Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSsusceptibility to cell wall perturbation. Through gene deletion analysis we have found that the WC-1 ortholog, LreA, is a bone fide blue light receptor in A.fumigatus that is required for the photopigmentation response. However, the DlreA mutant retains several blue light mediated responses, including thegermination and stress resistance phenotypes , suggesting other blue light receptors are operative in this fungus. We also show that the putative red lightsensing phytochrome, FphA, is involved with some, but not all, blue light specific phenotypes, indicating a complex interaction between red and blue lightphotosystems in A. fumigatus. Finally, whole genome microarray analysis has revealed that A. fumigatus displays broad patterns of gene induction andrepression upon exposure to light. Affected genes are largely metabolic and include those involved in lipid and sterol synthesis, respiration, carbohydratecatabolism, amino acid metabolism and metal ion homeostasis. Taken together, these data demonstrate the importance of the photic environment on thephysiology of A. fumigatus and provide a foundation for future studies into an unexplored area of this important pathogen.74. Analysis of critical domains in calcineurin A required for septal targeting and function in Aspergillus fumigatus. Praveen R. Juvvadi 1 , Jarrod R.Fortwendel 2 , Christopher Gehrke 1 , Frédéric Lamoth 1 , William J. Steinbach 1 . 1) Department of Pediatrics, Duke University Medical Center, Durham, NC; 2)Department of Microbiology and Immunology, University of South Alabama, Mobile, AL.Calcineurin, a calmodulin (CaM)-dependent protein phosphatase, is known to play key roles in virulence, growth and stress responses of pathogenicfungi. Critical understanding of calcineurin regulation and identifying the residues indispensable for calcineurin activity in vivo will pave the way fordevising new drug targets for combating invasive aspergillosis. Previous studies from our laboratory showed that the calcineurin complex (CnaA and CnaB)in Aspergillus fumigatus selectively localizes at the hyphal tip and septum to direct proper hyphal growth and regular septum formation. However, thedomains responsible for targeting and function of CnaA at the hyphal septum remain unknown. Here we performed extensive truncational and mutationalanalyses of the functional domains of CnaA to investigate the relevance of these domains for localization and function of CnaA at the septum. Importantlywe found that (i) CaM, the key protein known to activate calcineurin is not required for septal localization of CnaA but is required for its function at thehyphal septum, (ii) the PxIxIT substrate binding motif in CnaA is required for its localization at the hyphal septum, indicating it localizes at the septum byinteracting with other as yet unknown protein/s(iii) binding of CnaB subunit is not necessary for septal localization of CnaA but the regulatory subunit isrequired for its activation at the septum, and (iv) triple mutations in the catalytic active site do not affect septal localization of CnaA but completely blockhyphal growth revealing that both septal localization and activity of CnaA are required for proper hyphal growth.75. Calcium imaging and measurement during growth and response to stresses in Aspergillus fumigatus. Alberto Muñoz 1 , Margherita Bertuzzi 2 , JanBettgenhaeuser 1 , Elaine Bignell 2 , Nick Read 1 . 1) Fungal Cell Biology Group, University of Edinburgh, Edinburgh, United Kingdom; 2) Microbiology Section,Imperial College London, London, United Kingdom.Calcium signalling and homeostasis are essential for the growth, differentiation and virulence of filamentous fungi. During infection, A. fumigatus mustbalance concomitant demands to: (1) withstand toxic levels of exogenous calcium (3-5 mM) in the host environment which can be >100,000x that of thefungal cytosolic free calcium ([Ca 2+ ] c) concentration; (2) appropriately integrate homeostatic and stress-responsive adaptations; and (3) undergo normalcalcium signalling. There is evidence for calcium signalling regulating numerous processes including spore germination and hyphal tip growth. The lowresting level of [Ca 2+ ] c (50-100 nM) is maintained by Ca 2+ -pumps and -antiporters, and cytoplasmic Ca 2+ -buffering. However, [Ca 2+ ] c becomes an intracellularsignal when its concentration is transiently increased. We have developed two methods for measuring and imaging [Ca 2+ ] c: (1) 96-well plate luminometryusing the