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Comparative Genomic Analyses of the Human Fungal Pathogens Coccidioides and
Their Relatives
Thomas J. Sharpton, Jason E. Stajich, Bridget M. Barker, Bruce W. Birren, Garry T.
Cole, John N. Galgiani, Malcolm J. Gardner, Marcin Grynberg, Matthew R. Henn,
Chiung-Yu Hung, Vinita S. Jordar, Theo N. Kirkland, Chinnapa D. Kodira, Cody
McMahan, Rama Maiti, Anna Muszewska, Daniel E. Neafsey, Marc J. Orbach, Steven D.
Rounsley, Jennifer R. Wortman, Qiandong Zeng, John W. Taylor
RUNNING TITLE: Comparative Genomics of Coccidioides
Abstract
The dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary
pathogens that cause morbidity and mortality in immunocompetent humans and other
mammals. Infection results from exposure to asexual Coccidiodies spores
(arthroconidia), which are found in arid desert soil where the fungus has been considered
to grow as a soil saprophyte. Relatively little is known about the virulence mechanisms
and life history of this fungal lineage, which is thought to have become pathogenic in
recent evolutionary history because its closest known relatives are not pathogens. To
gain insight into these issues, we sequenced the genomes of two pathogenic Coccidioides
species, C. immitis and C. posadasii, a close, non-pathogenic relative, Uncinocarpus
reesii, and a more distantly related pathogenic fungus, Histoplasma capsulatum.
Through comparison of these four Onygenales genomes with those from 13 more
distantly related Pezizomycete fungi, we identified changes in gene family size associated
with a host/substrate shift from plants to animals in the Onygenales lineage. Comparing
these Onygenales genomes with each other, we further found gene gains and other
evolutionary changes in the Coccidioides lineage that may underlie its infectious
phenotype. These evolutionary changes include lineage-specific metabolic and
membrane-related gene gains, as well as rapidly evolving secondary metabolite genes.
Our results suggest that Coccidioides species are not soil saprophytes, but that they
remain associated with their dead animal hosts in soil. Furthermore, our analysis
identifies genes and proteins associated with the evolution of pathogenicity in
Coccidioides that may be targets for treatment and prevention of coccidioidomycosis.
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RUNNING TITLE: Comparative Genomics of Coccidioides
[Supplemental material is included in this manuscript submission. The sequence data
generated in this study for C. immitis and H. capsulatum has been deposited to GenBank
under the accession nos. AAEC02000001-AAEC02000053 and AAJI01000001AAJI01002873 respectively. The data sequence data generated for C. posadasii and U.
reesii is in the process of being submitted and is currently publicly available at
http://www.tigr.org/tdb/e2k1/cpa1/ and
http://www.broad.mit.edu/annotation/genome/uncinocarpus_reesii/ respectively.]
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RUNNING TITLE: Comparative Genomics of Coccidioides
Introduction
Coccidioides, the causal agent of coccidioidomycosis or ‘Valley Fever’, is one of
the few fungal pathogens capable of causing life-threatening disease in
immunocompetent adult humans. As such, it provides one of the best opportunities to
study the evolution of fungal pathogenesis without the complication of the loss of host
immunocompetency. The unusual virulence of this fungus and its potential for dispersal
and infection by airborne spores led to its listing as a U.S Health and Human Services
Select Agent (Dixon 2001). Coccidioides infects at least 150,000 people annually and
roughly 5% of these infections develop into severe disease for which existing treatments
can be prolonged and difficult to tolerate (Galgiani et al. 2005). Fortunately, because
infection confers life-long immunity (Cole et al. 2004) and selected gene knockouts have
been shown to reduce virulence in mice (Hung et al. 2005; Hung et al. 2002), it is likely
that an effective vaccine can be developed or new drug targets identified (Hector and
Rutherford 2007).
The medical relevance of Coccidioides has driven interest in its biology and
evolution. The genus is composed of two closely-related species, C. immitis and C.
posadasii, and is a member of the Onygenales, an unusual Ascomycete order in that its
species tend to associate with animals rather than plants. Despite their association with
animals, recently diverged fungal relatives of Coccidioides are not known to cause
disease (Untereiner et al. 2004). This observation has led to the theory that Coccidioides
recently acquired its pathogenic phenotype, a key aspect of which is the organism’s
dimorphic lifecycle. Coccidioides is reported to grow as saprobic filaments through the
arid, alkaline soil of New World deserts (Papagiannis 1967), (Fisher et al. 2007).
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RUNNING TITLE: Comparative Genomics of Coccidioides
Inhalation by a host of airborne Coccidioides asexual spores (arthroconidia) produced
during filamentous growth can yield infection. Once in the host lungs, the parasitic phase
is initiated as arthroconidia enlarge into spherules, documenting a morphological switch
from polar to isotrophic growth. Spherules subsequently differentiate to produce internal
spores (endospores) that are released upon spherule rupture. This latter morphology,
endospore-containing spherules, is unique to Coccidioides among all known
Ascomycota. Endospores are capable of disseminating in the host and re-initiating the
spherulation cycle, but the host can sequester spherules in a granuloma to prevent disease
dissemination. In the absence of a successful host response, chronic infection may persist
for at least 12 years (Hernandez et al. 1997), although human disease can progress to
death in a much shorter period. Upon death of the host, due to coccidioidomycosis or
not, the fungus reverts to filamentous growth and the production of arthroconidia.
Although a great deal is known about the clinical aspects of coccidioidomycosis
and the biology of this fungus in laboratory mice, relatively little is known about the life
history of Coccidioides between infections (Barker et al. 2007). Similarly, it is unclear
how the unique spherule stage in Coccidioides evolved or how Coccidioides causes
disease in immunocompetent humans while closely related taxa cannot. To address these
questions, we sequenced the genomes of Coccidioides immitis and Coccidioides
posadasii as well as their Onygenlean relatives Uncinocarpus reesii, a non-pathogenic
fungus, and Histoplasma capsulatum, a mammalian pathogen, and compared these
genomes to those of 13 more distantly related Ascomycota. Our comparison of
Coccidiodies genomes to those of relatives at various phylogenetic distances enabled the
identification of evolutionary changes at various points along the Coccidioides lineage.
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RUNNING TITLE: Comparative Genomics of Coccidioides
These changes include gene family expansions and contractions, changes in
chromosomal structure, lineage-specific gains and losses of genes, rapid evolution of
genes, and natural selection acting on genes, all of which provide insight into the
evolution of virulence and the life history of Coccidioides.
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RUNNING TITLE: Comparative Genomics of Coccidioides
Results
Genome Sequencing and Annotation
The Coccidioides posadasii C735 genome was sequenced at 8X coverage and was
assembled into 58 contigs, totaling 27 Mb. Sequenced at 14X coverage, the C. immitis
RS genome assembled into 7 contigs and totaled 28.9 Mb. Despite having similar
genome sizes, the number of annotated genes in the Coccidioides species, 10,355 in C.
immitis and 7,229 in C. posadasii, differs greatly (Table 1). However, 9,996 of the C.
immitis genes have BLASTN hits with greater than 90% identity in the C. posadasii
genome. Thus, the majority of the differences are likely the result of different annotation
methodologies used by the institutions that produced the data. One notable difference is
the decision to include predicted genes found in the repetitive regions of C. immitis and to
remove them in C. posadasii. Furthermore, a conservative approach was used to annotate
C. posadasii genes, whereby only those predictions supported by EST data were included
in the final dataset. Because of these annotation differences, we applied conservative
approaches to our subsequent comparative analyses, considering only those genes
annotated in both Coccidioides species.
