Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS17. Can-Hsp31 is important for Candida albicans growth and survival. S. Hasim, N. Ahmad hussin, K. Nickerson. Biological Science, Univesity of NebraskaLincoln, Lincoln, NE.Candida albicans is an opportunistic pathogen that is able to grow as budding yeast, pseudohyphae, and hyphae. A key feature of C. albicans is its abilityto grow in diverse microenvironments and develop complex and highly efficient responses in order to survive within the host environment. The C. albicansHsp31 (ORF19.251) gene encodes a protein that belongs to the DJ1/PfpI family with close homology to other fungal Hsp31-like proteins. Despite intensivestudy, the function of these fungal Hsp31 proteins is unknown. The crystal structure of Can-Hsp31 was solved to 1.6 Å resolution. Its structure is similar tothose of the E.coli and S. cerevisiae Hsp31 proteins except that Can-Hsp31 is a monomer in the crystal while all other known homologues are dimers. Inthis report, we show that the C. albicans’ Hsp31 is important for growth and survival under various stress conditions.18. Influence of N-glycans on a-/b-(1,3)-glucanase and a-(1,4)-amylase from Paracoccidioides brasiliensis yeast cells. Fausto Bruno Dos Reis Almeida 1 ,Valdirene Neves Monteiro 2 , Roberto Nascimento Silva 3 , Maria Cristina Roque-Barreira 1 . 1) Cellular and Molecular Biology, University of Sao Paulo, RibeiraoPreto, Brazil; 2) University of Goias, Anapolis, Brazil; 3) Biochemistry and Immunology, University of Sao Paulo, Ribeirao Preto, Brazil.Paracoccidioides brasiliensis (Pb) is a temperature-dependent dimorphic fungus and the causative agent of paracoccidioidomycosis, the most prevalentsystemic mycosis in Latin America. The cell wall (CW) of Pb is a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylatedproteins, that may perform several functions. N-glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection fromproteolytic degradation. Our group has been describing the role of N-acetylglucosaminidase (NAGase) in fungal growth, exerted through participation inchitin metabolism and CW remodeling. In addition, by assessing yeast cells cultured with tunicamycin (TM), we determined that N-glycans play importantroles in growth and morphogenesis of Pb yeasts and are required for the fungal NAGase function. In this study, we verify the influence of TM-mediatedinhibition of N-linked glycosylation on a- and b-(1,3)-glucanase, as well as the a-(1,4)-amylase, produced by Pb yeast cells. The treatment of Pb with 15 mgTM/ml did not interfere with a- and b-(1,3)-glucanase production, secretion or on enzyme structure. The absence of N-glycans did not affect pH optimum(5.5) or temperature optimum (45 °C). Moreover, the fully- and under-glycosylated forms of the enzymes had similar Km and Vmax values. On the otherhand, a-(1,4)-amylase demonstrated lower enzymatic activity when underglycosylated, although no difference was detected between the pH andtemperature optimums of the two forms. Our results corroborates with the recent observation that a-(1,4)-amylase from Pb plays important roles on thefungal CW a-(1,3)-glucan biosynthesis. However, interestingly the Pbaglucan gene, that encode to a-(1,3)-glucanase, had its expression increased by 2.5-fold in Pb cells treated with TM when evaluated by qRT-PCR, suggesting an indirect influence of TM on CW glucan synthesis. Genes encoding to UPR(Unfolding Protein Response) and CW synthesis showed their expression increased, corroborating with our data. Analyses investigating the effect of N-glycans in mycelium cells are under way in our laboratory. Our results suggest that N-glycans do not play direct effect on a- and b-(1,3)-glucanase activityproduced by yeasts cells but indirect effect by affecting a-(1,4)-amylase.19. Cell wall structure and biosynthesis in oomycetes and true fungi: a comparative analysis. Vincent Bulone. Sch Biotech, Royal Inst Biotech (KTH),Stockholm, Sweden.Cell wall polysaccharides play a central role in vital processes like the morphogenesis and growth of eukaryotic micro-organisms. Thus, the enzymesresponsible for their biosynthesis represent potential targets of drugs that can be used to control diseases provoked by pathogenic species. One of themost important features that distinguish oomycetes from true fungi is their specific cell wall composition. The cell wall of oomycetes essentially consists of(1®3)-b-glucans, (1®6)-b-glucans and cellulose whereas chitin, a