Teixeira et al. BMC Genomics 2014, 15:943
http://www.biomedcentral.com/1471-2164/15/943
RESEARCH ARTICLE
Open Access
Comparative genomics of the major fungal agents
of human and animal Sporotrichosis: Sporothrix
schenckii and Sporothrix brasiliensis
Marcus M Teixeira1, Luiz GP de Almeida2, Paula Kubitschek-Barreira3, Fernanda L Alves4,5, Érika S Kioshima1,6,
Ana KR Abadio1, Larissa Fernandes7, Lorena S Derengowski1, Karen S Ferreira8, Rangel C Souza2, Jeronimo C Ruiz5,
Nathalia C de Andrade3, Hugo C Paes1, André M Nicola9,10, Patrícia Albuquerque1,10, Alexandra L Gerber2,
Vicente P Martins1, Luisa DF Peconick1, Alan Viggiano Neto1, Claudia B Chaucanez1, Patrícia A Silva1,
Oberdan L Cunha2, Fabiana FM de Oliveira1, Tayná C dos Santos1, Amanda LN Barros1, Marco A Soares4,
Luciana M de Oliveira4,11, Marjorie M Marini12, Héctor Villalobos-Duno13, Marcel ML Cunha3, Sybren de Hoog14,
José F da Silveira12, Bernard Henrissat15, Gustavo A Niño-Vega13, Patrícia S Cisalpino5, Héctor M Mora-Montes16,
Sandro R Almeida17, Jason E Stajich18, Leila M Lopes-Bezerra3, Ana TR Vasconcelos2 and Maria SS Felipe1,9*
Abstract
Background: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species,
S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses
are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the
presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and
infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other
human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes.
Results: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total
of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic
showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA
suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii
and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive
elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I
intron encoding homing endonucleases (HE’s) exclusively in S. brasiliensis. Analysis of protein family expansions and
contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1
and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was
observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary
adaptations from a plant pathogenic or saprobic to an animal pathogenic life style.
(Continued on next page)
* Correspondence: msueliunb@gmail.com
1
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil
9
Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade
Católica de Brasília, Brasília, DF, Brazil
Full list of author information is available at the end of the article
© 2014 Teixeira et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
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(Continued from previous page)
Conclusions: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plantassociation to mammalian parasitism, which contributes to the understanding of how environmental interactions
may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi
revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.
Keywords: Sporothrix schenckii, Sporothrix brasiliensis, Comparative genomics, Fungal evolution
Background
The fungal genus Sporothrix includes about 60 species
found on all inhabited continents mainly occurring as
environmental saprobes, living in association with plants
or decaying matter. One lineage within the genus is
composed of at least four pathogenic species associated
with human and animal sporotrichosis: Sporothrix schenckii
sensu stricto, S. brasiliensis, S. globosa, and S. luriei [1-6].
Subcutaneous infections caused by S. schenckii are globally
endemic [1,2]. Additionally, outbreaks have been described
from South Africa, Australia, China and India [7-10].
During the last two decades, an ongoing zoonotic outbreak of sporotrichosis has been observed in Brazil.
Initially thought to be caused by Sporothrix schenckii,
detailed studies demonstrated that most outbreak isolates were actually S. brasiliensis [6].
Sporothricosis is classically associated with rural activities such as agriculture, floriculture or hunting, but
more recently felines have emerged as source of human
infection. The most common clinical form is a chronic
subcutaneous/lymphocutaneous disease acquired after
inoculation of fungal material into the skin. Extracutaneous and disseminated forms secondary to cutaneous
infection have been described in patients who are immunocompromised as a result of AIDS, chronic alcoholism and diabetes [11]. Rarely, severe cases involving
pulmonary infection are noted [3,4].
The pathogens of the genus Sporothrix exhibit a thermodimorphic phenotype: in its saprophytic stage or in in vitro
culture at 25°C the fungus grows with its filamentous form
characterized by hyaline, septate hyphae with sympodial
conidiogenous cells that produce two types of spores:
hyaline conidia that form clusters and brown, thickwalled spores that are distributed perpendicularly alongside the hyphae. During the parasitic stage, the fungus is
found as cigar-shaped yeast cells that can also be obtained
in vitro by switching the temperature to 37°C [12]. This
dimorphism is essential for virulence in the mammalian
host [13,14] and is also found in other human pathogenic fungi such as Blastomyces dermatitidis, B. gilchristii,
Histoplasma capsulatum, Paracoccidioides brasiliensis, P.
lutzii, Coccidioides immitis and C. posadasi. However, all
of these other dimorphic fungi are members of the order
Onygenales, phylogenetically distant from Sporothrix in
the Ophiostomatales [15]. The long genetic distance between these two orders suggests that thermo-dimorphism
is a convergent phenotype shared by only a few members
of these two orders. Genes such as histidine kinase (drk1)
regulates the transition of mycelium to yeast and consequently the maintenance of virulence in B. dermatitidis
and H. capsulatum [16]. This gene, also identified in
S. schenckii, shows 65% of identity with its ortholog in
B.dermatitidis and seems to be highly expressed during
the yeast stage [17].