The Coccidioides genomes also differ in their amount of repetitive DNA (Table
2). While these genomes have similar amounts of non-repetitive sequence, differing only
by 400 kb, the C. immitis genome, which is 17% repetitive, has an additional 1.84 Mb of
repetitive sequence compared to C. posadasii (12% repetitive). In both genomes, the
majority of repeats are in the high copy frequency class (greater than 20 copies), which
differs from U. reesii and H. capsulatum. The U. reesii genome contains relatively little
repetitive content (4%) and the majority of the repeats are of the medium copy number
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class (6-20 copies). The repeats in the H. capsulatum genome, which is 19% repetitive,
are equally divided among the three frequency classes.
A closer inspection of the sequence composition of the Coccidioides genomes
reveals a correlation between repeat containing regions and dinucleotide content. For
example, the GC content of repeats in both Coccidioides genomes is 14-15% lower than
the GC content of non-repetitive sequence (Fig 1). Furthermore, in C. immitis, CpG
dinucleotides are 19 times more abundant in non-repetitive sequence than in repetitive
sequence. In fact, in many repetitive regions the CpG frequency per kilobase drops to
zero for many kilobases and there are contiguous stretches of 100kb windows with a CpG
frequency as low as 0.08 per kilobase. Conversely, the count in non-repetitive sequence
averages 51 per kilobase. No other dinucleotide exhibits such a dramatic bias across
repeat classes.
Coccidioides Chromosome Structure and Conserved Synteny
Previous work using CHEF gel-electrophoresis estimated that Coccidioides
species have four chromosomes (Pan and Cole 1992b). However, chromosomes of
similar size are difficult to resolve with electrophoretic karyotyping (Taga et al. 1998).
Supercontigs three and four in the high quality C. immitis genome sequence assembly are
4.66 Mb and 4.32 Mb respectively, suggesting that these two linkage groups may have
been unresolved in prior karyotyping studies. The prediction that Coccidioides species
have five chromosomes is supported by an optical map and a whole genome alignment.
As shown in (SI1), an optical map of the C. posadasii genome identifies five distinct
linkage groups that accommodate 29 of its contigs. A NUCMER-generated whole
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genome sequence alignment (SI2) between C. immitis and C. posadasii suggests that,
despite having seven supercontigs, the C. immitis genome also comprises five
chromosomes. First, C. immitis supercontigs five and six likely constitute a single
chromosome because they are co-linear with C. posadasii contigs linked to its fifth
chromosome through the optical map data. Second, the extremely short seventh
supercontig (0.47 Mb long) in the C. immitis genome exhibits no unique, co-linear
homology to any of the C. posadasii contigs and likely represents an insertion in one of
the C. immitis chromosomes.
To explore the differences in chromosome structure between C. immitis and C.
posadasii, we compared 1kb windows of one genome against the other. Of the 23.8Mb
of non-repetitive sequence in C. immitis, 22.3Mb (93.5%) exhibits homology to the C.
posadasii genome with a median sequence identity of 98.3%, leaving 1.5Mb of nonrepetitive C. immitis DNA that do not have significant similarity to C. posadasii. The
reciprocal analysis confirms the 22.3Mb of matching sequence and finds 1.1Mb of nonrepetitive C. posadasii genome that is absent from C. immitis. Taken together, these
numbers suggest that the two species of Coccidioides share a core genome of 22.3Mb,
and that each has 1.1 to 1.5Mb of unique, non-repetitive sequence. A parallel analysis
using only the annotated protein coding genes from each Coccidioides genome identifies
282 genes from C. immitis that are absent from C. posadasii, and 66 genes from C.
posadasii that are absent in C. immitis (SI3).
The intersection of the window- and the gene-based analyses identifies 53
species-specific gene-containing regions in C. immitis and 45 gene-containing regions in
C. posadasii. In some cases, these regions are shorter than 2kb and contain just a single
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RUNNING TITLE: Comparative Genomics of Coccidioides
gene. In others, the regions are several hundred kilobases and contain several annotated
genes and substantial repetitive DNA. Analysis of EST datasets from the respective
species finds expression evidence for at least 132 C. immitis genes and 42 C. posadasii
genes in these regions. For 37 of the C. immitis regions, the corresponding location in the
C. posadasii genome is completely bounded within a sequence contig. This evidence
indicates that these regions are unlikely the result of assembly artifacts, but instead are
truly species-specific regions.
A few features of these regions are intriguing. Firstly, three of the four regions
greater than 100 kb in length are on a single chromosome (chromosome 4), and account
for 120 of the 282 C. immitis-specific genes. Secondly, many of the regions are very
close to the ends of the seven supercontigs and are likely to be close to the ends of the
five chromosomes. Finally, the genomic locations of six of the C. posadasii-specific
regions are syntenically identical to C. immitis-specific regions.
Eurotiomycetes Phylogeny and Divergence Times
We conducted a phylogenetic analysis on 2,891 aligned orthologs conserved between 16
Eurotiomycetes and the two Sordariomycetes used to root the tree, Sclerotinia
sclerotiorum and Fusarium graminearum. Three methods were used to determine
phylogenetic relationships between the taxa (Materials and Methods), each producing
trees of the same topology with 100% bootstrap support for all internal nodes. The
median branch lengths from the neighbor-joining trees were used as the final branch
lengths in the phylogeny (SI4). The phylogeny is consistent with previous studies (Geiser
et al. 2006), (James et al. 2006) and served as the basis for subsequent genome
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comparisons. In their analysis of Fungal kingdom divergence times, Taylor and Berbee
estimated that the Eurotiomycetes and Sordariomycetes split roughly 215 million years
ago (MYA) by calibrating the Basidiomycete/Ascomycete divergence at 400 MYA
(Taylor and Berbee 2006). Using this calibration point and the NPRS method in the r8s
phylogenetic software (Sanderson 2003), we estimated divergence times among the
Eurotiomycetes represented in the phylogeny (Figure 2 and SI5). Previous analyses
predicted that C. immitis and C. posadasii diverged approximately 12.8 MYA, but those
efforts were limited to nine microsatellite loci and five nuclear genes (Fisher et al. 2000),
(Koufopanou et al. 1998). Our whole genome analysis advances this estimate to
approximately 5.1 MYA, due principally to our choice of calibration to the fossil record.
In addition, our analysis predicts that the Coccidioides species diverged from U. reesii
roughly 75 MYA and that these taxa diverged from H. capsulatum approximately 150
MYA.
Hierarchical Comparative Genomics
The different methods for the discovery of genes involved in adaptive evolution
that we have employed are best applied to groups of taxa with specific evolutionary
divergences (Boffelli et al. 2004). For example, analyses of gene family expansions or
contractions can be performed over a much greater range of evolutionary divergence than
is possible for tests for positive selection. Fortunately, the wealth of available fungal
genome sequences permits whole genome comparisons over a large range of evolutionary
distances and, thus, enabled the appropriate employment of a variety of such methods. In
addition, comparisons across a highly populated, well-constructed phylogeny provide an
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evolutionary context for observed differences between the taxa. Ultimately, these
analyses facilitate an understanding of how Coccidioides lineage-specific changes
correlate with the ecology of the organism and its ability to infect immunocompetent
animals.