genetically encoded, bioluminescent aequorin; and (2) fluorescence microscopy using the genetically encoded calcium-sensitive, fluorescentprotein G-CaMP5. Aequorin is ideally suited for quantitative measurements of [Ca 2+ ] c calcium signatures in cell populations whereas fluorescence imagingof the G-CaMP5 is good for single cell and subcellular measurements of [Ca 2+ ] c. Using the aequorin methodology we have found that transient increases in[Ca 2+ ] c with specific, reproducible calcium signatures in A. fumigatus arise from exposure to stresses such as high external calcium. In our analysis, [Ca 2+ ] cspikes in actively growing hyphal tips have been imaged using G-CaMP5. Exposure of conidial germlings to high external calcium induces dramatic and verydynamic changes in [Ca 2+ ] c with the generation of localized [Ca 2+ ] c transients and waves. Furthermore, there is considerable heterogeneity in the [Ca 2+ ] cresponses of different germlings within the cell population. Calcium imaging and measurement using genetically encoded probes, particularly whencombined with pharmacological and genetic analyses, will provide major new insights into calcium signalling in filamentous fungi.76. WITHDRAWN77. The copper transporter ctpA in Aspergillus fumigatus is critical for conidial melanization and virulence in an invertebrate infection model. SrijanaUpadhyay, Xiaorong Lin. Biology, Texas A&M University, College Station, TX.Aspergillus fumigatus is an opportunistic pathogen that causes life-threatening invasive diseases in immunocompromised hosts. This fungus producesabundant, easily aerosolized, and heavily melanized conidia that are the infectious particles. The melanin, or the bluish green pigment coated on theconidial surface, is associated with fungal virulence and resistance to environmental stresses. This melanin is synthesized through the DHN melaninpathway by a cluster composed of six structural biosynthetic genes. Although all Aspergillus species produce conidial melanin, this DHN melanin genecluster found in A. fumigatus is not conserved in all species of this genus. In other species, laccases are critical for melanization and copper has beenshown to be critical for their activity. In A. nidulans, defective ygA that encodes a copper transporter results in reduction in conidial laccase activity andpoor conidial pigmentation. Whether copper is important for conidial melanization or whether it affects the function of the DHN gene cluster in A.fumigatus are not clear. In this study we have identified ctpA in A. fumigatus as the homolog of ygA in A nidulans and demonstrated its importance forconidial melanization under the copper limiting and the copper replete conditions. The defect in melanization caused by the deletion of the ctpA gene canbe remediated by addition of copper in the media or by the overexpression of the ctpA gene. Lack of melanin is caused by growing the wild type in thecopper-limiting conidiation or by the deletion of the ctpA gene. This renders the A. fumigatus conidia more immune-dominant, since these conidia cancause exacerbated immune-responses from the invertebrate host, larvae of Galleria mellonella. Furthermore, we have identified and characterizeregulators that play important roles in maintaining copper homeostasis and melanization in A. fumigatus.78. Aspergillus nidulans SNXA HRB1 is an SR/RRM family protein that rescues defects in the CDC2/CYCLINB pathway. Steven James 1 , Travis Banta 2 , JamesBarra 1 , Clifford Coile 2 , Ryan Day 2 , Cheshil Dixit 2 , Steven Eastlack 2 , Anh Giang 2 , Yulon Huff 2 , Julie Kobie 1 , Faustin Mwambutsa 2 , Mimi Nguyen 2 , AmandaOrzechowski 1 , Kristin Shingler 1 , Sarah Lea Anglin 2 . 1) Dept. Biology, Gettysburg College, Gettysburg, PA; 2) Dept. Biology, Millsaps College, Jackson, MS.Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated. To further characterize this regulation in Aspergillus nidulans, weconducted a screen for extragenic suppressors of nimX2 cdc2 that resulted in the identification of the cold-sensitive, G1-arresting snxA1 mutation. Our datashow that snxA1 suppresses defects in regulators of the G2/M transition, including nimX2 cdc2 , nimE6 cyclinB , and nimT23 cdc25 , but does not suppress the G1/SarrestingnimE10 cyclinB mutation or any of four S phase mutations. Furthermore, the snxA1 mutation or deletion of snxA alter localization patterns ofNIME CYCLINB at the restrictive temperatures for snxA1 and nimX2, supporting a role for SNXA in cell cycle control. snxA encodes the A. nidulans ortholog of140