Besides dimorphism and thermo-tolerance, current
knowledge about virulence factors of Sporothrix remains
scant. The cell surface of pathogenic fungi plays a key
role in the host-fungus interplay, mediating various processes associated with pathogenesis. The fungal cell wall
is mainly composed of glycoconjugates: structural polysaccharides such as chitin and β-glucans, and cell wall
glycoproteins [18]. Few proteins and glycoconjugates
have been identified so far in the S. schenckii cell wall
and their relevance for the host-fungus interaction and
stimulation of the host immune system was reinforced
by recent studies [19,20]. However, the identity of the
enzymes involved in biosynthetic pathways of cell wall
components is still lacking. Another cell wall virulence
factor, melanin, was found in S. schenckii conidia and
yeast cells being produced in vitro or in vivo during
infection [21]. Melanin pigments protect the fungus
from the mammalian host’s innate immune responses
providing resistance to oxidizing agents and fungal cell
death during phagocytosis [22,23].
Members of the pathogenic lineage in Sporothrix seem
to behave in the host remarkably different from Ophiostoma species, suggesting a fundamental habitat shift
from a plant- to a mammal-associated life style [24]. Remarkably, most fungi from the order Ophiostomatales
live in association with bark beetles in woody plants, displaying adaptation strategies for insect transmission that
are very different from those of S. schenckii and their relatives [2,25]. The main biological questions of this work
revolve around the dimorphic and pathogenic status of
the two Sporothrix species, which are phenotypically
similar to human/animal pathogenic Onygenales but
philogenetically closely related to plant-associated Sordariomycetes. To address these questions, we performed
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a comparative genomic analysis of the pathogens with 14
other fungi, either dimorphic pathogens or plant-associated
Sordariomycetes. Of these, we chose the closest relative to
the Sporothrix lineage, Grosmannia clavigera, for more
detailed comparison. G. clavigera is a tree-pathogenic and
insect-associated fungus from a related genus from the
Ophiostomatales order [26]. It is a haploid filamentous
Ascomycete and a symbiont of the bark beetle Dendroctonus ponderosae, which affects commercial conifer forests,
parks, protected areas and urban forests across North
America [27]. These genomic analyses allowed us to
identify the core genes for general and secondary metabolism as well genes related to autophagy, adhesion,
cell wall assembly and melanin biosynthetic processes.
We have also shown that genomic adaptation in the
Sporothrix lineage has led to expansion of some protein
domains and lack of genes associated with plant biomass decay when compared to other Sordariomycetes,
which can be interpreted as an adaptation from plant to
an animal associated life style.
Results and discussion
Genomes features, assemblies and synteny
The S. schenckii and S. brasiliensis genomes were each
pyrosequenced to ~20x coverage. The S. schenckii genome (strain 1099–18) yielded 16 scaffolds with N50 of
4.3 Mb, containing 237 contigs comprising a total size of
32.4 Mb. The S. brasiliensis genome (strain 5110) yielded
13 scaffolds with N50 of 3.8 Mb, containing 601 contigs,
had a total genome size of 33.2 Mb, and shared similar
genomic characteristics with G. clavigera [26] (Table 1).
Telomeric repeats (TTAGGG/CCCTAA)n were found at
5’ or 3’ terminal ends of 5 out of 13 scaffolds in the
S. schenckii and 7 out of 13 scaffolds in the S. brasiliensis
genome. Terminal repeats were found in both ends of 1 and
3 scaffolds of S. schenckii and S. brasiliensis respectively,
Table 1 Sporothrix genome characteristics
Characteristic
S. schenckii S. brasiliensis G. clavigera*
Genome size
32.4 Mb
33.2 Mb
29.8 Mb
Coverage
17X
20X
64X
Supercontig number
16
13
18
N50 supercontig
4.3 Mb
3.8 Mb
1.2 Mb
G + C content
62%
62%
53.4%
Protein coding genes
10,293
9,091
8,314
Median Transcript length
1,522 bp
1,602 bp
1,641 bp
Introns per gene
1.0
1.1
1.9
Median Intron length
91.2 bp
123.4 bp
70 bp
Median Intergenic distance 1,530 bp
1,913 bp
1,466 bp
tRNA
140
268
139
*Genomic information collected according previously published G. clavirera
genome [26].
revealing the presence of complete linear chromosomes.
Pair-wise genome alignments showed that both Sporothrix species are highly syntenic sharing 97.5% average
sequence identity (Figure 1A). According to the genomic alignments long inverted segments were found in
the two Sporothrix genomes (Figure 1A-C). S. schenckii
and S. brasiliensis were predicted to have 10,293 and 9,091
protein coding genes respectively, similar to other Eurotiomycetes and Sordariomycetes, and slightly higher than
G. clavigera (Table 1). The G + C content in S. schenckii
and S. brasiliensis genomes is one of the highest in Ascomycota. S. schenckii and S. brasiliensis genomes display
62% of G + C contents in both species, which is considerably higher than G. clavigera (53.4%) [26] and 50–52% in
most other fungi in Pezizomycotina [28]. We detected
similar distributions of transcript lengths, but we found
less introns per gene in Sporothrix genomes than in those
of G. clavigera. The tRNA contents revealed a great discrepancy among the analyzed fungi; G. clavigera harbors
at least 2-fold more tRNAs than Sporothrix genomes
(Table 1). We analyzed the homology relationships
among fungi from the Ophiostomataceae family, comparing the gene content of S. schenckii, S. brasiliensis
and G. clavigera by Bidirectional-best Blast Hits (BBH).