As mentioned, at the broadest evolutionary distance, we evaluated changes in
gene family size between nine Eurotiomycetes and Sordariomycetes. At an intermediate
evolutionary distance, we compared the 4,499 orthologs conserved among four
Onygenales genomes – C. immitis, C. posadasii, U. reesii, and H. capsulatum – to study
rates of protein evolution and gene gain and loss. Finally, among the two most closely
related genomes, those of C. immitis and C. posadasii, we compared 6,763 orthologs to
identify genes with signatures of positive selection.
Gene Family Expansions and Contractions
Changes in gene family size may accompany changes in lifestyle (Lespinet et al.
2002). Unlike most other Pezizomycotina, which tend to be associated with plants, the
Onygenales are generally capable of growth on animal tissue and exhibit dimorphic life
cycles. Related to their growth on animals, the Onygenales are also noted for their ability
to grow on keratin and other animal proteins. We evaluated the evolution of gene family
size in the Pezizomycotina to identify changes that may be associated with these
characteristics.
Many of the gene family size reductions unique to the Onygenales involve genes
coding for enzymes associated with growth on plant material (Figure 3). For example, in
the four Onygenales genomes, there are no proteins containing the fungal cellulose
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binding domain (CBM1), no tannases, no pectate lyases, no pectinesterases, and no
copies of NPP1, a necrosis-inducing protein found in many plant pathogens (Gijzen and
Nurnberger 2006). Furthermore, the four Onygenales genomes analyzed here have very
few (1-2) copies of the enzymes cellulase and cutinase while the Eurotiales genomes
contain many copies (8-16 and 4-5, respectively). Also reduced in Onygenales are
several distinct glycosyl hydrolase families and the melibiase family, genes that also are
involved in the catabolism of plant carbohydrates (DEY and Pridham 1972).
There are several additional gene families that are significantly reduced in size in
the Onygenales relative to the Eurotiales (SI6). For example, the Onygenales have
relatively small sizes for many families that make up the NADP Rossman clan, including
short chain dehydrogenases, zinc-binding dehydrogenases, FAD dependent
oxidoreductases, NAD dependent epimerases, GMC oxidoreductase and shikimate
dehydrogenases. Furthermore, the relative paucity of p450, condensation and KR protein
families in the Onygenales indicates that this order did not experience a radiation of
secondary metabolite genes similar to that previously reported for the Eurotiales (Cramer
et al. 2006). Finally, the Onygenales possess relatively few heterokaryon incompatibility
(HET) proteins (1-3 in Onygenales compared to 8-40 in Eurotiales), homologs of which
have been shown to play a role in prohibiting vegetative fusion between genetically
incompatible individuals (Espagne et al. 2002).
We identify six gene families that have grown in size within the Onygenales, five
of which are involved in protein digestion (Figure 3). For example, the peptidase S8
family more than tripled in size in the lineage leading to Coccidioides and Uncinocarpus.
More dramatically, the subtilisin N family experienced a seven-fold increase in the size
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along the same lineage. Both families are related to subtilisins which are extracellular
serine-proteases implicated in the pathogenic activity of several fungi (da Silva et al.
2006; Segers et al. 1999) including Aspergillus fumigatus (Monod et al. 2002) and are an
important virulence factors in many prokaryotes (Henderson and Nataro 2001). In
addition, peptidase S8 expansion involves homologs of the S8A subfamily, which
includes several well-described keratinolytic subtilases (keratinases) (Cao et al. 2008;
Descamps 2005). Futhermore, Coccidioides and U. reesii share a roughly two foldexpansion in the deuterolysin metalloprotease (M35) family, and Coccidioides species,
alone, show an additional expansion of three genes in the their lineage. One of these three
genes is MEP1, a known virulence factor in Coccidioides, which is thought to be
involved in host immune system evasion (Hung et al. 2005). The fungalysin
metallopeptidase (M36) family also doubled in size in Coccidioides and U. reesii. This
family contains secreted proteins that are involved in lung colonization in Aspergillus
fumigatus (Monod et al. 1993) and are thought to contribute to the invasion of keratinized
tissue by several dermatophytes (Jousson et al. 2004). Additionally, we observe an
expansion of the APH phosphotransferase family in the Onygenales. In prokaryotes, in
which this family is best described, these proteins are involved in aminoglycoside
antibiotic inactivation. While they are not as well understood in eukaryotes, their
function in these organisms is associated with protein kinase activity, specifically
homoserine kinase, fructosamine kinase and tyrosine kinase activity. Finally,
Coccidioides and U. reesii have 2-3 times as many CFEM domain (Common in several
Fungal Extracellular Membrane proteins) containing proteins as H. capsulatum and the
Eurotiales. Though their mode of function is not well understood, members of this
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family have been implicated in the pathogenic activity of Magnaporthe grisea (Kulkarni
et al. 2003), and appear to be associated with two virulence traits - iron acquisition
(Weissman and Kornitzer 2004) and biofilm formation (Perez et al. 2006) - in Candida
albicans. Overall, the major differences in gene family size were found between the
Onygenales and the other Eurotiomycetes; there were few significant differences in gene
family size between the Coccidioides and U. reesii lineages.
Gene Gain and Loss
Lineage specific gene gain can result in adaptation and changes in virulence
(Censini et al. 1996), (Lawrence 2005). To document changes in gene content, we
focused on closely related Onygenales and analyzed the 5,280 orthologs that are shared
between U. reesii and both Coccidioides species, as well as the 5,976 orthologs shared
between U. reesii and at least one Coccidioides species. These orthologs represent the
minimum set of core genes present in the ancestor of Coccidioides and U. reesii. To
identify changes in gene complement that could contribute to the phenotypic differences
between the Coccidioides pathogens and the non-pathogen U. reesii, we conducted a
parsimony-based analysis of lineage specific gene gain and loss using H. capsulatum as
an outgroup (Figure 4). The U. reesii lineage exhibits a greater rate of gene turnover
(1,075 gains and 399 losses) than the Coccidioides lineage (280-291 gains and 109
losses). It is important to note that gene gains and losses inferred from a set of four taxa
may alternatively represent ancestral genes retained in the ingroup, such as the
Coccidioides species, that were independently lost in both the sister and outgroup
lineages, in this example U. reesii and H. capsulatum, respectively.
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We performed functional enrichment analysis of genes gained in the Coccidioides
lineage and found two functions potentially related to virulence: heme/tetrapyrrole
binding (P = 3.5x10-4) and transcription factor activity (P = 0.039). Heme and
tetrapyrrole binding have been shown to be important for scavenging iron within the host
in several fungal pathogens (Howard 1999). Transcription factor activity and DNA
binding proteins obviously play roles in many cellular processes, and they are certainly
involved in fungal morphogenesis (Johannesson et al. 2006) (Ernst 2000). A key
difference between Coccidioides species and U. reesii is the morphogenesis of spherules
and endospores, and we can search among the gene gains for those that may be important
to this process by examining transcription during spherule development.
