Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTScommonly treated with azole inhibitors of ergosterol biosynthesis. Prophylactic treatment in immuncompromised patients2,3 often leads to thedevelopment of drug resistance. Since C. albicans is diploid and lacks a complete sexual cycle, conventional genetic analysis is challenging. An alternativeapproach is to study the mutations that arise naturally during the evolution of drug resistance in vivo, using isolates sampled consecutively from the samepatient. Studies in evolved isolates have implicated multiple mechanisms in drug resistance, but have focused on large-scale aberrations or candidategenes, and do not comprehensively chart the genetic basis of adaptation5. Here, we leveraged next-generation sequencing to systematically analyze 43isolates from 11 oral candidiasis patients, collected sequentially at two to 16 time points per patient. Because most isolates from an individual patientwere clonal, we could detect newly acquired mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss ofheterozygosity (LOH) events. Focusing on new mutations that were both persistent within a patient and recurrent across patients, we found that LOHevents were commonly associated with acquired resistance, and that persistent and recurrent point mutations in over 150 genes may be related to thecomplex process of adaptation to the host. Conversely, most aneuploidies were transient and did not directly correlate with changes in drug resistance.Our work sheds new light on the molecular mechanisms underlying the evolution of drug resistance and host adaptation.503. Yeast-Hypha transition and immune recognition of Candida albicans influenced by defects in cell signal transduction pathways. Pankaj Mehrotra,Rebecca A Hall, Jeanette Wagener, Neil A.R. Gow. Aberdeen Fungal Group, Aberdeen.During the infection process C. albicans has to respond to various stresses imposed by the host environment including oxidative and osmolarity stressgenerated by phagocytic cells such as macrophages and neutrophils, and also the cell wall stress agents such as exposure to caspofungin and otherantifungal antibiotics. These stress responses area orchestrated through the activation of multiple stress pathways including the cAMP-PKA, several MAPKpathways and the Ca 2+ -calcineurin pathway influence the cell wall shape and composition. We are investigating the effect of the activation or inhibition ofthese pathways on immune recognition mechanisms. We therefore determined the importance of the MAPK, cAMP-PKA and Ca 2+ -calcineurin pathways onthe fungal innate immune response by examining uptake, phagocytosis, and cytokine profile induced by mononuclear and polynuclear lymphocytes inresponse to a library of mutants in each of the above pathways under stressed and non-stressed conditions. We find that the activation and inhibition ofthese pathways plays a important role in remodeling of cell wall and hence the immunological profile. For example, deletion of TPK1 and CNA1 resulted inlower pro-inflammatory cytokine production. Immune- recognition was also affected by the exposure of C. albicans signaling mutants with Calcofluorwhite,caspofungin , oxidative and osmotic stress and changes in temperature. These results suggest that stress signaling pathways act in a co-ordinatedfashion to regulate yeast-hypha morphogenesis and the changes in the cell wall which in turn affects the immunological signature of the cell. Thusexposure to different microenvironments significantly modifies the immunological response to fungal cells, suggesting that responses to local stressesmakes the fungal cell surface is a moving target for immunological surveillance.504. GPI PbPga1 of Paracoccidioides brasiliensis is a surface antigen that activates macrophages and mast cells through the NFkB signaling pathway. C.X. R. Valim, L. K. Arruda, P. S. R. Coelho, C. Oliver, M. C. Jamur. Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil.Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensiscell wall components interact with host cells and influence the pathogenesis of PCM. PbPga1 is a GPI anchored protein that is up-regulated in the yeastpathogenic form. GPI anchored proteins are involved in cell-cell and cell-tissue adhesion and have a key role in the interaction between fungal and hostcells. PbPga1 is an O-glycosylated protein that is localized on the yeast cell surface. Recombinant PbPga1 (rPbPga1) induces nitric oxide (NO) productionand TNF-a release in murine peritoneal macrophages (Valim et al.Plos One, 2012). In the present study, rPbPga1 was able to activate NFkB in macrophagelikeRaw cells that had been transfected with NFkB luciferase as well as in a reporter cell line for NFkB activation derived from RBL-2H3 mast cells. Theresults show that like macrophages, rPbPga1 also activates the transcription factor NFkB in mast cells. However, rPbPga1 does not activate NFAT nor is itable to induce liberation of beta hexosaminidase . The lack of beta hexosaminidase release suggests the PbPga1 is not able to activate RBL-2H3 mast cellsvia the high affinity IgE receptor. Mast cell activation by rPbPga1 does result in activation of the transcription factor NFkB suggesting stimulation ofcytokine production. Taken together these results indicate that the surface antigen PbPga1 may play an important role in PCM pathogenesis by activatingmacrophages and mast cells.505. Cladosporium fulvum effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Andrea Sánchez-Vallet 1 , Raspudin Saleem-Batcha 2 , Anja Kombrink 1 , Guido Hansen 2 , Dirk-Jan Valkenburg 1 , Jeroen R. Mesters 2 , Bart P.H.J. Thomma 1 . 1) Laboratory ofPhytopathology, Wageningen University, Wageningen, Netherlands; 2) Institute of Biochemistry, Center for Structural and Cell Biology in Medicine,University of Lübeck, Lübeck, Germany.Successful pathogens secrete effector proteins to deregulate host immunity which is triggered upon detection of pathogen-associated molecularpatterns (PAMPs). Several fungal pathogens employ LysM effectors, such as Ecp6 from Cladosporium fulvum, to sequester fungal cell wall-derived chitinoligomers which act as PAMP and would otherwise be recognized by host immune receptors and trigger defense responses. The mechanism by whichLysM effectors scavenge chitin molecules remained unknown thus far. Based on crystal structure analysis of Ecp6, we reveal a novel mechanism for chitinbinding by intrachain LysM dimerization, leading to a binding groove in which chitin is deeply buried in the effector protein. Isothermal titrationcalorimetry experiments show that the concerted action of two LysM domains mediates a single chitin binding event with ultra-high (pM) affinity.506. Genotypic and phenotypic characterization of Setosphaeria turcica reveals population diversity and a candidate virulence gene location. SantiagoMideros 1 , Chia-Lin Chung 1,3 , Jesse Poland 2,4 , Gillian Turgeon 1 , Rebecca Nelson 1,2 . 1) Cornell University, Dept. of Plant Pathology and Plant-Microbe Biology,Ithaca, NY, USA; 2) Cornell University, Dept. of Plant Breeding and Genetics, Ithaca, NY, USA; 3) National Taiwan University, Dept. of Plant Pathology andMicrobiology, Taipei, Taiwan; 4) USDA-ARS, Hard Winter Wheat Genetics Research Unit, Kansas State University, Manhattan, KS, USA.The dothideomycete maize pathogen Setosphaeria turcica (anamorph Exserohilum turcicum) causes Northern Leaf Blight, one of the most commonfungal diseases of maize worldwide. Little is known about the genetic basis of virulence and aggressiveness in this pathogen, although several races havebeen described based on their compatibility with maize resistance genes Ht1, Ht2, Ht3 and HtN. To study the genetic basis of virulence and aggressiveness,we generated a F1 population consisting of 221 monosporic progeny of a cross between a race 1 strain and a race 23N strain. Genotyping-by-sequencing(GBS) was conducted on the population and an additional 13 diverse isolates that included the parental lines. We obtained between 341,000 and 428,000sequence tags for each of the 234 isolates. Alignment to the S. turcica Et28A v1.0 genomic sequence(http://genome.jgi.doe.gov/Settu1/Settu1.home.html) yielded 27,174 single nucleotide polymorphisms (SNPs) at a density of 0.63 SNPs per kb. In the 13isolates, using 9,526 filtered SNPs, we found an average nucleotide diversity (p) of 0.297. Using 564 polymorphic markers with less than 35% missing calls,we created a high-density genetic map that resulted in 23 linkage groups and a total length of 1,686 cM. The Et28A sequence has 407 scaffolds, fourscaffolds formed a single linkage group in our genetic map. The rest of the genome remains fragmented. To identify genomic regions controlling virulence27th Fungal Genetics Conference | 245