A total of 4,788 genes were found in all three genomes
and 2,001 were found to be Sporothrix-restricted genes,
indicating a high content of specific genes in the Sporothrix lineage. A total of 1,549 and 508 genes were
considered orphan sequences in S. schenckii and S. brasiliensis, respectively (Figure 1B). We have performed
the comparative analysis of core genes involved in general
and secondary metabolism, as well genes involved in
transport and catabolism showing a high degree of
conservation when compared to those present in other
Ascomycetes (Additional file 1: Text 1). Genomes from
S. schenckii and S. brasiliensis were deposited in the Genbank under respectively accession numbers: AXCR00000000
and AWTV00000000.
Phylogenomic analysis
A total of 395 orthologous protein clusters were identified by BBH after searching 25 fungal genomes, including Ascomycetes, Basidiomycetes and Chytridiomycetes
(Additional file 2: Table S1). A Maximum Likelihood
phylogenomic tree was generated using a 153,436 amino
acids position alignment and calibrated with the origin
of Ascomycota clade around 500–650 MYA. The phylogenomic tree, as expected, placed S. schenckii and
S. brasiliensis in a monophyletic clade closest to G. clavigera being apart from other Sordariomycetes (Figure 2).
According to the phylogenomic tree, S. schenckii and
S. brasiliensis diverged about 3.8-4.9 MYA suggesting a
recent event of speciation in the genus Sporothrix.
Additionally, evolutionary origin of the ophiostomatoid
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A
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B
C
Figure 1 Genomic alignments, synteny and homology of S. schenckii and S. brasiliensis. A) Dot-plot of S. schenckii and S. brasiliensis using
ordered scaffold sequences. B) Predicted proteins in S. schenckii and S. brasiliensis were compared with the predicted proteins of G. clavigera. The
Venn diagram was built using minimum query/subject coverage of 50% and e-value of E ≤ 1×10−20. C) Genomic alignments of S. schenckii (bottom)
and S. brasiliensis (top) showing chromosomal inversions in the genomes of these pathogens.
fungi was dated to 69.1-89.9 MYA, being highly divergent from the plant pathogen G. clavigera (Figure 2).
The divergence time varied across sister species of fungal pathogens along the Ascomycota phylum, such as C.
immitis vs. C. posadasii diverged about 5.1 Mya [29]
and P. brasiliensis vs. P. lutzii about 11 to 32 Mya [30].
Mitochondrial genomic comparisons
The mitochondrial genome assembly of S. schenckii strain
1099–18 is 26.5 Kb in size and shares 99-100% average
sequence identity and 97-100% coverage in comparison to
that of previously published S. schenckii strains ATCC
10268 (AB568599) and KMU2052 (AB568600) (data not
shown). The mitochondrial genome assembly of S. brasiliensis strain 5110 spans 36 Kb but covers only 71-75% of
the three S. schenckii mentioned genomes before. Despite
the high similarity between the two analyzed mitochondrial genomes (99% of identity), S. brasiliensis harbors
parasitic group-I intron encoding homing endonucleases
(HE’s) which is responsible for the higher mitochondrial
genome size in this species. Those elements were detected
in the cytochrome C oxidase 1, ATP synthase subunit 6
and between NADH dehydrogenase subunits 2 and 3
ORF’s (Figure 3). These HE’s found in the S. brasiliensis
mitochondrial genome were classified into two families
according Interpro domain screening: LAGLIDADG
and GIY-YIG. Mitochondrial LAGLIDADG HE’s from S.
brasiliensis (SPBR09268, SPBR09281 and SPBR09282)
shared 75%, 84% and 78% of identity to Madurella
mycetomatis (YP_006576197), Fusarium graminearum
(YP_001249331) and F. solani (YP_005088115), respectively.
The S. brasiliensis mitochondrial GIY-YIG HE (SPBR09426
and SPBR09429) is highly conserved among other
Sordarimycetes, sharing 72% and 77% of identity to Podospora anserina (NP_074919) and Ceratocystis cacofunesta
(YP_007507073), respectively . C. cacofunesta contains 37
intronic ORFs, thus being responsible for one of the largest mitochondrial genomes among Sordariomycetes [31].
Fungal mitochondrial genomes present a constant genetic
mobility, probably due to the activity of group-I intron
encoding homing endonucleases. Mitochondrial introns
and their ORFs have been associated with mitochondrial
parasitism and genomic size changes thus causing genomic instability, which was reported before in S. cereviseae, P. anserina, Neurospora crassa, Ophiostoma and
Aspergillus [32-36]. According to the phylogenetic tree,
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Figure 2 Phylogenomic relationships of Sporothrix species and other thermodimorphic fungal pathogens. The phylogenetic tree was
constructed from an alignment of 153,436 amino acids of 395 orthologous protein clusters. The tree was inferred by Maximum Likelihood method
implemented in RAxML and Dayhoff amino acid substitution was used as the best protein substitution model. The tree was calibrated with the origin
of the Ascomycota clade around 500–650 MYA.
no common ancestor was found in Sordariomycetes
class, suggesting an independent or convergent evolution of group-I intron encoding LAGLIDADG and GIYYIG elements in Ascomycota (Figure 3).
Transposable elements expansions in S. brasiliensis
genome
Transposable elements (TEs) comprise 0.34% and 0.62%
of the S. schenckii and S. brasiliensis genomes, respectively (Table 2). Fungal genomes contain substantially
different amounts of repetitive DNA sequences. The
assembled genome of Magnaporthe oryzae contains 10.8%
of repetitive DNA sequences, in M. grisea it is 4.2%, in N.
crassa is 10% and in S. cerevisiae almost 6% [37-40]. Differences in the TE contents are observed even between
closely related species, e.g. in the genus Paracoccidioides.