Spherule specific genes
The endosporulating spherule stage of the Coccidioides life cycle is a unique
Ascomycete phenotype and is necessary for pathogenicity (Rappleye and Goldman
2006). We compared expressed sequence tag (EST) libraries generated from
Coccidioides growing as mycelia or as spherules and identified 1201 genes with putative
spherule-specific expression. Of these, 93 have been gained in the Coccidioides lineage
since divergence with U. reesii, or retained in Coccidioides species and lost in other
Onygenales (SI7). These gained or retained spherule-specific genes are enriched for
several functions, including generation of precursor metabolites and energy (P = 0.002),
mitochondrion related (P = 0.048), and cation-transporting ATPase activity (P = 0.025).
In addition, 24 of these 93 genes exhibit signal peptides or membrane anchors, suggesting
that they may be involved in cell surface/membrane related biology. One interesting
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spherule-specific retained gene is CIMG_06184, which shares homology to the
Aspergillus fumigatus toxin Asp-hemolysin and is involved in iron acquisition (Ebina et
al. 1994). Another is CIMG_02178, a ureidoglycolate hydrolase that plays a key role in
metabolism of allantoin, the nitrogen-rich compound produced as a by-product of freeradical attack in mammals and that is known to be metabolized by some pathogens when
nitrogen is limiting (Divon and Fluhr 2007).
Rapidly evolving Onygenales proteins
Orthologs conserved between Coccidioides and U. reesii and subject to positive natural
selection in the Coccidioides lineage may have contributed to Coccidioides’ adaptation to
surviving within a mammalian host. Comparing the ratio of dN to dS is a conventional
means of identifying signatures of positive selection (Nielsen 2005), but because the
median dS between U. reesii and Coccidioides is 1.60 (calculated from 5,820 3-way
orthologs), mutations at synonymous nucleotide positions are saturated between these
taxa and they are thus too diverged to use this methodology (Figure 5). An alternative
approach is to estimate the evolutionary rate for proteins in a phylogeny with the
expectation that genes under positive selection will have an increased substitution rate
relative to genes without this selective pressure. Increases in the derived protein
substitution rate can be visualized as a relatively long branch in a phylogeny.
Referencing H. capsulatum as an outgroup and using maximum likelihood analysis to
infer ancestral character states, an analysis of 4,499 four-way ortholog clusters finds 60
genes in Coccidioides and 283 genes in U. reesii that have evolved with a significantly
faster amino acid substitution rate (P < 0.05, (Table 3)). A functional enrichment test
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finds that the rapidly evolving Coccidioides genes are enriched for biopolymer metabolic
processes (P = 0.017) and RNA metabolic process (P = 0.022).
One rapidly evolving Coccidioides protein is coded by the gene CIMG_07738. It
shares distant homology (31% identical, 43% similar) to the expression library
immunization antigen 1 protein, which, when used as a vaccine, has been shown to
provide protection against coccidioidomycosis to BALB/c mice (Ivey et al. 2003). In
addition, two membrane associated transporters (CIMG_11858 – transmembrane amino
acid transporter, CIMG_09822– major facilitator superfamily protein) are evolving faster
in Coccidioides compared to their ortholog in U. reesii. Major facilitator superfamily
transporters in particular have been shown to provide multidrug resistance to azoles and
other fungicides (Del Sorbo et al. 2000) and amino acid transporters in Candida albicans
appear to be important to virulence because their deletion reduces the acquisition of the
limiting metabolite Nitrogen (Biswas and Monideepa 2003). Finally, we identify a
rapidly evolving Coccidioides lipase (CIMG_05493), which is a member of a protein
family important to virulence in several pathogenic fungi, including C. albicans, Candida
glabrata, Cryptococcus neoformans and Aspergillus fumigatus (Ghannoum 2000)
(Gacser et al. 2007).
Adaptive evolution in Coccidioides
Antigenic pathogen genes subject to host immune selection pressure often exhibit
signatures of positive natural selection. The 6,763 orthologs conserved between C.
posadasii and C. immitis were screened to identify genes subject to positive selection
since the species diverged. The low median dS value of 0.023 indicates that the two taxa
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are recently diverged and the orthologs are not subject to mutational saturation. As
observed in other organisms (Nielsen et al. 2005), the median dN/dS value is low (0.25),
suggesting that most genes are under purifying selection (Figure 6). However, there are
57 genes that exhibit signatures of positive natural selection with dN/dS significantly
greater than 1 (P < 0.05, (Table 4)). These genes are enriched for several GO categories,
including metabolic process (P = 2.6x10-4), phosphorylation (P = 0.012), and Sadenosylmethionine-dependent methyltransferase activity (P = 9.7x10-3).
S-adenosylmethionine-dependent methyltransferase activity is important in the
production of secondary metabolites, which may include toxins or other compounds
important to pathogenic lifestyle. In A. flavus and A. parasiticus, this methyltransferase
is responsible for converting sterigmatocystin to O-methylsterigmatocystin, the
penultimate step in aflatoxin biosynthesis (Yu et al. 1993). Genetic manipulation of the
global regulator of secondary metabolites, LaeA, which can be bound by this
methyltransferase via a conserved thiopurine S-methyltransferase binding site, has led to
the discovery of new toxins in Aspergillus nidulans (Bok et al. 2005). One gene under
positive selection, CIMG_02025, contains a thiopurine S-methyltransferase domain,
suggesting it may interact with LaeA in Coccidioides to control secondary metabolism
and perhaps mediate infection.
Secreted proteins under positive selection are considered important candidate
antigens because the selection may involve an “evolutionary arms race” via direct
interaction with the host immune system (Dietsch et al. 1997). While in vivo analysis
must confirm host interaction, five secreted proteins exhibit signatures of positive
selection (CIMG_03148, CIMG_06700, CIMG_02887, CIMG_09119, CIMG_05660)
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and, although their functions are unknown, they may be good candidates for vaccine
discovery.
Proline Rich Antigens
The proline-rich protein, Prp1 (also known as antigen 2 or the proline-rich
antigen, rAG2/Pra) has been found to provide insufficient protection from infection as a
monovalent recombinant vaccine (Herr et al. 2007). However, it has been shown that a
recombinant vaccine of the Coccidioides paralogs Prp1 and Prp2 (divalent vaccine)
provides better protection than either protein alone (Herr et al. 2007). It is believed that
this improved performance is due to host exposure to a more heterogeneous set of T-cell
epitopes which enhances the Th1 response to infection and provides a more robust and
sustained immune response. Searching the C. posadasii genome for additional Prp
homologs identifies six additional genes in this family, which were subjected to the
ProPred algorithm to predict the presence of MHC class II epitopes. Of these six
additional Prp paralogs, Prp5 is the most interesting because it contains the highest
number of promiscuous T-cell epitopes, which bind to numerous histocompatibility
alleles (Table 5, SI8). In addition, Prp 5 shows the highest reactivity in IFN-gamma
ELISPOT assays (SI9). As the best-predicted vaccine candidate, it is proposed for
inclusion in a trivalent vaccine (Prp1 + Prp2 + Prp5) against coccidioidomycosis.
