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Fungal Genomics. Gustavo H. Goldman Universidade de São Paulo, Brazil. Brief overview on fungal genomics Fungal genomics: a tool to explore central metabolism of A. fumigatus and its role in virulence

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Fungal Genomics

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    1. Fungal Genomics Gustavo H. Goldman Universidade de São Paulo, Brazil

    2. Brief overview on fungal genomics • Fungal genomics: a tool to explore central metabolism of • A. fumigatus and its role in virulence • Highlights on genes and pathways possibly important for the interaction A. fumigatus-host

    3. - The Fungi represent a single eukaryotic kingdom, characterized • by an osmotrophic growth habitat in which extracellular enzymes • are secreted to break down complex substrates, the resulting • simple sugars and amino acids being taken up by the growing • fungus. • -Fungi exist in two distinct morphological growth forms: • the unicellular yeasts (which grow by budding or simple fission) • and the filamentous fungi (which produce polarized hyphal • strands that aggregate to form a network called a mycelium). • Theosmotrophic growth habit of fungi is extremely effective for • colonizingdiverse habitats and has made the fungi the principal • degradersof biomass in all terrestrial ecosystems and also • important pathogens of both plants and animals.

    4. - The yeasts and filamentous fungi cover a huge evolutionary • range. The Pezizomycotina (filamentous ascomycetes) and the • Saccharomycotina (budding yeasts), for example, diverged from • one another some 900–1000 million years ago (Mya), and the • Saccharomycotina alone are more evolutionarilydiverged than • the Chordate phylum of the animal kingdom. • Of the eukaryotic genome sequences currently available, more • than half come from the kingdom Fungi

    5. e-fungi: a data resource for comparative analysis of fungal genomes (Hedeler et al., 2007. BMC Genomics 8: 426; - Several Ascomycete species, two sequenced Basidiomycete fungi, Ustilago maydis and Phanerochaete chrysosporium, plus the Zygomycete Rhizopus oryzae and the Microsporidian Encephalitozoon cuniculi. In addition, two non-fungal species, the Oomycetes Phytophthora sojae and Phytophthora ramorum. Cornell et al., 2008. Genome Research, 15: 1620-1631

    6. Oomycetes Cornell et al., 2008. Genome Research, 15: 1620-1631

    7. (A) Broad species tree based on concatenated sequences from 30 universal protein clusters using maximum likehood approach (B) Basidiomycete and Ascomycete species tree based on 60 universal fungal proteins Taphrinomycotina Saccharomycotina Pezizomycotina

    8. Gene duplications - In the evolution of the S. cerevisiae and Candida glabrata genomes, following a whole-genome duplication (WGD), the majority of the duplicated genes have been lost - For Sacharomycotina genomes, results are consistent with those of the previously published study (Dujon et al., 2004). S. cerevisiae (438 duplication-containing clusters) has more than twice as many duplications as K. lactis and Kluyveromyces waltii (206 and 181 clusters, respectively), which diverged prior to the WGD event

    9. C. glabrata appears to have fewer duplication-containing clusters • than S. cerevisiae. Only 325 were identified, indicating greater • gene loss pos-WGD. • Y. lipolytica possesses the greatest number of highly duplicated • clusters. Forty-seven clusters containing more than five proteins • were identified, compared with only 10 for S. cerevisiae.

    10. - Gene duplications among the Pezizomycotina, in general, appears to be slightly higher than among the Sacharomycotina. The exceptions are N. crassa and C. immitis (372 and 374 clusters, respectively), which both possess fewer duplication- containing clusters than S. cerevisiae. - Among the Aspergillus genomes, A. niger and A. oryzae possess the most duplication-containing clusters

    11. In the Basidiomycetes, duplication in the P. chrysosporium • genome (885 clusters) appears much higher than in that of • U. maydis (300 clusters) • The Zygomycete R. oryzae possesses by far the most duplication • clusters (2,481) of all the fungi analysed, almost three times as • many as the next highest, P. chrysosporium.