In P. brasiliensis TEs correspond to 8-9% of the genome
and twice this amount in P. lutzii (16%) [41]. Although
less common, lower TE contents have been described in
other fungi, for example 0.48% of Trichoderma and 0.1%
of Fusarium graminearum genomes assemblies [39,42].
Despite their lower content in Sporothrix genome,
all classes of TEs have been detected with large variation in number and diversity between S. schenckii and
S. brasiliensis. Two major types of retrotransposons,
LINEs and LTRs, were found in S. schenckii and S. brasiliensis, but no SINE elements were found (Table 2).
In S. brasiliensis, a five-fold expansion of Gypsy-like
elements was observed compared to S. schenckii and
both genomes contain more Gypsy-like elements than
Copia-like elements. We have observed a 4 and 19 foldchange between Gypsy-like and Copia-like elements for
S. schenckii and S. brasiliensis respectively, as usual for
fungal genomes [38]. The overall 2-fold expansion of
repetitive sequence content between Sporothrix species
may reflect an expansion of retrotransposons in the
S. brasiliensis genome or either a contraction in that of
S. schenckii (Figure 4). According to population genomics
studies and mating type distribution, the S. brasiliensis
population appears to be clonal and no recombination
events were detected [43]. We observed a predominance
of a single mating clone in epidemics of sporotrichosis
and no or limiting sex could favor the accumulation of
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A
B
Figure 3 Comparative analysis of mitochondrial genomes of S. schenckii and S. brasiliensis. (A) Gene content and order in mitochondrial
genomes of S. schenckii vs. S. brasiliensis showing high synteny despite the considerable difference in size. Insertions of LAGLIDADG and GIY-YIG
intron I type homing endonucleases in S. brasiliensis are present inside cytochrome C oxidase 1, ATP synthase subunit 6 and NADH dehydrogenase
subunits 2 and 3 genes. (B) Phylogenetic analysis was performed using the five LAGLIDADG or GIY-YIG elements found in S. brasiliensis showing
no pattern of ancestry with other close related species.
repetitive elements in microorganisms genomes [44,45].
Assexual propagation could lead extinction due inability
to control proliferation of vertically transmitted TEs, as
accumulation occurs due to the inefficiency of purifying
selection in clonal species [46].
Equivalent numbers of DNA transposons were found in
S. schenckii and S. brasiliensis genomes. hAT-like elements
were the most prevalent in both species (Table 2). Transposons from Tc1/mariner and Mutator superfamilies were
also found in both genomes. Four copies of Helitrons were
identified in S. brasiliensis while a PiggyBac-like element
was detected only in S. schenckii. Almost all TEs identified
in Sporothrix genomes corresponded to defective and
truncated copies, with exception of one LINE-like element
found in S. schenckii, suggesting that Sporothrix species are
able to control their proliferation. This finding suggests that
TEs are present in Sporothrix but with low transposition
activity. We discuss why this could be below.
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Table 2 Transposable element composition in Sporothrix
genomes
Type
S. schenckii
S. brasiliensis
LTR - Copia-like
3
3
LTR - Gypsy-like
12
57
LTR - BelPao-like
1
0
LINE
14 (1)a
7
Tc1/mariner-like
9
6
hAT-like
12
9
Mutator-like
1
4
PiggyBac –like
1
0
Helitron
0
4
Total elements
53
90
Percent of assembly
0.34%
0.62%
a
The number of potentially functional elements is shown in parentheses.
Protein family expansion and contraction in the
Sporothrix lineages
Gene duplications are an important source of evolutionary innovation and new gene copies can evolve new
adaptive functions shaping an organism’s gene content.
The differences among gene families have been related
to emerging processes due to differential degrees of
genetic drift, and thus the effectiveness of selection,
acting on genomes [47]. The pathogenic phenotypes of
S. schenckii and S. brasiliensis could result of expansion
in specific gene families that confer advantages in the
interaction with human/animal hosts. On the other hand,
gene families that ate necessary for the plant-associated
lifestyles of other Sordariomycetes could be contracted.
To test these hypotheses, we have compared the genomes
from closely related Sordariomycetes and dimorphic
fungal pathogens. Changes in S. schenckii and S. brasiliensis gene families were inferred based on domain
expansions or contractions assigned by Interpro, Pfam
and SMART databases and statistically tested by hypergeometric comparisons (P < 0.05 - Figure 5, Additional
file 3: Figure S2, Additional file 4: Figure S3 and Additional
file 5: Figure S4) and the reported p-values were used for
multiple testing using q-value.
We have not observed the enrichment of peptidases
genes in Sporothrix lineage, specifically the MEROPS
families M35 or M36, which are expanded in dimorphic
fungal pathogens as adaptation to mammalian hosts
[29,41]. On the other hand, we observe a lack of polysaccharide lyase genes which are associated with decay of
plants (CAZy PL family) when compared to other Sordariomycetes (Figure 6A, Additional file 2: Table S5).