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Discussion
To gain insight into the biology and evolution of the primary human pathogens
Coccidioides immitis and C. posadasii, we sequenced the genomes of these two species
in addition to a nonpathogenic relative, Uncinocarpus reesii, and a more diverged
pathogen from the Onygenales, Histoplasma capsulatum, and compared these genomes to
those of various Pezizomycotina. Specifically, gene family size changes, lineage specific
gene gain and loss, non-syntenic chromosomal regions and proteins subjected to positive
selection were identified through the approach. This investigation improves our
understanding of the Coccidioides life history, by identifying genes important to
adaptation and possibly to virulence, both of which provide insight into how these fungi
evolved a close association with animals and a pathogenic phenotype.
The genomes of the Onygenales are similar in structure to other filamentous
Ascomycete genomes. For example, most Ascomycete genomes have between five and
seven chromosomes. Based on an optical map and whole genome alignments, we predict
that the Coccidioides species have five chromosomes. In addition, the size of the
Onygenales genomes (22-33 Mb) is similar to the Eurotiales A. nidulans (30Mb), A.
fumigatus (29 Mb) and A. terreus (29Mb). However, both orders possess smaller
genomes than the well-characterized Sordariomycetes N. crassa (39 Mb), F.
graminearum (36 Mb), and M. grisea (42 Mb), possibly indicating that genome size may
correlate more strongly with phylogenetic signal than with lifestyle. Furthermore, as
observed in other fungal lineages (Cuomo et al. 2007; Fedorova et al. 2008), the
Coccidioides genomes contain species specific ‘genomic islands’, many of which are
located in chromosomal sub-telomeric regions. Interestingly, eight of these islands
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occupy identical syntenic positions in C. posadasii and C. immitis, suggesting that they
originate through nonrandom processes.
Some filamentous fungi are subject to RIP (repeat induced point-mutation), a
process that detects duplicated DNA and introduces C:G to T:A transitions into the
repeats (Selker and Garrett 1988) (Montiel et al. 2006). Experimental observations in the
Sordariomycete fungi Neurospora crassa (Selker and Garrett 1988), Fusarium
graminearum (Cuomo et al. 2007) and Magnaporthe grisea (Ikeda et al. 2002) suggest
that the RIP process preferentially targets CpA dinucleotides, though additional
dinucleotides may be mutated (Galagan and Selker 2004). Consequently, RIP produces a
signature of biased dinucleotide frequencies, most notably a paucity of CpA
dinucleotides. A RIP-like signature was observed in the Afut1 transposon of the
Eurotiomycete A. fumigatus in such that CpG dinucleotides exhibited a biased transition
frequency (Neuveglise et al. 1996). However, subsequent work identified additional A.
fumigatus transposons exhibiting biased CpA frequencies, indicating that if RIP occurs in
A. fumigatus, at least some repeats are subject to preferential CpA mutation (Gardiner
and Howlett 2006). An analysis of C. posadasii crypton transposons identified the same
Afut1 RIP-like mutational signature in these transposable elements (Goodwin et al. 2003).
We surveyed the Coccidioides genomes for biased dinucleotide frequencies and observed
a genome-wide correlation between a paucity of CpG dinucleotides and repetitive content
(Figure 1). While such a correlation is suggestive of RIP, because its canonical signature
of a skewed CpA distribution is not observed, we cannot strongly conclude that RIP, at
least as it is defined for Sordariomycetes, has shaped the evolution of Coccidioides
repeats. The repeat associated CpG bias observed in Coccidioides and A. fumigatus
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could be the result of a wholly different mutational process or RIP has evolved a
heightened preference for CpG dinucleotides as a mutational target in these fungi.
Comparing genomes across the Pezizomycotina enables investigation of
substantial gene family size changes in Coccidioides and related fungi. In Coccidioides,
families of plant cell wall degrading enzymes are reduced in number compared to those
of fungi that are known to decay plant matter. In contrast to the Aspergillus species, for
example, Coccidioides exhibits few or no cellulases, cutinases, tannases, or
pectinesterases, and exhibits reductions in numbers of genes involved in sugar
metabolism. Coupled with these losses in plant degrading enzymes are expansions of
protease families in Coccidioides relative to other filamentous Ascomycetes, the most
notable being keratinase. Taken together, these gene family changes support our
speculation that Coccidioides species are not typical soil fungi in that they maintain a
close association with keratin-rich animals both during infection of a living host and after
the host has died by growing through the carcass as mycelium. Additional evidence is
found in the relative paucity of dehydrogenases and HET proteins in Coccidioides
relative to Aspergillus, which suggests, respectively, that Coccidioides utilizes fewer
metabolites and encounters fewer genetically distinct individuals than their soil saprobe
counterparts. Our speculation is consistent with previous research showing an
association of Coccidioides with living animals (Emmons 1942), the difficulty of
recovering Coccidioides species from soil, and the narrowly patchy distribution of
Coccidioides in soil (Maddy 1967) (Green et al. 2000).
The gene family size changes observed in Coccidioides are paralleled in U. reesii,
indicating that adaptation by gene family expansion or contraction related to the shift
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from plant to animal substrate must have occurred before the divergence of these two
taxa. However, it was a surprise that another pathogenic member of the Onygenales, H.
capsulatum, did not show the same gene family changes. Histoplasma exhibits the same
paucity of genes that code for proteins that decay plant material, but does not share the
expansion of proteases observed in Coccidioides. Although Coccidioides and
Histoplasma are both associated with living mammals and can cause systemic disease in
humans, their niche is different. H. capsulatum is associated with bird and bat guano,
and perhaps, unlike Coccidioides, this fungus is not equipped to survive in dead hosts but
rather inhabits their excrement. If correct, this thinking suggests that adaptation to
remain associated with dead hosts evolved after the divergence of H. capsulatum from
the Coccidioides/Uncinocarpus lineage.
The similarities in gene family size between the Coccidioides species and U.
reesii suggest that gene family expansion and contraction alone are not responsible for
the pathogenic phenotype observed in Coccidioides. Although U. reesii can grow on
animal tissue and can be cultured from animal organs and hair (Sigler and Carmichael
1976), this organism has never been known to cause disease, cannot be grown at 37º C
and, to our knowledge, only grows in a filamentous morphology. Therefore, genomic
differences other than gene family size must account for the phenotypic differences
observed between Coccidioides and U. reesii. The comparative analyses we have
performed point to several interesting candidates for this role.
First, we identified metabolism related proteins gained in Coccidioides that may
underlie its infectious life history. For example, following divergence from U. reesii,
Coccidioides species either acquired or retained a suite of genes enriched for several
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functions, notably including heme/tetrapyrrole binding, an attribute of a class of proteins
shown to be important for scavenging iron within a host (Howard 1999). In addition, the
set of Coccidioides-specific genes with spherule-specific expression are enriched for
metabolism-related functions such as the generation of precursor metabolites and energy.
Included in this set is a key enzyme involved in allantoin metabolism, a nitrogen rich
compound utilized by some pathogens when nitrogen is limited (Divon and Fluhr 2007).
These findings suggest that Coccidioides has acquired or retained genes that enable the
fungus to grow on metabolites found within a host, such as iron and nitrogen.