    12. - Many of the motifs expanded in the Pezizomycotina indicate increased metabolic flexibility compared to the Sacharomycotina. For example, there are expansions in protein families involved in transport into and out of cell, alcohol dehydrogenase domains, and P450 proteins. - In addition to the expansion in Pfam motifs associated with responses environmental stresses and resources, there is an expansion in the motifs associated with regulation of gene expression. Analysis of 84 Pfam motifs associated with DNA binding shows that, on average, Pezizomycotina species possess almost twice as many proteins containing these motifs as Saccharomycotina species.

    13. Fungal genomics: a tool to explore central metabolism of A. fumigatus and its role in virulence • Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. • Fedorova, N.D., Khaldi, N., Joardar, V.S., Maiti, R., Amedeo, P., Anderson, M.J., • Crabtree, J., Silva, J.C., Badger, J.H., Albarraq, A., Angiuoli, S., Bussey, H., • Bowyer, P., Cotty, P.J., Dyer, P.S., Egan, A., Galens, K., Fraser-Liggett, C.M., • Haas, B.J., Inman, J.M., Kent, R., Lemieux, S., Malavazi, I., Orvis, J., Roemer, T., • Ronning, C.M., Sundaram, J.P., Sutton, G., Turner, G., Venter, J.C., White, O.R., • Whitty, B.R., Youngman, P., Wolfe, K.H., Goldman, G.H., Wortman, J.R., Jiang, B., • Denning, D.W., and Nierman, W.C. • PLoS Genet. 2008 Apr 11;4(4):e1000046.

    14. The genus Aspergillus was named by P. A. Micheli in 1729 • after a holy water sprinkler, or aspergillum, which resembled the • genus-characteristic conidia forming structure of these fungi. • It includes over 200 species of mostly asexual fungi found • ubiquitously in soil as well as in forage products, food, dust, • organic debris, and decomposing vegetation

    15. Most of them are saprophytes, but a surprising number of • species are able to infect wounded plants and animals. • - Aspergillus fumigatus is exceptional amongst the aspergilli in • being both a primary and opportunistic pathogen as well as a • major allergen associated with severe asthma and sinusitis • - SUPREME OPPORTUNISTS !!!!!!!!!!!!!!

    16. The ability of several species to cause disease in an • immunosuppressed individual implicates that under appropriate • conditions any Aspergillus species can provoke different forms of • Aspergillosis. • - However, the fact that A. fumigatus is by far the most • commonly identified species in pulmonary mycosis although its • relative abundance among environmental Aspergillus conidiais • low, is in favor of the existence of specific cellular attributes that • support its growth inside the ecological niche • “immunocompromised host”.

    17. From: Krappman, S. 2007. Pathogenicity determinants and allergens. In Goldman, G.H. and Osmani, S.A. (in press)

    18. From: Krappman, S. 2007. Pathogenicity determinants and allergens. In Goldman, G.H. and Osmani, S.A. (in press)

    19. From: Krappman, S. 2007. Pathogenicity determinants and allergens. In Goldman, G.H. and Osmani, S.A. (in press)

    20. Factors that determine virulence of fungal opportunistic • pathogens are hard to define, as the host’s immune status is • crucial for the outcome of infection; moreover, general as well as • specific cellular attributes of the fungus have a a large impact on • its survival inside the hostile environment of an infected individual. • Here, the term “virulence determinant” is used a broad sense to • describe gene products and cellular aspects of Aspergillus that • were characterized to support its capacity to cause disease in an • immunocopromised host. This includes common traits that account • for the physiological versatility of this fungus or its saprobic • lifestyle, although these features represent factors that are • required for growth in general.

    21. Virulence genes (What is virulence ?) • The ability to survive in a human host is not the consequence of • the presence of true virulence genes but of the metabolic • capabilities. It has evolved to succeed as a saprophyte, including • its temperature versatility, defense mechanisms against oxidative • stress, and ability to effectively export potentially harmful • chemicals present in the its environment. In support of this • hypothesis is the observation that no genomic components are • shared and exclusively by A. fumigatus and other human • pathogens such as the Candida or Cryptococcus species.