The subfamilies PL1, PL3 and PL4 are broadly distributed among Sordariomycetes, but were not observed in
the Sporothrix species. Interestingly loss of plant degrading enzymes were also observed in other dimorphic fungal pathogens such as H. capsulatum and C. immitis
(Figure 6A, Additional file 2: Table S5), which has also
been previously interpreted as adaptation from plants to
animals [29,41]. As an alternative for the absence of PL
Figure 4 Transposable element content in Sporothrix schenckii and S. brasiliensis genomes. The number of potentially functional elements
is shown in parentheses.
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Figure 5 Interpro domains most enriched (A) or depleted (B) in Sporothrix lineages compared to other thermo dimorphic fungi and
close related Sordariomycetes. SBRA - S. brasiliensis, SSCH – S. schenckii, GCLA – G. clavigera, MORY – M. oryzae, PANS – P. anserina, NCRA – N.
crassa, VDAH - V. dahliae, FGRA – F. graminearum, AFUM – A. fumigatus, PMAR – P. marneffei, CIM – C. immitis, PBRA – P. brasiliensis, PLUT – P. lutzii,
BDER – B. dermatitidis, HCAP – H. capsulatum. The reported p-values were used for multiple testing using q-value.
proteins, it may be hypothesized that Sporothrix may
digest pectin by using polygalactouronase (CAZy GH28)
as a replacement strategy. Additionally, we didn’t detect any GH72 genes in Sporothrix genomes although
those genes were found in all remaining analyzed fungi
(Additional file 2: Table S5).
LysM domain-containing proteins display carbohydrate
binding modules, usually 42–48 amino acids residues in
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A
B
Figure 6 Gene expansion/contraction of carbohydrate-active enzymes (CAZy) in the genus Sporothrix. (A) Overall comparison of
Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), Auxiliary Activities (AAs) and
Carbohydrate-Binding Modules (CBMs) against other thermo dimorphic fungi and closely related Sordariomycetes. (B) CBM 50 gene family is
highly expanded in the Sporothrix lineage.
length, found in prokaryotes and eukaryotes. These proteins are classified in the CAZy database as CBM family
50 and harbor N-acetylglucosamine (GlcNAc) binding
characteristics. According to PFAM and CAZy counts, a
marked expansion of the LysM domain PF01476 (CAZy
CBM family 50) was detected in the Sporothrix lineage.
The phylogenetic tree clearly showed highly expanded
branches in the Sporothrix lineage when compared to
other Sordariomycetes and thermo dimorphic fungal
pathogens (Figure 7). Moreover, the comparative analysis of CAZy CBM family 50 also revealed a high expansion of this domain (Figure 6B, Additional file 2:
Table S5). Recent events of duplications of LysM homologs subsequent to a speciation event were detected in
the Sporothrix lineage using reconciled tree approach
(Additional file 6: Figure S1). The first duplication (Figure 7,
clade I) is related to genes containing single or multiple
repetitions of LysM domains, chitin-binding module
type 1 (CBM18) plus glycoside hydrolase (GH18). The
second event of duplication was observed in genes harboring multiple copies of LysM, providing evidence of
recent intergene duplication of this domain within paralogues (Figure 7, clade II). The structure of Sporothrix
LysM domain-containing genes are presented separately
in Additional file 3: Figure S2.
Chitin is a linear polymer of β-(1,4)-linked GlcNAc,
and is one of the major components of fungal cell wall.
The absence of chitin in mammalian cells makes this
polymer a potential targets for the innate immune system [48]. Chitin can be recognized by mammalian cells,
and is bound and degraded by chitin-binding proteins
GH18, playing an important role in inflammation and
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Figure 7 Unrooted maximum likelihood tree revealing LysM expansions in the Sporothrix lineage. Two major expansions were detected:
The first duplication is related to genes containing single or multiple repetitions of LysM domains (CBM50), chitin binding module type 1 (CBM18)
plus glycoside hydrolase (GH18) – clade I. The second event of duplication was observed in genes presenting multiple copies of LysM (CBM50),
and is evidence of recent intergene duplication of this domain within paralogous - clade II.
innate and adaptive immunity based on their modulation
on various disease states [49]. Chitin can mask immune
recognition by blocking dectin-1-mediated interaction
with fungal cell walls [50]. Also, chitin modulates epithelial immunity of the skin expressing high levels of
cytokine and chemokine and increases TLR4 expression on keratinocytes [51]. Possibly these proteins are
present to bind the own Sporothrix chitin exposed
upon cell damage, and in this way it may protect itself
against recognition of this polymer by keratinocytes.
LysM effector (Ecp6) from Cladosporium fulvum was
characterized as a virulence factor of this phytopathogenic fungus on tomato plants. Carbohydrate binding
assays have shown that Ecp6 specifically binds to chitin,
the major constituent of the fungal cell wall, acting as a
PAMP upon recognition by the plant during fungal
invasion. The presence of chitin-binding effector Ecp6
in the apoplast masks the perception of chitin by plant
receptors preventing the activation of defense responses
[52]. In addition significant expansion of the LysM domain
was detected in dermatophytes, compared to the thermodimorphic fungi [53]. Dermatophytosis and sporotrichosis
are characterized as cutaneous mycoses and the infection
is acquired after contact and/or trauma of skin. LysM
domain containing proteins have not been characterized
as virulence factors in human or animal fungal pathogens so far, and the role of those proteins requires
further investigation and could be a novel mechanism
for fungal evasion in mammalian host tissue.
