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

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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). -The osmotrophic growth habit of fungi is extremely effective for colonizing diverse habitats and has made the fungi the principal degraders of 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 evolutionarily diverged than the Chordate phylum of the animal kingdom. - Of the eukaryotic genome sequences currently available, more than half come from the kingdom Fungi

5 - 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 e-fungi: a data resource for comparative analysis of fungal genomes (Hedeler et al., 2007. BMC Genomics 8: 426; www..e-fungi.org.uk)

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

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 Saccharomycotina Pezizomycotina Taphrinomycotina

8 - 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 Gene duplications

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.

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14 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.

15 -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

16 - 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 !!!!!!!!!!!!!!

17 -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 conidia is low, is in favor of the existence of specific cellular attributes that support its growth inside the ecological niche “immunocompromised host”.

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

21 - 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.

22 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.

23 - 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.

24 From: Gallagan, J. et al. 2005. Nature, 438: 1105-1115 - 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.

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

26 - 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.

27 Phylogenetic Tree: (90 Concatenated Proteins) Affc

28 - 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.

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

30 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

31 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 37 o C 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

32 At least 12 copies of mitochondrial genome per nuclear genome Affc lineage (A. fumigatus, N. fischeri, and A. clavatus) Sequenced organisms Af293A1163N. fischeriA. clavatus Length (Mp)28.81029.20532.55227.859 Assemblies > 100 Kb18111316 GC content50%49% No. of genes96319906104079125 Mean gene length (Bp)1478145514661483 % Genes with introns79%80% 81% % Coding49% 47%49%

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

34 Alignment of the A1163, N. fischeri, and A. clavatus assemblies against the eight Af293 chromosomes A1163 N. fischeri A. clavatus Sec. Met. Clu. TE Ribosomal DNA Centromere 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.

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36 - 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.

37 - 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.

38 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

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

40 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. Main features of the interaction A. fumigatus-host

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

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

43 Thermophily

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

45 From: Nierman, W. et al. 2005. Nature, 1151-1156 - 323 genes (clusters 1 and 2) higher expression at 48 o C than at 37 o C -135 genes (cluster 3) higher expression at 37 o C than at 48 o C -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 o C than at 48 o C -Conclusion: Host temperature alone (37 o C) is insufficient to turn on many virulence-related genes

46 Secondary metabolites

47 4.9 Mb 1 98 -> centromere sec met cluster LaeA or iron ‘up-expressed’ clusters cluster conserved in at least one more species 4.0 Mb 3 3.9 Mb 4 5 3.6 Mb 6 2.0 Mb 7 1.8 Mb 8 2 4.8 Mb #1 (NRPS Pes1) New cluster (NRPS SidC); #2 (PKS) #14 (PKS); rDNA #4 (PKS, pigment); #5 (DMATs, fumigaclavine) #6 (PKS); #7 (PKS); #8-9 (NRPS SidD/E) #10 (ETP toxin), #11 (NRPS), #12 (PKS), #13 (NRPS) #15 (PKS) 107 ##16, 17 (NRPS-like) ? #18 (NRPS) #19 (NRPS) #20 (NRPS, gliotoxin); #21 (NRPS); #22 (PKS) #23 (PKS, DMAT) #24 (fumitremorgin, pseurotin); #25? Distribution of SMP Clusters along the 8 A. fumigatus chromosomes 95 -> 57 ->92 -> 100 -> <- 93 <- 96 <- 53 <- 94 <-103 <-99 Perrin, R.M, 2007. PLoS Pathogens 3: e50.

48 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.

49 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 The A. fumigatus gliotoxin cluster

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

51 Gliotoxin received considerable attention for over two decades as a putative virulence factor based on the following observations: (i)Up to 93 % of A. fumigatus strains recovered from cancer patients with IA produced gliotoxin compared to less than 20 % of the environmental isolates; (ii) A. fumigatus is the most prolific producer of gliotoxin among the pathogenic Aspergillus species tested; (iii) Gliotoxin is immunosuppressive and proapoptotic for mammalian cells; (iv) Gliotoxin inhibits the NADPH oxidase activity responsible for the neutrophil oxidative burst; (v) Circulating gliotoxin is readily detected in experimental aspergillosis and in sera from patients with IA, while it is only occasionally detected in patients without any evidence of IA; (vi) Mice infected with a non-gliotoxin-producing strain survived longer than those infected with a genetically unrelated gliotoxin producer; and (vii) Sugui et al. (2007) have shown that  gliP was unable to infect immunosupressed mouse strains.

52 LaeA From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582 -In a screen in search for A. nidulans mutants impaired in the production of the carcinogen sterigmatocystin, a global regulator of the production of secondary metabolites could be identified, the laeA gene product (Bok and Keller, 2004) - LaeA is a transcriptional regulator, perhaps acting at a chromatin remodelling level (probably it is a methyl transferase)

53 From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582

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58 Transcriptional regulation of chemical diversity in Aspergillus fumigatus by LaeA (Perrin et al., PLoS Pathogens, 2007, 3: e50) -Of the 943 genes showing significant differences in expression between  laeA and wild type, 415 showed increased expression in  laeA and 528 showed decreased expression -The most remarkable discovery was the near-global suppression of secondary metabolite gene expression in the  laeA mutant. Nearly all (97 %) of the secondary metabolite gene cluster loci showed decreased expression in  laeA, with a mere three genes in this category showing increased expression in  laeA - The authors suggest the possibility that virulence attributes are not influenced as much by individual metabolites as by the blend of LaeA-regulated toxins, which, in combination, may confer an advantage to the pathogen.