Second, we find evidence that membrane-related genes in the Coccidioides
lineage may also contribute to its infectious phenotype. For example, the genes uniquely
gained along the Coccidioides lineage are enriched for integral membrane function (P =
1.7e-4). Of these Coccidioides specific genes that are also uniquely expressed in the
parasitic morphology, roughly one-third exhibit signal peptides or signal anchors,
indicating they may be involved in cell surface or membrane biology. In addition, one of
the proteins found to be rapidly evolving in the Coccidioides lineage is a lipase, which is
a member of a protein family implicated in several fungal virulence mechanisms
involving the cell membrane. This finding suggests that lipases may be important to the
adaptation of Coccidioides, possibly by contributing to the development of the
membrane-rich endosporulating spherules, the morphology required for disseminated
infection. Also, because morphological differentiation requires transcriptional alteration
and because the endospore producing parasitic morphology is unique to Coccidioides,
some of its recently acquired transcription factors may contribute to the development of
endospores in spherules. Because mutations that prevent endospore formation are
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avirulent, proteins involved in differentiation of the cell wall and membrane associated
with endospore formation may warrant further consideration as potential drug targets.
Finally, the results suggest that secondary metabolism has contributed to the
evolution of virulence in Coccidioides. A gene that shares homology to the Aspergillus
fumigatus mycotoxin Asp-hemolysin was either gained or retained in Coccidioides
relative to other Onygenales and, based on our analysis of ESTs obtained from spherules
and mycelium, is uniquely expressed in the pathogenic morphology. In addition, we
identify a functional enrichment of S-adenosylmethionine-dependent methyltranserase
activity among proteins found to be rapidly, or adaptively, evolving in Coccidioides. In
A. flavus and A. parasiticus, this function is responsible for the penultimate step in
aflatoxin biosynthesis. While experimental verification is required, these findings
suggest that Coccidioides may produce secondary metabolites, such as mycotoxins, that
facilitate the infection process.
Taken together, these findings suggest a time line for the evolution of the
pathogenic phenotype of Coccidioides (Figure 7). The first step in this evolutionary
transition was the shift from a nutritional association with plants to animals, an event that
occurred before Coccidioides and U. reesii diverged and is revealed through our analysis
of gene family size. Because U. reesii and other closely related fungi grow in association
with animals but are not known to cause disease, it is unlikely that this nutritional
association directly yielded virulence. Rather, the common ancestor of Coccidioides and
U. reesii likely grew on dead or decaying animal matter or on external surfaces of an
animal host, such as hair. However, once this association with animals was established,
ongoing lineage specific evolution in Coccidioides – most notably in metabolism,
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membrane and mycotoxin genes – enabled the adaptation to survive and grow within an
animal host, ultimately yielding disease. The two Coccidioides species diverged
approximately 5.7 MYA, well before humans arrived in the New World (Pitulko et al.
2004), so this evolutionary transition to a pathogenic phenotype likely involved primarily
rodent hosts and not humans.
An alternative hypothesis for the subsequent differentiation between Coccidioides
and U. reesii is that a rapid evolutionary burst may have yielded a relatively immediate
pathogenic phenotype in Coccidioides. However, historical observations challenge this
hypothesis and suggest that pathogenesis in Coccidioides may be an emerging phenotype.
While many isolates suggest that Coccidioides individuals are generally primary
pathogens, most of these experimental strains of Coccidioides have been isolated via
dead animal intermediates (Elconin et al. 1964; Green et al. 2000). Thus the isolate
collection process biases the strains available for analysis. In vivo, Coccidioides often
forms granulomas within a host’s lung tissue, whereby the fungus is walled-off within the
host and neither the host nor pathogen are killed. Work by Emmons suggests that this
occurs frequently in what is likely the major host reservoir for Coccidioides: Kangaroo
Rats (Dipodomys) and Pocket Mice (Perognathus) (Emmons 1942). So while some
strains of Coccidioides are capable of growing within and killing a host, the predominant
strategy of most strains may be to 'sit' as a granuloma and 'wait' for an optimal time, such
as suppression of the immune system or host death, to colonize the animal tissue.
Ongoing adaptation within Coccidioides may have yielded the virulent and hypervirulent
strains capable of circumventing the formation of granulomas in the host.
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In summary, we speculate that the pathogenic phenotype of Coccidioides is the
result of adaptive changes to existing genes and the acquisition of small numbers of new
genes that began with a shift from plant-based to animal-based nutrition. Though the
hierarchical comparative genomics methodology presented here identifies significant
evolutionary changes in the Coccidioides lineage, improving the phylogenetic resolution
during relatively recent Coccidioides evolution and the practical knowledge regarding its
functional genetics and ecology would facilitate a superior understanding of the
organisms’ life history and evolution. Future studies that engage in population level
analyses within Coccidioides, that compare Coccidioides to the genomes of other closely
related, non-pathogenic Onygenales, and that clarify the functional genetics and ecology
of Coccidioides will be necessary to test the hypotheses that we have proposed and, thus,
improve our knowledge regarding the evolutionary origins and treatment of
coccidioidomycosis.
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Materials and Methods
Genome Sequences and Annotations Utilized
Genome sequence at 8X coverage was generated via Sanger shotgun sequencing
and annotated by the J. Craig Venter Institute (formerly The Institute for Genome
Research) for Coccidioides posadasii strain C735∆SOWgp (March 2006 release,
http://www.tigr.org/tdb/e2k1/cpa1/). Genome annotation was conducted using a multitiered approach. First, the AAT alignment package identified genome sequence
homologous to proteins in the NCBI non-redundant database (NR) and custom fungal
protein databases. In addition, the ab-initio gene prediction tools tigrscans and
glimmerHMM were trained via high confidence, manually curated gene models and used
to predict genes. Finally, 53,664 ESTs, 26,201 of which were derived from mycelium
and 27,463 of which were derived from spherules (SI10), were assembled and aligned to
the genome using PASA (Haas et al. 2003). Combiner subsequently used this evidence
to produce a final set of annotation predictions (Allen et al. 2004).
Predicted C. posadasii proteins were functionally annotated via comparison to the
following protein, domain and profile databases: NR, Pfam, TIGRfam HMMs, Prosite
and Interpro. In addition, SignalP, TMHMM and TargetP were used to predict protein
membrane localization. Where appropriate, predicted proteins were first categorized into
JCVI’s domain based paralogous families whereby the annotation prediction could be
performed in the context of related genes.
The Coccidioides immitis strain RS (June 2008, AAEC02000000), Uncinocarpus
reesii strain 1704 (May 2006 release,
http://www.broad.mit.edu/annotation/genome/uncinocarpus_reesii/), and the Histoplasma
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capsulatum strain WU24 (May 2006; AAJI01000000) genome sequence and annotations
were generated by the Broad Institute as described in (Galagan et al. 2003). All three
genomes were produced using Sanger shotgun sequencing. The U. reesii and H.
capsulatum annotations were generated using automated pipelines. The C. immitis
annotation employed ESTs for gene model construction and was manually reviewed. A
total of 65,754 ESTs were sequenced for C. immitis RS, including 22,382 mycelia ESTs
and 43,372 spherule ESTs constructed from stage-specific genetic template. Sequence
coverage levels for C. immitis, U. reesii, and H. capsulatum were respectively 14.4X, 5X,
and 7X.
