Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSCenter for Genome Research and Biocomputing, Oregon State University, Corvallis, OR; 5) Department of Botany and Plant Pathology, Oregon StateUniversity, Corvallis, OR.Fungal infections have become an increasingly significant problem for immunocompromised individuals, transplant recipients, the elderly, several casesinvolving healthy individuals. There is a significant growth in incidences of morbidity and mortality associated with medically important fungi, specificallyAspergillus species. Aspergillus fumigatus virulence has been attributed to production of pigments, adhesins on the surface of the cell wall, secretedproteases, and mycotoxins. Current treatments consist of oral corticosteroids, antifungal medications, and/or surgery to remove aspergillomas. Many ofthese treatments have substantial shortcomings. Detection and diagnosis is also weighty problem as most clinical tests take weeks for results allowing theinfection to proceed. Appropriately, the paradigm for human fungal interactions has been focused on the host deficiencies mediating virulence ofopportunistic pathogenic fungi. There has been substantial progress in identifying and characterizing secreted proteins (effectors) from bacterial,oomycete, and fungal plant pathogens. A subset of these effector proteins are able to enter host cells and modulate host intracellular functions. Using ourbioinformatics pipeline we have been able to identify a family of secreted proteins from A. fumigatus sharing a conserved N-terminal RXLR-like motif. Wefound this family is expanded amongst primary fungal pathogens. The RXLR and RXLR-like motifs from known intracellular effectors of plant pathogenicand mutualistic oomycetes and fungi have been shown to facilitate effector entry into plant cells via binding external phosphatidylinositol-3-phosphate(PI3P). Here we describe AF2, a candidate effector from A. fumigatus that contains a N-terminal RxLR-like motif. Through the use of confocal microscopyand flow cytometry we show AF2 is rapidly able to enter several primary and immortalized mammalian cell lines. Through the use of isothermal titrationcalorimetry and liposome binding assays we show AF2 has nanomolar binding affinity for PI3P, and does not bind other mono or poly-PIPs that we havetested thus far. Based on our bioinformatics and biochemical analysis we postulate AF2 is a secreted effector protein capable of rapidly translocating intomammalian cells. We will present our latest findings on the physiological relevance of AF2.484. A role for PalH-mediated signal transduction in A. fumigatus virulence and cell wall integrity: An exploitable target for combination therapy? M.Bertuzzi 1 , C.M. Grice 1 , L. Alcazar-Fuoli 2 , A.M. Calcagno-Pizarelli 1 , J. Kalchschmidt 1 , S. Gill 1 , K. Fox 1 , A. Cheverton 1 , Hong Liu 3 , V. Valiante 4 , E.A. Espeso 5 , S.GFiller 3 , A. Brakhage 4 , E.M. Bignell 1 . 1) Centre for Molecular Bacteriology & Infection , Imperial College London , London (UK); 2) Mycology Referencelaboratory, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid (Spain); 3) David Geffen School of Medicine at UCLA, Division ofInfectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center (USA); 4) Leibniz-Institute for Natural Product Research andInfection Biology Hans Knöll Institute, Molecular and Applied Microbiology, Jena (Germany); 5) Dept. of Cellular and Molecular Biology, AspergillusMolecular Genetics Unit, Centro de Investigaciones Biológicas (C.S.I.C.), Madrid (Spain).Adaptation to host-imposed stress is a crucial requirement for persistence of Aspergillus fumigatus in the mammalian lung. In Aspergillus species, PacCsignalling promotes tolerance of alkaline environments via signal-dependent proteolytic processing of the transcription factor PacC. The aim of this studywas to test the requirement for A. fumigatus PalH during infection and to decipher its role in PacC-mediated signalling. The role of PalH in alkalinemediatedPacC processing was tested using electrophoretic mobility shift assay, and A. fumigatus virulence was examined in a murine neutropenic modelof pulmonary aspergillosis. To probe the mechanistic basis of PalH-mediated signalling, we utilised a split-ubiquitin Membrane Yeast Two-Hybrid (MYTH)assay to assess protein interactions amongst candidate A. fumigatus signalling proteins of this pathway. A. fumigatus isolates expressing epitope-taggedPalH protein were constructed to assess the relevance of PalH oligomerisation. Analysis