key cell wall component of fungi, occurs in minute amounts in the walls of some oomycetespecies only. Thus, the cell walls of oomycetes share structural features with both plants [cellulose; (1®3)-b-glucans] and true fungi [(1®3)-b-glucans, (1®6)-b-glucans and chitin in some cases]. However, as opposed to the fungal and plant carbohydrate synthases, the oomycete enzymes exhibit specific domaincompositions that may reflect polyfunctionality. In addition to summarizing the major structural differences between oomycete and fungal cell walls, thispresentation will compare the specific properties of the oomycete carbohydrate synthases with the properties of their fungal and plant counterparts, withparticular emphasis on chitin, cellulose and (1®3)-b-glucan synthases. The significance of the association of these carbohydrate synthases with membranemicrodomains similar to lipid rafts in animal cells will be discussed. In addition, distinguishing structural features within the oomycete class will behighlighted with the description of our recent classification of oomycete cell walls in three different major types. Genomic and proteomic analyses ofselected oomycete and fungal species will be correlated with their cell wall structural features and the corresponding biosynthetic pathways.20. Investigating the function of a putative chitin synthase from Phytophthora infestans. Stefan Klinter, Laura Grenville-Briggs, Hugo Mélida, VincentBulone. School of Biotechnology, Division of Glycoscience, Royal Institute of Technology (KTH), Stockholm, Sweden.The oomycete Phytophthora infestans is a plant pathogen that causes potato late blight, a devastating disease associated with tremendous economiclosses. In contrast to true fungi, oomycetes are traditionally described as cellulosic micro-organisms. Indeed, in addition to other b-glucans, cellulose is amajor polysaccharide in the mycelial cell wall of P. infestans while chitin and other N-acetylglucosamine (GlcNAc)-based carbohydrates are absent fromhyphal walls. However, a putative chitin synthase gene (chs) is present in the genome. Bioinformatic analysis identified the C-terminal region of thepredicted protein to be highly similar to glycosyltransferase family 2 proteins, such as fungal chitin synthases, while the N-terminal domain is moredivergent. Orthologous putative chs genes are present in all sequenced oomycete genomes and phylogenetic analysis shows the oomycete gene productsform a new clade separate from the fungal lineage. The P. infestans chs transcript is highly abundant in older mycelium. However, no chitin synthaseactivity was detectable in microsomal fractions assayed with radioactively-labeled UDP-GlcNAc, the natural substrate of chitin synthase. Suprisingly, hyphalgrowth was severely retarded in the presence of low micromolar concentrations of the chitin synthase inhibitor nikkomycin Z, a structural analogue ofUDP-GlcNAc. Microscopic analysis of nikkomycin Z-treated hyphae revealed frequent tip swelling and bursting. Similarly, transient RNA-mediated silencingof the chs gene resulted in severely reduced growth, and hyphae showed a hyper-branched morphology with swollen tips. As a first step to determine theprecise function of the P. infestans chs gene, we have cloned and expressed it in Saccharomyces cerevisiae.21. Deciphering cell wall structure and biosynthesis in oomycetes using carbohydrate analyses and plasma membrane proteomics. Hugo Melida 1 ,Vaibhav Srivastava 1 , Erik Malm 1 , J. Vladimir Sandoval-Sierra 2 , Javier Dieguez-Uribeondo 2 , Vincent Bulone 1 . 1) Division of Glycoscience, Royal Institute ofTechnology (KTH), Stockholm, Sweden; 2) Mycology Department, Royal Botanical Garden (CSIC), Madrid, Spain.Some oomycete species are severe pathogens of economically important animals or plants. Proteins involved in cell wall metabolism represent excellenttargets for disease control. The objective of our work was to determine the fine cell wall polysaccharide composition of selected species and identify thecorresponding membrane-bound biosynthetic enzymes and other proteins involved in cell wall remodeling. In the first instance, we performed a detailedcarbohydrate analysis of the mycelial cell walls of 11 oomycete species from 2 major orders, the Saprolegniales and Peronosporales. We then selected thefish pathogen Saprolegnia parasitica for in-depth proteomics analysis. Our results indicate the existence of 3 clearly different cell wall types. This126