    22. Its basic lifestyle is that of a saprophyte, raising the question • whether A. fumigatus represents a true pathogen at all. The • answer to this may lie in the viewpoint on at the interplay of the • fungus and its environment: Pathogenicity strictly relies on a host • to be infected and damaged, therefore in the setting of Aspergillus • colonizing this specific ecological niche it has to be regarded as • a pathogen; in case of fungal proliferation in the absence of a host, • saprophytic propagation might be used as proper description.

    23. - Its comparison with the genomes of two distantly related species, A. nidulans and A. oryzae, has led to many unexpected discoveries, including the possibility of a hidden sexual cycle in A. fumigatus and A. oryzae. From: Gallagan, J. et al. 2005. Nature, 438: 1105-1115

    24. Comparison of molecular divergence in aspergilli and yeasts 60 Kluyveromyces lactis Candida glabrata A. nidulans fish A. oryzae 70 A. terreus birds 80 A. clavatus S. uvarum Average protein sequence identity 90 mice S. paradoxus N. fischeri humans 100 A. fumigatus S. cerevisiae

    25. - This significant phylogenetic distance has hindered some aspects of comparative genomic analysis such as identification of differential genetic traits responsible for the differences in virulence, sexual, and physiological properties of A. fumigatus.

    26. Phylogenetic Tree: (90 Concatenated Proteins) Affc

    27. - To maximize the resolving power of whole-genome comparative analysis, we selected a very closely related sexual species, Neosartorya fischeri NRRL181 (A. fischerianus), and a more distantly related asexual species, A. clavatus NRRL1, for complete sequencing.

    28. A. fumigatus Chromosomes Size (MB) 1 4.891 4.834 4.018 3.933 3.922 3.779 2.021 1.789 2 3 ~35 copies rDNA 4 5 6 7 8 Centromeric area Telomere From: Nierman, W. et al. 2005. Nature, 438: 1151-1156

    29. N. fischeri [A. fischerianus] • Apart from sister taxa, A. fumigatus var ellipticus, N. fischeri is the most closely related species to A. fumigatus • N. fischeri is the teleomorph of A. fischerianus • Rarely identified as a pathogen with only two medical cases reported in literature • Scarcity in environment • Misidentification in the laboratory • Relative lack of virulence • Role in food spoilage • Homothallic with thermoresistant ascospores • Reduced growth at 42ºC relative to 37ºC and no radial growth at 48ºC in contrast to A. fumigatus which shows increased growth at 42ºC relative to 37ºC and measurable growth at 48º

    30. A. clavatus • A. clavatus is a very rare human pathogen with only one medical case reported in literature (post-surgery endocarditis) • Grows more slowly at 37oC than A. fumigatus • Bigger spore size may prevent lung penetration • Although not a common pathogen, it is probably an important allergen and has been shown to be the cause of an extrinsic allergic alveolitis known as malt worker's lung • Produces a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins • Causes neurotoxicosis in sheep and cattle fed infected grain

    31. Sequenced organisms  Af293 A1163 N. fischeri A. clavatus Length (Mp) 28.810 29.205 32.552 27.859 Assemblies > 100 Kb 18 11 13 16 GC content 50% 49% 49% 49% No. of genes 9631 9906 10407 9125 Mean gene length (Bp) 1478 1455 1466 1483 % Genes with introns 79% 80% 80% 81% % Coding 49% 49% 47% 49% Affc lineage (A. fumigatus, N. fischeri, and A. clavatus) At least 12 copies of mitochondrial genome per nuclear genome

    32. The Af293 gene set has been classified into four groups after comparison with the six other sequenced aspergilli

    33. Alignment of the A1163, N. fischeri, and A. clavatus assemblies against the eight Af293 chromosomes A1163 N. fischeri A. clavatus Sec. Met. Clu. TE Centromere Ribosomal DNA For each pair of genomes, syntenic blocks were defined as a minimum of 5 adjacent matching genes with a maximum of 20 intervening non-matching genes in the reference and target genomes.

    34. - Together these regions, referred to here as A. fumigatus-specific • islands, comprise over 5.9 % (1.7 Mb) of the Af293 genome. The • islands show a significant telomeric bias with larger blocks found • at chromosome ends, while smaller ones tend to reside in central • chromosomal areas. Notably these small blocks often contain • gene clusters involved in secondary metabolism or detoxification. • In addition to non-syntenic genes, species-specific islands • harbour a disproportionate number of TEs and other repeat • elements in comparison with the syntenic areas of the genome.

