg25 transcriptional regulation by SrbA and management of C4-methyl sterol intermediateaccumulation is highly important for hypoxia stress adaptation in A. fumigatus. Future studies will explore the impact of reducing C4-methyl sterol levels inDsrbA on A. fumigatus virulence.356. VeA Regulates Conidiation, Gliotoxin Production and Protease Activity in the Opportunistic Human Pathogen Aspergillus fumigatus. SourabhDhingra 1 , David Andes 2 , Ana M. Calvo 1 . 1) Biological Sciences, Northern Illinois University, DeKalb, IL; 2) Medical Mycology and immunology, University ofWisconsin-Madison, Madison, WI.Aspergillus fumigatus is the causative organism of invasive aspergillosis. Our study shows that normal levels of veA expression are necessary for wildtypemorphological differentiation in this medically important fungus. Specifically, deletion or overexpression of veA reduce conidiation. Parallely, brlAexpression was also affected by alterations in veA transcription levels. In addition, our studies revealed that veA regulates gliotoxin production in A.fumigatus. Gliotoxin production was decreased in the deletion veA and over-expression veA strains, where gliZ and gliP expression was altered.Interestingly, veA also controls hydrolytic enzyme activity in this human pathogen. Deletion of veA resulted in a reduction of protease activity; this is thefirst report of a veA homolog with a role in controlling fungal hydrolytic activity. Although veA affects several cellular processes in A. fumigatus,pathogenicity studies in a neutropenic mouse infection model indicated that veA is dispensable for virulence. More research will be conducted to studythe effect of veA in non-neutropenic mice.357. HapXcess and C-terminal truncation impairs Aspergillus fumigatus' iron homeostasis. Fabio Gsaller 1 , Veronika Klammer 1 , Beatrix E. Lechner 1 , PeterHortschansky 2 , Axel A. Brakhage 2 , Ernst R. Werner 3 , Hubertus Haas 1 . 1) Division of Molecular Biology, Medical University of Innsbruck, Austria; 2)Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany; 3) Divisionof Biological Chemistry, Medical University of Innsbruck, Austria.The maintenance of iron homeostasis is indispensable as iron is essential for various cellular processes but can be toxic at iron excess. In A. fumigatus thebZIP-like transcription factor HapX is important for adaption to iron starvation and consequently virulence due to its role in repression of iron consumingpathways (i.e. heme biosynthesis, TCA cycle, respiration) and activation of iron uptake (i.e. siderophore biosynthesis and uptake, reductive ironassimilation). In this study we demonstrate that conditional hapX overexpression using the xylose-inducible xylP promoter leads to repression of genesinvolved in iron consumption (i.e. heme biosynthetic hemA and leucine-biosynthetic leuA) and activation of iron acquisition-related genes (i.e.siderophore-biosynthetic sidG and siderophore transporter-encoding mirB) within one hour of induction. In agreement, elevated hapX expressiondecreased the cellular accumulation of protoporphyrin IX, the iron-free precursor of heme, and increased production of the extracellular siderophoreTAFC. HapX-truncation studies revealed that the C-terminal 93 amino acid residues are essential for its activating as well as repressing functions. HapX N-terminally tagged with Venus green fluorescent protein localized to the nucleus during iron starvation but was undetectable after an one hour-shift to ironsufficiency. These data demonstrate tight iron-regulation of hapX expression at the protein level as previously shown at the transcript level. Consistently,HapX-deficiency is detrimental only during iron limitation. Two hapX copies and in particular xylP promoter-mediated overexpression of hapX causedgrowth defects independent of the iron availability, which underscores the importance of a precisely regulated HapX level. This work was supported by theAustrian Science Foundation grant FWF P21643-B11 to HH.358. The CCAAT-Binding-Complex mediates Iron Regulation in Aspergillus fumigatus. Hubertus Haas 1 , Christoph Joechl 1 , Thorsten Heinekamp 2 , Ilse D.Jacobsen 2 , Markus Schrettl 1 , Axel A. Brakhage 2 , Lukas Schafferer 1 . 1) Division of Molecular Biology, Biocenter, Innsbruck Medical University, Austria; 2)Department for Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany.Iron is essential for a wide range of cellular processes but its excess is toxic. Therefore, microorganisms evolved fine-tuned mechanisms for uptake andstorage of iron, to sustain iron homeostasis. In the opportunistic fungal pathogen Aspergillus fumigatus, the bZIP-type transcription factor HapX mediatesadaption to iron starvation by activating siderophore biosynthesis and repressing iron-dependent pathways. HapX-deficiency attenuates the virulence of A.fumigatus underlining the importance of adaptation to iron starvation in pathogenicity. The HapX N-terminal amino acid sequence predicts interactionwith the DNA-binding, heterotrimeric CCAAT-binding complex (CBC), which is conserved in all eukaryotes and believed to co-regulate up to 30% of allgenes. Here, we characterized the role of the CBC in iron regulation of A. fumigatus by analysis of the phenotypic consequences of genetic inactivation ofthe CBC subunit HapC. HapC-deficiency was deleterious during both iron starvation as well as iron sufficiency, demonstrating iron-independent regulatoryfunctions of the CBC. In contrast, HapX is important during iron starvation only. As shown previously for HapX-deficiency, HapC-deficiency derepressedgenes involved in iron-consuming pathways during iron starvation but decreased siderophore metabolism at transcriptional and metabolic levels.Inhibition of reductive iron assimilation by ferrous iron chelation blocked colony formation of both HapC-deficient and HapX-deficient conidia. Moreover,inactivation of HapC was epistatic to HapX-deficiency. Taken together, these data indicate that the CBC mediates both the activating and the repressingfunctions of the iron-regulatory transcription factor HapX. The central role of the CBC in environmental adaptation is underlined by HapC-deficiencyrendering A. fumigatus avirulent in a murine model of aspergillosis. This work was supported by the Austrian Science Foundation grant FWF I282-B09 toHH.359. Protein kinase A signaling in Aspergillus fumigatus: Identification of downstream targets. Juliane Macheleidt 1,4 , Wolfgang Schmidt-Heck 2 , Ilse D.208