59 Perrin, R.M, 2007. PLoS Pathogens 3: e50.

60 Positional bias of LaeA-regulated gene expression -54 % of the clusters showing differential expression in  laeA were found within 300 kb of telomeres

61 Sub-telomere directed gene expression during initiation of invasive aspergillosis Andrew McDonagh, Natalie D. Fedorova, Jonathan Crabtree, Yan Yu, Stanley Kim, Dan Chen, Omar Loss, Timothy Cairns, Gustavo H. Goldman, Darius Armstrong-James, Ken Haynes, Hubertus Haas, Markus Schrettl, Gregory May, William C. Nierman, and Elaine Bignell PLoS Pathogens (in press)

62 - The Eberwine method of mRNA amplification involves reverse transcription of mRNA with an oligo dT primer bearing a T7 RNA polymerase promoter site, to direct in vitro transcription of antisense RNA (aRNA) after double stranded cDNA synthesis and is favoured for linear mRNA amplification from limited quantities of starting material. - To identify fungal attributes preferentially employed during adaptation to the host niche, and thus contributing to the virulence of the saprophytic parasite A. fumigatus, we compared the transcriptomes of developmentally matched A. fumigatus isolates following laboratory culture or initiation of infection in the neutropenic murine lung.

63 Comparative time-course of A. fumigatus Af293 germination and hyphal development in the murine lung, and laboratory culture

64 - At 12-14 hours post-infection >80% of A. fumigatus conidia had undergone germination and primary hyphal production. At this time point recovery of germlings in BAL fluid was routinely achievable in the order of 10 3 germlings per lavaged lung. - Pools of 24 neutropenic CD1 male mice with 10 8 conidiospores. Bronchoalveolar lavage was performed immediately using pre-warmed sterile saline and samples (BALFs) were snap frozen prior to RNA extraction and amplification. - Total RNA yields from pooled BALFs ranged from 108 – 800 ng and yielded up to 258 μg aRNA after 2 rounds of linear amplification. In vitro reference RNA samples were similarly prepared from developmentally matched A. fumigatus germlings

65 A genome-wide transcriptional snapshot of A. fumigatus Af293 during initiation of murine infection. Of 2180 genes (22.6 % of the whole genome) having a fold-change in log intensity ratio of 2 or greater, 1281 were up-regulated and 897 were down-regulated. a region of ribosomal DNA. Centromere

66 - we could identify a minimum of eleven siderophore biosynthesis/transport genes as important during growth in the murine lung including two ferric-chelate reductases. Thirteen amino acid permease genes were more abundantly represented during host-adaptation than growth in YPD. Nine genes annotated as maltose permeases or transporters in the current Af293 annotation were also more abundantly represented during initiation of murine infection. - Our analysis identified increased abundance of transcripts from the elastinolytic metalloprotease, an aorsin-like serine protease, and three dipeptidylpeptidases. Thus transcription of this subset of A fumigatus proteases is significantly higher in the murine lung relative to rich laboratory culture.

67 - Functional categories of ergosterol biosynthesis, heme biosynthesis and aerobic respiration were significant among genes underrepresented during infection, relative to laboratory culture as well as multiple functional categories representing ribosome biogenesis and assembly, and protein biosynthesis and processing. - This may reflect the poor nutritional value of murine lung relative to YPD and/or reduced growth (due to any number of stresses) during host-adaptation compared to broth culture. This trend is evidenced on multiple levels within our dataset, comprising repression of genes directing ribosomal protein synthesis, rRNA synthesis, RNA polymerase I and II activity, translation initiation and elongation, tRNA processing and synthesis, intracellular trafficking, secretion and vesicular trafficking.

68 Distribution of lineage specific and telomere-proximal genes among differentially expressed host adaptation dataset.

69 Overlap between murine adaptation and in vitro stress datasets alkaline adaptation, iron deprivation and nutrient starvation

70 Comparative analysis of A. fumigatus gene expression datsets

71 - Out of 415 genes down-regulated in the absence of LaeA we identified 99 genes having increased abundance during initiation of murine infection. - Functional categorisation of shared genes revealed that 40% (n=40) were involved in secondary metabolite biosynthesis, among these we could identify three complete secondary metabolite clusters, those directing gliotoxin and pseurotin biosynthesis. - We identified 49 and 40 genes, having subtelomeric locations and secondary metabolite biosynthetic functions respectively.

72 Summary - Among the ~150 fungal species that are able to cause disease in a mammalian host, Aspergilli are exceptional because they can elicit allergic responses or harm immunocompromised individuals, in most severe cases with a fatal outcome. - Their default way of life, however, is that of a saprophyte, which is characterized by the uptake of nutrients from a decaying organic substrate, and it is likely that the pathogenicity of this fungal species is based to a great extent on its saprophytic lifestyle.

73 -One possible explanation to this high level of conservation among previously identified virulence-associated genes is that A. fumigatus virulence may be a combinatorial process, dependent on a pool of virulence genes, which interact in various combinations in different genetic backgrounds. Alternatively, yet unknown A. fumigatus-specific genes (or other differential genetic traits) may contribute to its ability to survive in the human host. Summary

74 - Alkaline adaptive capability,previously found to be essential for A. nidulans virulence in neutropenic mice, is likely to be important for growth of A. fumigatus spores at physiological pH. Accordingly we identified 102 genes preferentially expressed during both murine infection and in vitro alkaline adaptation Among them are 36 genes having unknown function, two sodium ATPases, the plasma membrane zinc ion transporter and an alkaline phosphatase. Summary

75 - Taken together these analyses indicate that a significant component of the LaeA regulon, comprised mainly of secondary metabolism genes, is represented among transcripts more abundant during infection. Furthermore the subtelomeric bias observed among differentially expressed murine adaptation genes extends beyond secondary metabolite biosynthesis and does not appear to be a general feature of adaptation to environmental change. Summary


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