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
- Highlights on genes and pathways possibly important for the
interaction A. fumigatus-host
- 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.
- 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
e-fungi: a data resource for comparative analysis of fungal genomes
(Hedeler et al., 2007. BMC Genomics 8: 426; www..e-fungi.org.uk)
- 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
Oomycetes
Cornell et al., 2008. Genome Research, 15: 1620-1631
(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
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
-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.
- 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 duplicationcontaining clusters than S. cerevisiae.
- Among the Aspergillus genomes, A. niger and A. oryzae possess
the most duplication-containing clusters
- 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.
- 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.
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.
-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
- 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 !!!!!!!!!!!!!!
-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”.
From: Krappman, S. 2007. Pathogenicity determinants and allergens.
In Goldman, G.H. and Osmani, S.A. (in press)
From: Krappman, S. 2007. Pathogenicity determinants and allergens.
In Goldman, G.H. and Osmani, S.A. (in press)
From: Krappman, S. 2007. Pathogenicity determinants and allergens.
In Goldman, G.H. and Osmani, S.A. (in press)
- 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.
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.
- 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.
- 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
Comparison of molecular divergence
in aspergilli and yeasts
Average protein sequence identity
60
Candida glabrata
A. nidulans
A. oryzae
Kluyveromyces lactis
70
A. terreus
A. clavatus
fish
birds
80
S. uvarum
90
N. fischeri
A. fumigatus
100
S. paradoxus
mice
S. cerevisiae
humans
- 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.
Phylogenetic Tree:
(90 Concatenated Proteins)
Affc
- 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.
A. fumigatus Chromosomes
Size (MB)
1
4.891
2
4.834
3
4.018
~35 copies rDNA
4
3.933
5
3.922
6
3.779
7
2.021
8
Centromeric area
Telomere
1.789
From: Nierman, W. et al. 2005. Nature, 438: 1151-1156
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
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
Affc lineage (A. fumigatus, N. fischeri, and A. clavatus)
Sequenced organisms
Af293
A1163
N. fischeri
A. clavatus
28.810
29.205
32.552
27.859
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%
Length (Mp)
Assemblies > 100 Kb
At least 12 copies of mitochondrial genome per nuclear genome
The Af293 gene set has been classified into four groups
after comparison with the six other sequenced aspergilli
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.
- 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.
- 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.
Biased Distribution of Biological Processes b/w
Core and Species-specific Genes (GO)
Species-specific Genes:
Core Genes:
secondary metabolism
carbohydrate metabolism
transmembrane transport
information processing
cellular processes
cell wall biogenesis
Highlights on genes and pathways possibly important for the
interaction A. fumigatus-host
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.
From: Krappman, S. 2007. Pathogenicity determinants and allergens.
In Goldman, G.H. and Osmani, S.A.
From: Krappman, S. 2007. A Comparative View of the Genome of
Aspergillus fumigatus. In Goldman, G.H. and Osmani, S.A.
Thermophily
From: Nierman, W. et al. 2005. Nature, 438: 1151-1156
- 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
Secondary metabolites
Distribution of SMP Clusters along the
8 A. fumigatus chromosomes
#1 (NRPS Pes1)
1
New cluster (NRPS SidC); #2 (PKS)
98 ->
4.9 Mb
95 ->
#4 (PKS, pigment); #5 (DMATs, fumigaclavine)
2
57 ->
107
#6 (PKS); #7 (PKS); #8-9 (NRPS SidD/E)
3
#10 (ETP toxin), #11 (NRPS), #12 (PKS), #13 (NRPS)
100 ->
#15 (PKS)
<-109
104 ->
#18 (NRPS)
<- 53
<-106
#20 (NRPS, gliotoxin); #21 (NRPS); #22 (PKS)
<-101
108 ->
#23 (PKS, DMAT)
7
<-103
3.9 Mb
<- 94
#19 (NRPS)
6
3.9 Mb
<- 96
##16, 17 (NRPS-like) ?
5
4.0 Mb
<- 93
#14 (PKS); rDNA
4
4.8 Mb
92 ->
3.6 Mb
centromere
<-99
2.0 Mb
sec met cluster
#24 (fumitremorgin, pseurotin); #25?
8
<-102
55 ->
1.8 Mb
LaeA or iron ‘up-expressed’ clusters
cluster conserved in at least one more species
Perrin, R.M, 2007. PLoS Pathogens 3: e50.
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.
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
The A. fumigatus gliotoxin cluster
Gardiner and Howlett, 2005, FEMS Microbiol. Lett. 248: 241-248
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.
LaeA
-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)
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
From: Bok, J.W. et al., 2005. Eukaryotic Cell, 4: 1574–1582
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.
Perrin, R.M, 2007. PLoS Pathogens 3: e50.
Positional bias of LaeA-regulated gene expression
-54 % of the clusters showing
differential expression in laeA
were found within 300 kb of
telomeres
Perrin, R.M, 2007. PLoS Pathogens 3: e50.
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)
- 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.
Comparative time-course of A. fumigatus Af293 germination and
hyphal development in the murine lung, and laboratory culture
- 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 103 germlings per lavaged lung.
- Pools of 24 neutropenic CD1 male mice with 108 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
A genome-wide transcriptional snapshot of A. fumigatus
Af293 during initiation of murine infection.
Centromere
a region of ribosomal DNA.
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.
- 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.
- 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.
Distribution of lineage specific and telomere-proximal genes
among differentially expressed host adaptation dataset.
Overlap between murine adaptation and in vitro stress datasets
alkaline adaptation, iron deprivation and nutrient starvation
Comparative analysis of A. fumigatus gene expression datsets
- 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.
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.
Summary
-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
- 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
- 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.