Small GTPases are an independent superfamily of
GTP-binding proteins, sharing a common enzymatic
activity and producing GDP by the hydrolysis of GTP,
and play pivotal roles in cell division and signaling,
vesicle fusion and protein synthesis [54]. These proteins are also involved in filamentation, mating, growth
at 37°C and virulence in C. neoformans [55,56]. Significant expansions in Ras, Rho and Rab Small GTPase
superfamilies (IPR020849, IPR003578 and IPR003579)
were observed in the Sporothrix lineage when compared to other Ascomycetes (Figure 5, Additional file 2:
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Table S4). Judging from the phylogenetic trees of small
GTPases, the majority of branches harboring Sporothrix homologues suggests a higher diversity among the
Ascomycetes we analyzed (Additional file 4: Figures S3,
Additional file 5: Figure S4, Additional file 7: Figure S5).
Reconciliation of small GTPases gene trees and species
tree do not indicate Sporothrix-specific duplications,
that instead independent gene losses in other species
explain the increased copy number in Sporothrix (Additional
file 8: Figure S6, Additional file 9: Figure S7, Additional
file 10: Figure S8). An alternative hypothesis is that the
Sporothrix Ras, Rho and Rab genes have duplicated recently and rapidly diverged creating the observed long
branch lengths. In addition we detected highly supported clades containing Sporothrix and other pathogenic dimorphic fungi, suggesting convergent evolution
of small GTPases, reinforcing the high plasticity of signal transduction in the Sporothrix lineage (Additional
file 4: Figure S3, Additional file 5: Figure S4, Additional
file 7: Figure S5). To date, such a high diversity of Small
GTPase proteins as found in the Sporothrix lineage has
not been reported from any other class of fungi.
Another group that was expanded by INTERPRO and
SMART domain counts in the Sporothrix lineage is the
polyketide synthase (PKS), enoylreductase (IPR020843)
family (Figure 5, Additional file 2: Tables S3, S4). Polyketides comprises diverse fungal secondary metabolites
such as antibiotics, pigments, and mycotoxins that are
formed from simple carbon precursor acids catalyzed by
polyketide synthases (PKSs) [57]. Filamentous fungi are
producers of polyketide metabolites, several of which of
pharmacological or agricultural interest [58,59]. Fungal
PKSs are in general a linear succession of ketosynthetase
(KS), acyltransferase (AT), dehydratase (DH), enoyl
reductase (ER), ketoreductase (KR), acyl carrier protein
(ACP), and thioesterase (TE) domains [60]. ER domains reduce enoyl groups to alkyl groups (saturated)
during production of secondary metabolites. Among
fungal genomes, few potential PKS orthologous genes
are shared, even between closely related taxa [61]. We
identified various paralogous duplications in the phylogenetic analysis of PKS-containing protein, and the
Sporothrix lineage appears to have of PKS-encoding
genes that is at least 3-fold larger than that of the other
species analyzed (Additional file 2: Table S4, Figure 8).
This was confirmed using the reconciled approach of PKS
gene tree with the species tree analyzed (Additional
file 10: Figure S8). Convergent branches linking Sporothrix and other pathogenic dimorphic fungi were also
observed. We identified discontinuous distributions of
PKS homologs among the analyzed fungal species,
with low bootstrap values, that can be explained by
gene duplication, divergence, and gene loss [61]. We
also identified expanded clades harboring Sporothrix
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and dimorphic fungi with high branch support values,
suggesting a great diversity of this protein family.
Another large expansion found in the Sporothrix lineage
is a fungal trichothecene efflux pump (Pfam PF06609), an
evidence of detoxification via mycotoxin pump [62]. Apart
from that, fungal genomes generally harbor a lower
content of Leucine-rich repeat (LRR) proteins than other
ophistokonts [63]. Gene expansions of several LRR
superfamilies were identified in the Sporothrix lineage
(Additional file 2: Tables S3, S4). LRR proteins expansions are commonly found in eukaryotic parasites such
as Trypanosoma and Giardia [64,65]. These domains
consist of 2–45 motifs of 20–30 amino acids in length
providing a structural framework for protein-protein
interactions. LRR proteins are involved in a variety of
biological processes and are source of genetic variation
for the ongoing process of antigenic variability in pathogens [66,67]. In addition expansion of LRR proteins was
described for Candida species as a virulence factor [68].
The role of these proteins should be further investigated
in the genus Sporothrix.
Cellular processes and dimorphism
The basic mechanisms of DNA repair in Sporothrix do
not deviate from the eukaryotic consensus, as expected,
but neither S. brasiliensis nor S. schenckii seem to have a
homolog to the Neurospora crassa RIP-defective (RID)
methyl-transferase [69]. This suggests that RIP-like mutation patterns found in transposable elements in this fungus are not generated by the classical, RID-dependent
mechanism. This is not a surprise, given that at least one
previous report [70] pointed to the possibility of alternative pathways for repetitive DNA quelling via RIP. This
is in contrast to other Sordariomycetes and even more
distantly related members of the Ascomycota, such
as P. brasiliensis and H. capsulatum, for which the
tBLASTn tool yielded hits with high homology. Since
the number of degenerate TEs and overall evidence of
transposon activity in Sporothrix spp. genomes is low, it
would be interesting to identify which pathway is
responsible for TE suppression in this genus instead of
RIP. We also note the absence of a mus-18 homologue
for the alternative, UV damage-related nucleotide excision repair pathway [71], found in Neurospora crassa.