For the purposes of identifying phylogenetic relationships between taxa, the abinitio gene annotation tool AUGUSTUS was used to predict genes in the following
genome sequences: Aspergillus flavus strain NRRL3357 (AAIH00000000), Blastomyces
dermatitidis strain ATCC 26199
(http://genome.wustl.edu/pub/organism/Fungi/Blastomyces_dermatitidis/assembly/Blasto
myces_dermatitidis-3.0/), Histoplasma capsulatum strain 186AR
(http://genome.wustl.edu/pub/organism/Fungi/Histoplasma_capsulatum/assembly/Histopl
asma_capsulatum_G186AR-3.0/), H. capsulatum strain 217B
(http://genome.wustl.edu/pub/organism/Fungi/Histoplasma_capsulatum/assembly/Histopl
asma_capsulatum_G217B-3.0/), Paracoccidioides brasiliensis strain pb03
(http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis/MultiHome.
html), and Penicillium marneffei strain ATCC 18224 (ABAR00000000). Genome
sequence and annotation data for Aspergillus clavatus strain NRRL (AAKD00000000),
A. fumigatus strain Af293 (Nierman et al. 2005), A. nidulans strain A4 (Galagan et al.
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2005), A. niger strain CBS51388 (Pel et al. 2007), A. oryzae strain RIB40 (Machida et al.
2005), A. terreus strain NIH2642 (AABT01000000), Fusarium graminearum strain PH-1
(Cuomo et al. 2007), Neurospora crassa strain OR74A (Galagan et al. 2003) and
Sclerotinia sclerotiorum strain 1980 (AAGT01000000) were downloaded from GenBank.
C. posadasii Optical Map Generation
Genomic DNA was prepared in agarose plugs using a modification of the Schartz
protocol (Pan and Cole 1992a; Schwartz and Cantor 1984). The mycelial phase of C.
posadasii C735 was grown in liquid medium which contained 1% glucose plus 0.5%
yeast extract (GYE) at 30°C for 48 h in a gyratory shaker-incubator. Approximately 40
mg (wet weight) of mycelia was resuspended in 1.0 ml of 125 mM EDTA-50 mM
sodium citrate to which 10 mg of chitinase (200 to 600 units of activity per g; Sigma) was
added. The suspension was immediately mixed with 1.5 ml of molten 1% low-meltingpoint agarose (at 55°C in 125 mM EDTA-50 mM sodium citrate), which was added to
plug molds and cooled to 4°C for 20 min. Spheroplasts were produced in the agarose by
incubation of the plugs in 50 mM sodium citrate (pH 5.7)-0.4 M EDTA (pH 8.0)-1% 2mercaptoethanol at 37°C for 24 h. The plugs were then placed in fresh solution as
described above and incubated under the same conditions for an additional 24 h. To lyse
the spheroplasts, plugs were washed three times with 50 mM EDTA (pH 8.0) and then
incubated in NDS buffer (0.5 M EDTA [pH 8.0], 10 mM Tris-HCl [pH 9.5], 1%
lauroylsarcosine) which contained 2 mg of proteinase K per ml at 50°C for 24 h. The
plugs were subsequently rinsed three times with 50 mM EDTA (pH 8.0) and stored at
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4°C in 0.5 M EDTA. The plugs were shipped to OpGen, Inc. (Madison, WI) where a
MluI optical restriction map (Schwartz et al. 1993) was prepared.
Ortholog Detection and Alignment
Several orthology datasets were generated including 18-way clusters among the
Pezizomycotina, four-way orthology clusters between Onygenalean fungi C. immitis, C.
posadasii, U. reesii and H. capsulatum, three-way clusters between C. immitis, C.
posadasii and U. reesii and pairwise orthologs between the two Coccidioides species.
For each dataset, all predicted protein sequences from the appropriate genomes were
searched against each other with BLASTP and clustered into orthologous groups using
OrthoMCL (Li et al. 2003) with the following criteria: the alignment must cover 60% of
the query sequence and the E-value must be less than 10-5. Single copy orthologs for use
in the estimation of rates of molecular evolution were identified as the clusters with
exactly one member per species. Multiple sequence alignments of ortholog clusters were
constructed with PROBCONS (Do et al. 2005) and MUSCLE (Edgar 2004).
Phylogenetic Analysis
Phylogenetic relationships were determined for the following 18 taxa: C. immitis,
C. posadasii, U. reesii, H. capsulatum (strains 186R, 217B and WU24), Blastomyces
dermatitidis, Paracoccidioides brasiliensis, Aspergillus fumigatus, A. clavatus, A. flavus,
A. niger, A. oryzae, A. terreus, A. nidulans, Penicillium marneffei, Sclerotinia
sclerotiorum and Fusarium graminearum. Gene sequences were selected by identifying
single-copy orthologs from the OrthoMCL clusters. Clusters were aligned with
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ProbCons, alignments were pruned with Gblocks (Castresana 2000), and data were
combined into a single alignment file containing 823,457 characters. Eighteen random
sub-selections were chosen without replacement and used to construct phylogenetic trees
with three methods: Neighbor-Joining (NJTREE), Bayesian with MrBayes (Ronquist and
Huelsenbeck 2003), and Maximum Likelihood (ML) with RAxML(Stamatakis et al.
2005).
Divergence time estimation was performed using Nonparametric rate smoothing
(NPRS) (Sanderson 2003) with the ML tree as a starting point and calibration of 215
million years for the Pezizomycotina origin.
Analysis of repeat-content
To estimate the amount of repetitive sequence in each genome in the absence of a
curated repeat library, we divided each genome into 1kb non-overlapping windows and
used BLASTN to align each window against its respective genome. The number of
matches greater than 500bp in length was totaled for each window. Repetitive sequence
was partially characterized by searching each window against the Repbase Fungal repeat
database using TBLASTN and a e-value cutoff of 1x10-2. Repeats were subsequently
divided into three frequency classes: small (n < 6), medium (n = 6-20) and large (n > 20).
Chromosome Structure Analysis
Whole genome alignments were generated with the MUMmer package (Delcher
et al. 2002). The C. immitis genome was used as the reference in all alignments due to its
depth of coverage and high quality assembly. Because of the differing phylogenetic
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distances between the species being compared, NUCMER was used to identify syntenic
regions between C. immitis and C. posadasii while PROMER was used between C.
immitis and U. reesii.
Gene Family Expansion and Contraction Analysis
To identify changes in the size of gene families, we determined the number of
copies of genes in sequenced genomes of the following Pezizomycetes: Aspergillus
terreus, A. oryzea, A. niger, A. fumigatus, A. nidulans, C. posadasii, C. immitis, U. reesii,
H. capsulatum, Stagonospora nodorum, Chaetomium globosum, Podospora anserina,
Neurospora crassa, Magnaporthe grisea, Fusarium graminearum, F. verticillioides, and
F. solani. For each genome sequence, Pfam protein domains (Finn et al. 2008) were
identified via HMMER using a cutoff value of E < 10-2and the number of genes
containing at least one copy of each Pfam protein domain was determined. Using a
multigene phylogeny, gene family sizes were associated with each taxon to determine the
magnitude of gene family expansions and contractions.
