Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSenhanced fungal nitrogen metabolism. Two types of ammonium transporter encoding genes, AMET and MEP, are expressed during pathogenicity. Genedisruption of AMET- a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruits,suggesting a contribution to pathogenicity. MEPB a gene modulating ammonium transport is expressed by C. gloeosporioides during pathogenicity andstarvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPAand MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulationby DmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant vs. WTstrains. Ammonia uptake by the WT germinating spores, but not by the DmepB strain with compromised ammonium transport, activated cAMP andtranscription of PKA subunits PKAR and PKA2. DmepB mutants showed 75% less appressorium formation and colonization than the WT, which waspartially restored by 10 mM exogenous ammonia. Thus while both AMET and MEPB genes modulate ammonia secretion, only MEPB contribute toammonia accumulation by mycelia and germinating spores that activates the cAMP pathways, inducing the morphogenetic processes contributing to C.gloeosporioides pathogenicity.480. Functional analysis of Nbs1 of Magnaporthe oryzae. K. Sasaki 1 , K. Amano 1 , T. Sone 2 , M. Narukawa 1 , T. Kamakura 1 . 1) Applied Biological Science,Tokyo Univ. of Science, Noda, Chiba, Japan; 2) Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.The filamentous fungus Magnaporthe oryzae causes rice blast, the most serious disease that affects global rice production. On the surface of host plant,a specialized infection structure called appressorium is formed on tip of germ tube. Induction of the development of appressorium requires severalexternal stimulants and a complete cycle of cell division. Although many studies have revealed some of process of appressorium formation in M. oryzae,the complete mechanism is still obscure. We selected Nbs1 from germ tube expressing cDNA library and made Nbs1 disruptants. The cDNA library mainlycontains the genes that express in the period of germ tube development and/or appressorium formation. Nbs1 is presumed to have forkhead associated(FHA) domain, which is contained in many proteins that are involved in DNA repair and cell cycle. In our previous study, Nbs1 disruptants showed growthdelay, abnormality of conidia formation and nuclear division, reduction of germination rate and appressorium formation rate, abnormal pigmentation andhigh sensitivity to DNA-damaging agents. Although Neurospora crassa knock-out mutants of rcaA, which share sequence similarities with Nbs1, showedsimilar phenotypes to Nbs1 disruptants, rcaA did not seem to contain FHA domain. Toward further study of the function of Nbs1, we induced a plasmidcarrying an rcaA (pNB51) or FHA domain-deleted Nbs1 (pCB51dF) into Nbs1 disruptants. Consequently, pNB51 and pCB51dF were able to partiallycomplement phenotypes of Nbs1 disruptants. This result suggested that rcaA has at least partial similar functions of Nbs1 in N. crassa and anotherfunctional domain exists in Nbs1.481. Influence of hypoxia on antifungal susceptibility, sterole pattern and biomarker release of Aspergillus spp. Ulrike Binder 1 , Elisabeth Maurer 1 ,Christoph Müller 2 , Franz Bracher 2 , Cornelia Lass-Flörl 1 . 1) Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Tirol,Austria; 2) Department of Pharmacy, Ludwig Maximilians University Munich, Germany.Invasive aspergillosis (IA) is a major life-threatening disease in immunocompromised patients, with mortality rates from 40% up to 90% in high-riskpopulations. The most common species causing aspergillosis is Aspergillus (A.) fumigatus, accounting for approximately 90% of infections. Depending onregional distinctions, A. flavus and A. terreus are frequently reported. During infection, fungal pathogens must adapt to microenvironmental stresses,including hypoxia as well as high CO2 levels. Such oxystress conditions are usually not taken into account in current in vitro models of infection, theassessment of antifungal sensitivities or the release of biomarkers used for diagnosis. Therefore, we compared the in vitro activity of