of PacC processing identified the requirement for PalH to initiatealkaline-mediated PacC signalling. A DpalH mutant is somewhat sensitive to alkaline pH, and attenuated for virulence in a murine model of pulmonaryaspergillosis. The mutant is also sensitive to cell wall-perturbing agents, and in the presence of the cell wall-active antifungal caspofungin undergoesextensive hyphal branching and ballooning compared to the parental and reconstituted strains. In the absence of PalH A. fumigatus-mediated damage ofepithelial cells is abrogated in vitro. By using a MYTH assay a significant interaction between A. fumigatus PalH and PalF was detected in Saccharomycescerevisiae. In A. fumigatus PalH-mediated PacC signalling, likely implemented in a (PalF) arrestin-like manner, commands a central role in the expression ofvirulence-determining functions. The impairment of PacC signalling exerts a synergistically inhibitory effect upon fungal viability in the presence of cellwall-active antifungal drugs and therefore represents an attractive target for the development of novel antifungal mono- and combination therapies. Ourresults support a scenario whereby PalH is an oligomerising receptor, responsive to extracellular pH, and required for virulence and echinocandintolerance. Future studies will focus upon the mechanism of PalH-mediated pH sensing.485. Aspergillus fumigatus trehalose-6-phosphate regulates innate immune responses and virulence through modulation of fungal cell wallcomposition. Srisombat Puttikamonkul 2 , Vishu K. Amanianda 3 , Jean-Paul Latge 3 , Kelly M. Shepardson 2 , John R. Perfect 4 , Nora Grahl 2 , Bridget M. Barker 2 ,Robert A. Cramer 1 . 1) Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH; 2) Immunology and Infectious Diseases,Montana State University; 3) Unite des Aspergillus, Institut Pasteur; 4) Medicine, Division of Infectious Diseases, Duke University Medical Center.Mechanism(s) behind the attenuated fungal virulence of trehalose biosynthesis pathway mutants are not fully understood. We observed previously thatTPS2/OrlA, a key enzyme in TPS1/TPS2 trehalose biosynthesis is required for cell wall integrity and fungal virulence in A. fumigatus. In this study, we testedthe hypothesis that the significant in vivo attenuated virulence and in vitro impaired cell wall integrity of DorlA is due to accumulation of Trehalose-6-Phosphate (T6P). Our data suggest that the mechanism behind the attenuated virulence of the A. fumigatus TPS2 null mutant, DorlA, in a murine model ofX-linked chronic granulomatous disease (X-CGD) is mediated by an increased susceptibility of DorlA to polymorphonuclear leukocyte (PMN) killing. In theabsence of PMNs in the xCGD murine model, DorlA exhibited restored fungal burden and virulence similar to wild-type inoculated animals. Null mutationsin putative trehalose biosynthesis proteins TslA and TslB in the DorlA background were able to ameliorate T6P accumulation and restore cell wall integrityand virulence strongly suggesting that accumulation of T6P is the key factor associated with DorlA virulence. Our results identify a previously unknownmechanism of immune modulation by the fungal carbohydrate metabolite T6P that has significant implications for targeting trehalose biosynthesis as anantifungal drug target.486. Fungal lipoxygenases: a novel instigator of asthma? Gregory J. Fischer 1 , Katharyn Affeldt 3 , Erwin Berthier 2 , Nancy P. Keller 1,2,3 . 1) Department ofGenetics, University of Wisconsin-Madison, Madison, WI; 2) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison,Madison, WI; 3) Department of Bacteriology, University of Wisconsin-Madison, Madison, WI.Statement of Purpose: Fungi have long been associated with asthmatic diseases, yet the exact mechanism(s) by which fungi induce asthma is unknown.We propose that fungal lipoxygenase enzymes and their eicosanoid products are involved in asthmatic diseases. Human 5-lipoxygenase derivedleukotrienes induce inflammation, mucus secretion, vasodilation, and bronchial constriction. We hypothesize that the fungal pathogen Aspergillusfumigatus is capable of secreting a 5-lipoxygenase homolog, LoxB, that participates in eicosanoid production, including leukotrienes. This secretedhomolog is translocated into lung epithelial cells, participates in the production of leukotriene and other eicosanoids, and exacerbates asthmaticresponses, such as bronchoconstriction. Together, this work will help delineate the role fungal products play in asthmatic diseases. Methods: We are240