Among transcription factors, the recent discovery of
the Ryp1 protein in H. capsulatum [72] is of interest for
the study of adaptive processes in fungi. Ryp1 and its
homologues in C. albicans and S. cerevisiae [73] are all
implicated in morphogenetic changes of their respective
species in response to environmental stimuli. We have
also found an ortholog of Ryp1 in Sporothrix. Given that
Ryp1 and the hybrid histidine-kinase Drk1 are two
determinants of mold-to-yeast transition in dimorphic
fungi, and that Sporothrix also has homologues for the
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Figure 8 Unrooted maximum likelihood tree of Polyketide synthase (PKS) enoylreductase (IPR020843) family showing expansions in
the Sporothrix lineage. Paralogous duplications are displayed in the related branches suggesting vast genomic apparatus of PKS containing
genes in the Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi are displayed in red.
latter [16], it seems reasonable to speculate that both
also have been coopted by this genus to coordinate dimorphic transition.
of those components, little is known about the surface
protein composition of S. schenckii and S. brasiliensis.
a) Adhesins and/or cell surface proteins
Cell wall assembly
The cell wall of fungi is a dynamic organelle, which is
constantly adjusted depending on environmental insults.
Fungal cell wall components are considered relevant for
virulence, have antigenic properties and participate in
the modulation of the host immune response by being
recognized by innate immunity receptors [14]. Current
models propose an arrangement of several stratified layers
composed of structural polysaccharides, mainly β-glucans
and chitin, proteins and glycoproteins, generally known
as mannoproteins, and other minor components. Cell
wall proteins (CWP) are either covalently linked to the
cell wall β (1,6)-glucans by a glycophosphatidylinositol
(GPI) moiety or linked via an alkali sensitive linkage to
β (1,3)-glucans. The sugar moieties in CWP are N– and/or
O- linked to the protein core [74]. Despite the importance
Previous studies searching for adhesins and antigens in
cell extracts of Sporothrix demonstrated the presence of
a main antigen on both species, known as Gp70, a
secreted antigen that is also present on the cell surface acting as an adhesin [75-77]. The genomes of S. schenckii and
S. brasiliensis were investigated with different predictors
to ascertain the presence of these cell wall components.
An in silico comparative analysis was performed in
order to determine the putative adhesins and/or cell
surface proteins bearing a GPI-anchor. According to
ProFASTA and FungalRV, S. schenckii harbors 68 and 61
surface proteins, respectively, that have adhesin properties
(n = 129). Of these, 12 were found by both predictors
(Additional file 11: Figure S9A, Additional file 2: Table S6)
totalizing 117 unique predicted proteins in the cell wall of
S. schenckii. For S. brasiliensis we have identified 54 and
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63 cell wall proteins (n = 117), respectively, 11 of which
were predicted by both algorithms. For this species,
a total of 106 unique predicted proteins (Additional file
11: Figure S9B, Additional file 2: Table S7). The protein
sequence relative to the previously proven cell surface/
adhesin Gp70 was not predicted to possess this function
or to be present in the surface location by any of the predictors used [77]. Previous studies described several other
important, non-classical surface proteins present in other
fungi but which were not recognized in Sporothrix by
FungalRV and ProFASTA [78-80]. The major classes of
proteins predicted in S. schenckii and S. brasiliensis, by
both ProFASTA and FungalRV, are currently annotated
either as hypothetical proteins or belonging to a protein
family with unknown function (Figure 9). This indicates
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that proteomic studies are needed to validate the expression of such proteins and biochemical functional studies
are necessary to clarify their role on the fungal cell surface.
The cell wall proteins and/or adhesins predicted for these
species were blasted against 11 fungi for comparative analysis, and each protein was blasted against the two Sporothrix species (Additional file 10: Table S8). Eight proteins
were found exclusively in S. brasiliensis and eight proteins were present only in S. schenckii. Sixteen Sporothrix-specific proteins were annotated as hypothetical
proteins. None of the proteins described are specific to
the group of human pathogenic fungi, but interestingly
there are some proteins putatively present in the cell
wall of Sporothrix that share homology with those of
plant and insect-associated fungi.
Figure 9 Putative adhesins identified and/or cell wall GPI-anchored proteins in S. schenckii (A) and S. brasiliensis (B) predicted by
ProFASTA and FungalRV. Putative adhesins were classified by their biological function using Gene Ontology (GO) and InterPro databases.
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b) Glucan and chitin metabolism
In S. schenckii, β-glucans are major components of the
cell wall [81,82], and are present as alkali-insoluble
and alkali-soluble glucans, containing predominantly β(1,3)-linkages in both cases [82]. In S. schenckii, no
genes related to the synthesis or degradation of βglucans have been reported to date. Genomic data analysis revealed a single FKS ortholog in S. schenckii and
S. brasiliensis genomes, as well as single orthologs in
the genomes of the other 14 fungal species studied here
(Additional file 11: Table S9). No genes related to the
synthesis of either β-1,6- or β-1,4-glucans were identified, although hydrolase orthologs for the three types
of β-glucan linkages in the S. schenckii cell wall, are
present in both genomes (Additional file 11: Table S9).
Chitin synthesis in fungi is a rather complex process,
regulated by multigene families encoding chitin synthase
isoenzymes, whose activities may be spatially regulated
to fulfill the multitude of roles ascribed to them [83].