amphotericin B(amB), different azoles and echinocandins in hypoxic conditions (1% O2, 5% CO2) to their activity in normoxic conditions against isolates of A. fumigatusand A. terreus and other aspergilli. Using Etest strips, we found a reduction of the minimal inhibitory concentration (MIC) for amB for all aspergilli inhypoxic conditions. Similarly, a significant reduction in the MIC for all tested azoles was demonstrated for A. terreus isolates, while for A. fumigatusisolates differences were less pronounced. For echinocandins, little or no change in the MEC (minimal effective concentration) was detected betweenhypoxic and normoxic conditions for all aspergilli. Most interestingly, A. terreus strains, that are resistant to amB in normoxia, exhibited sensitivity to amBin hypoxic conditions, defining a breakpoint of > 2 mg/ml. Notably, for none of the strains tested, MIC/MEC values increased in hypoxia. Currently we areinvestigating if changes in the sterole pattern or the amount of ergosterol contribute to these changes in antifungal susceptibility in hypoxia. The detectionof circulating fungal antigens in serum for Aspergillus galactomannan or b-D-glucan has become an accepted diagnostic strategy. However, sensitivity andspecificity vary extremely and the reasons are only partially clear; therefore, we are currently checking whether hypoxia influences the physiologicalkinetics of GM and b-glucan release.482. Sit and wait: Special features of Aspergillus terreus in macrophage interactions and virulence. M. Brock 1 , I.D. Jacobsen 2 . 1) MicrobialBiochemistry/Physiology, Friedrich Schiller University and Hans Knoell Institute, Jena, Germany; 2) Molecular Pathogenicity Mechanisms, Hans KnoellInstitute Jena, Germany.While Aspergillus fumigatus is known as the main cause of invasive pulmonary aspergillosis in immunocompromised patients, Aspergillus terreus is anemerging pathogen prevalent in some local hot spots. When tested in embryonated egg or murine infection models A. terreus required substantiallyhigher infectious doses compared to A. fumigatus to cause high mortality rates. Furthermore, when A. fumigatus and A. terreus infections were followedby in vivo imaging using bioluminescent reporter strains, germination and tissue invasion of A. terreus was significantly delayed. To elucidate differences inmore detail, the interaction of A. terreus and A. fumigatus with macrophages was compared. A. terreus was phagocytosed significantly faster, whichappears mainly due to higher exposure of galactomannan and glucans on the surface of conidia. Additionally, although phagocytosis of both speciesresulted in phagolysosome maturation, A. fumigatus efficiently inhibited acidification, which was not the case for A. terreus. However, within this acidicenvironment of phagolysosomes A. terreus showed long-term persistence without significant inactivation of conidia. Further analyses revealed thatinefficient blocking of acidification by A. terreus was due to differences in the spore colour pigment of both species. Recombinant production of anaphthopyrone synthase from Aspergillus nidulans enabled A. terreus to inhibit the acidification to a similar extent as observed for A. fumigatus. Thisalteration of the phagolysosomal environment resulted in an increased escape from macrophages and was accompanied by increased virulence in amurine infection model. We speculate that the long-term persistence of A. terreus wild-type strains in acidified phagolysosomes might be responsible forhigh dissemination rates observed in infected human patients, because A. terreus might hitchhike inside immune effector cells to reach secondary sites ofinfection.483. Identification and characterization of an RXLR-like effector family from medically relevant fungi. Shiv D. Kale 1* , Kelly C. Drews 1,2 , Helen R. Clark 1,3 ,Hua Wise 1,4 , Vincenzo Antignani 1 , Tristan A. Hayes 1,2 , Christopher B. Lawrence 1,2 , Brett M. Tyler 4,5 . 1) Virginia Bioinformatics Institute, Virginia Tech.,Blacksburg, VA; 2) Department of Biological Sciences, Virginia Tech., Blacksburg, VA; 3) Department of Biochemistry, Virginia Tech., Blacksburg, VA; 4)27th Fungal Genetics Conference | 239