Detection of Positive Selection
To identify genes subject to positive selection since the divergence of C. immitis
and C. posadasii, we compared the nonsynonymous substitution rate (dN) to the
synonymous substitution rate (dS) for all orthologous coding sequence pairs (ω = dN/dS).
A gene with ω = 1.0 is considered to be evolving neutrally. Values of ω significantly less
than 1 indicate purifying selection while values significantly greater than 1.0 are
consistent with positive directional selection. The value for ω was estimated using
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PAML (v3.15) (Yang 1997) under two evolutionary models: a null model where ω was
fixed (ω = 1) and another where ω was unconstrained and estimated from the data. For
each sequence pair, the maximum likelihood values generated for each model were
subject to a likelihood ratio test to identify orthologs for which the null hypothesis,
ω=1.0, could be rejected (p < 0.05). Genes under positive selection were identified as
ortholog pairs where ω is significantly greater than 1 after correcting for multiple tests
using Q-value (p < 0.05) (Storey and Tibshirani 2003). Processing of alignments, trees,
and PAML results was handled through Perl scripts utilizing the modules in the BioPerl
package (Stajich et al. 2002).
Relative Rates Test
A maximum likelihood approach was used to identify differences in the protein
substitution rate between orthologs in clusters containing single representatives from C.
immitis, C. posadasii, U. reesii, and H. capsulatum. Using H. capsulatum as the root,
protein phylogenies were constructed and substitution rates were estimated using the M0
model in PAML. The substitution rates were used to calculate the number of derived
mutations in the Coccidioides and U. reesii lineages since the taxa diverged. A
chi-square approximation was then used to determine if any two lineages displayed
significantly different substitution rates (p<0.05), after accounting for multiple testing via
a Bonferroni correction.
GO Enrichment Analysis
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Pfam protein domains were identified as described above and used to assign
proteins to GO categories via PFAM2GO data provided by the GO consortium
(Ashburner et al. 2000). Proteins with multiple domains corresponding to different
functions were allowed to belong to multiple GO categories. A particular GO function
was considered enriched if its representation among a selected subset of a taxon’s genes
is significantly greater than its representation among all genes in that taxon’s genome
(p<0.05 after a Bonferroni multiple test correction).
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Figures
Figure 1: Evidence of mutational bias associated with repetitive regions of
the Coccidioides genomes.
For both the C. immitis and C. posadasii genomes, each 1kb non-overlapping window of
sequence was categorized into a repeat (Rpt) class. Classification is based on the number
of homologous copies (n) of that window identified within the genome: non-repetitive,
low (n<6), mid (n=6-20), and high (n>20). The mean GC percentage, the mean CpG
frequency and the fraction of elements with a CpG frequency less than 0.010 was
measured for each repeat class. The results indicate that repeats in Coccidioides tend to
exhibit a paucity of CpG dinucleotides compared to non-repetitive regions.
Figure 2: Pezizomycotina Divergence Times
A RAxML generated maximum likelihood phylogeny based on an alignment of 1,148
concatenated genes that comprise 46,890 unambiguously aligned amino acid sites
following the removal of gaps (SI9) was used as a starting point to perform
nonparametric rate smoothing. Divergence times were subsequently estimated from the
smoothed phylogeny by applying a calibration of 215 MYA for the Pezizomycotina
origin as in (Taylor and Berbee 2006).
Figure 3: The Evolution of Gene Family Size in the Eurotiomycetes
Gene family size evolution was evaluated across seven Eurotiomycetes and two
Sordariomycete out groups. The columns capture the size of selected gene families across
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the taxa with italicized numbers representing gene family contractions and bold numbers
representing expansions (specific information regarding gene family function can be
found in the main text). Taxa are listed on the left-hand side according to their
phylogenetic orientation and represented by four letter identifiers - Anid: Aspergillus
nidulans; Afum: A. fumigatus; Ater: A. terreus; Hcap: Histoplasma capsulatum; Uree:
Uncinocarpus reesii; Cimm: Coccidioides immitis; Cpos: Coccidioides posadasii; Ncra:
Neurospora crassa; Fgra: Fusarium graminearum.
Figure 4: Lineage specific gene gain and loss across the Onygenales
The species phylogeny illustrates the number of gene gains (blue, +) and gene losses (red,
-) that occurred along specific lineages. Orthologous protein sequences from the C.
immitis, C. posadasii, U. reesii and H. capsulatum genomes were clustered using
OrthoMCL. Parsimony analysis of each cluster’s phylogenetic profile was used to infer
gene gains and gene losses. Coccidioides lineage gene gains vary depending on whether
the C. posadasii or C. immitis annotation is used (280 and 291 respectively). Changes in
the H. capsulatum lineage cannot be inferred as gain or loss due to the lack of an
outgroup.
Figure 5: Neutral mutation rates of conserved C. immitis, C. posadasii, and U. reesii
genes
Each histogram represents the synonymous substitution rate (dS) for each pair of
orthologs shared between the two species in question. Median values are indicated via
vertical red lines. A) A histogram of dS calculated from 5,820 orthologs conserved
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RUNNING TITLE: Comparative Genomics of Coccidioides
between C. immitis and U. reesii. The estimated median value (1.60) indicates that the
taxa are too highly diverged to identify the selection profile via dN/dS analysis. B) A
similar histogram calculated from 6,763 orthologs shared between C. immitis and C.
posadasii (median = 0.023).
Figure 6: Distribution of dN/dS values for all pairwise orthologs between C. immitis and
C. posadasii
Histograms illustrating the dN/dS value for each of the 6,763 pairwise orthologs
conserved between the C. immitis and C. posadasii genomes. The median value of 0.25
is indicated with a vertical red line and suggests that most genes have evolved under
purifying selection. The plot is bounded by dN/dS = 5, so all greater values are
represented in the right hand most bin.
Figure 7: A Timeline of genomic changes that underlie the evolution of pathogenecity in
Coccidioides
The significant evolutionary events identified in the hierarchical comparative genomic
analysis of the Coccidioides lineage are overlaid onto the Eurotiomycetes species
phylogeny. Taken together, they suggest that a gradual progression of evolutionary
events ultimately yielded the pathogenic phenotype of Coccidioides. A) Gene family
reductions associated with the metabolism of plant material were coupled with a growth
substrate transition from plant to animal material. B) Subsequent expansions in proteases
and keritinases led to a nutritional association with mammals in a non-pathogenic
fashion. C) The acquisition and adaptation of genes involved in metabolism, membrane
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biology and mycotoxin production led to metabolic and morphological phenotypes that
enabled survival within a live host, ultimately resulting in disease. D) Secreted proteins,
metabolism genes and secondary metabolism genes that are shared between the two
Coccidioides species have been subject to positive selection since they diverged. This
suggests that Coccidioides experiences on-going adaptation in response to host immune
system selection pressures.
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Table Titles
Table 1: Genome Statistics for Sequenced Onygenales
Table 2: Distribution of Repetitive Genomic Content
Table 3: Rapidly evolving Coccidioides proteins relative to U. reesii orthologs
Table 4: Pairwise Orthologs between C. immitis and C. posadasii with Ka/Ks >> 1
Table 5: Human MHC class II binding epitope prediction of C. posadasii PRP proteins
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