    35. - Coincidentally core and lineage-specific genes have a biased distribution along A. fumigatus chromosomes. Lineage-specific genes are disproportionately overrepresented among telomere-proximal genes defined here as genes located within 300 Kb from chromosome ends. About 38% of Affc-specific genes are telomere-proximal in comparison to 6% of Asp-core and 9% Affc-core genes.

    36. Biased Distribution of Biological Processes b/w Core and Species-specific Genes (GO) • Species-specific Genes: • secondary metabolism • carbohydrate metabolism • transmembrane transport • Core Genes: • information processing • cellular processes • cell wall biogenesis

    37. Highlights on genes and pathways possibly important for the • interaction A. fumigatus-host

    38. Main features of the interaction A. fumigatus-host 1. Contacting the host 2. Sensing the host 3. Feeding from the host 4. Damaging and fighting the host 5. Sensitizing the host From: Krappman, S. 2007. Pathogenicity determinants and allergens. In Goldman, G.H. and Osmani, S.A.

    39. From: Krappman, S. 2007. Pathogenicity determinants and allergens. In Goldman, G.H. and Osmani, S.A.

    40. From: Krappman, S. 2007. A Comparative View of the Genome of Aspergillus fumigatus. In Goldman, G.H. and Osmani, S.A.

    41. Thermophily

    42. From: Nierman, W. et al. 2005. Nature, 438: 1151-1156

    43. - 323 genes (clusters 1 and 2) higher expression at 48 oC than at 37 oC • -135 genes (cluster 3) higher expression at 37 oC than at 48 oC • Only 11 genes from the 551 homologues of the S. cerevisiae general • stress-response genes • Except for catalase B, no know genes implicated in pathogenicity • showed higher expression at 37 oC than at 48 oC • Conclusion: Host temperature alone (37 oC) is insufficient to turn • on many virulence-related genes From: Nierman, W. et al. 2005. Nature, 1151-1156

    44. Secondary metabolites

    45. Distribution of SMP Clustersalong the 8 A. fumigatus chromosomes #1 (NRPS Pes1) New cluster (NRPS SidC); #2 (PKS) 1 4.9 Mb 95 -> 98 -> #4 (PKS, pigment); #5 (DMATs, fumigaclavine) 2 107 4.8 Mb 57 -> 92 -> #6 (PKS); #7 (PKS); #8-9 (NRPS SidD/E) #10 (ETP toxin), #11 (NRPS), #12 (PKS), #13 (NRPS) 3 4.0 Mb 100 -> <- 93 #14 (PKS); rDNA #15 (PKS) 4 3.9 Mb <-109 104 -> <- 96 ##16, 17 (NRPS-like) ? #18 (NRPS) 5 3.9 Mb <- 53 <- 94 #19 (NRPS) #20 (NRPS, gliotoxin); #21 (NRPS); #22 (PKS) 6 3.6 Mb <-106 <-101 108 -> centromere sec met cluster LaeA or iron ‘up-expressed’ clusters cluster conserved in at least one more species #23 (PKS, DMAT) 7 2.0 Mb <-103 <-99 #24 (fumitremorgin, pseurotin); #25? 8 1.8 Mb <-102 55 -> Perrin, R.M, 2007. PLoS Pathogens 3: e50.

    46. From: Chamilos, G. and Kontoyiannis, D.P. 2007. The emerging role of mini-host models in the study of aspergillosis. In Goldman, G.H. and Osmani, S.A.

    47. The A. fumigatus gliotoxin cluster Of all the mycotoxins produced by A. fumigatus, five immunosuppressive ones – gliotoxin, fumagillin, helvolic acid, fumitremorgin A and Asp-hemolysin – could be identified up to now. From: Gardiner et al., 2005, Microbiology,151:1021-1032

    48. The A. fumigatus gliotoxin cluster Gardiner and Howlett, 2005, FEMS Microbiol. Lett. 248: 241-248