Based on differences in regions of high sequence conservation, chitin synthases have been attributed to seven
classes [83,84], whose functional implications are not yet
clear in all cases. Despite the low chitin content reported
for S. schenckii [81], genomic analysis showed the presence of seven CHS genes, in S. schenckii as well as in
S. brasiliensis genomes. A cluster analysis of their putative products, including 36 fungal chitin synthases,
revealed that each of the translation products of the
seven CHS Sporothrix genes identified in genomic databases (CHS1 to CHS7), could be ascribed to each of the
seven chitin synthase classes known (I to VII) (Additional
file 12: Figure S10, Additional file 2: Table S10). It is worth
noting that class III chitin synthases were thought to
occurr exclusively in filamentous fungi [85]. In our analysis, genes for class III chitin synthases were found in
the Sporothrix genomes as well as the genomes of other
dimorphic fungi (Additional file 2: Table S10), an indication that class III fungal chitin synthases might be
more widespread in fungi. Another interesting finding
was the head-to-head arrangement in the S. schenckii
and S. brasiliensis genomes of the CHS4 and CHS5
genes, whose putative translated products are class V
and VII chitin synthases (Additional file 8: Figure S6,
Additional file 2: Table S10). A similar arrangement was
reported for genes coding for classes V and VII of chitin
synthases in P. brasiliensis, A. nidulans, C. posadasii
and F. oxysporum [84,86-88]. The meaning of such
arrangement is unclear, although a common transcriptional regulation for these genes has been suggested for
A. nidulans and P. brasiliensis [84,88,89]. In agreement
with the large number of CHS genes in the Sporothrix
genomes, genomic analysis showed the presence of ten
and nine chitinase genes, respectively, in the S. schenckii
and S. brasiliensis genomes (Additional file 2: Table S10).
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Polysaccharide synthesis and hydrolysis-related genes
identified in S. schenckii and S. brasiliensis genomes
correlate with the biochemical composition of the cell
wall as reported by Previato et al. [81,82]. It remains
to be determined which individual synthase and/or
hydrolase gene might be involved in shaping the yeast,
mycelial and spore (conidial) walls of Sporothrix species, or even whether any of them might have any role
in survival and would provide potential targets for the
development of specific antifungal drugs.
c) Protein glycosylation
Glycoproteins are key components of the S. schenckii
cell wall, but thus far little is known about their biosynthetic pathways [23,24]. The genomes of S. schenckii and
S. brasiliensis contained the orthologs involved in elaboration of the N-linked glycan core, its transference
to proteins and in early trimming. These genes are also
known to be involved in glycoprotein endoplasmic
reticulum-associated degradation, a quality control system for proteins synthesized within the secretory pathway [90-92] (Additional file 2: Table S11). Furthermore,
the genomes contain the putative orthologs encoding
Golgi-resident glycosidases and glycosyltransferases that
further modify N-linked glycans, generating both hybrid
and complex N-linked glycans. The presence of a gene
with significant similarity to those encoding the Nacetylglucosaminidase III (Additional file 2: Table S11),
which adds the bisecting GlcNAc residue found in both
hybrid and complex N-linked glycans [93], suggests an
ability to elaborate more complex oligosaccharides than
those found in S. cerevisiae [94]. Moreover, our analysis
revealed genes encoding putative Golgi UDP-galactose
and CMP-sialic acid transporters, suggesting the ability
of these fungi to add these sugars to their glycans.
S. schenckii and S. brasiliensis also contain an ortholog
of A. nidulans ugmA, whose product generates the
galactomannan-building sugar donor [95], and some putative galactosyltransferases (Additional file 2: Table S12).
However, it remains to be addressed whether these
enzymes participate in elaboration of glycoproteins and/
or glycolipids. Sialic acid has previously been reported
as a component of S. schenckii cell wall glycolipids [96],
so it is likely that the putative Golgi CMP-sialic acid
transporter is involved in modification of such lipids.
The biosynthetic pathway for O-linked glycans can
also be predicted from the analysis of S. schenckii and
S. brasiliensis genomes (Additional file 2: Table S13).
Optimal characterization of O-linked glycans is via isolation from peptide-rhamnomannans [97]. They contain an
α1,2.mannobiose core, an α1,2-glucuronic acid unit, and
one or two rhamnose residues. The S. schenckii and
S. brasiliensis genomes contain three putative glucuronosyl transferases that might participate in the elaboration of
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this O-linked glycan (Additional file 2: Table S13). Our
genomic analysis could not find any obvious ortholog
for rhamnosyl transferases, but Sporothrix contains all
the required genes for synthesis of UDP-L-rhamnose
(Additional file 2: Table S13) [98,99], the sugar donor in
the enzyme reaction catalyzed by rhamnosyltransferases
[100]. Synthesis of GPI is quite conserved in eukaryotic
cells and the Sporothrix genome contains all genes to
elaborate this glycolipid (Additional file 2: Table S14).
Melanin metabolism
Melanins are dark pigments formed by phenolic and indolic oxidation. These biopolymers are produced by a wide
range of organisms, possibly contributing to the maintenance of several species throughout evolution [101]. In
fungi, the expression of these pigments has been associated with virulence [102]. Fungi may synthesize melanin
by several pathways: in pathogenic fungi, most commonly
from endogenous substrate via the 1,8-dihydroxynaphthalene (DHN) pathway or the L-3,4-dihydroxyphenylalanine
(L-DOPA) pathways [102]. The latter type is prevalent in
Basidiomycetes. However, evidence of both pathways has
been found in S. schenckii by means of specific substrate
supplementation or drug-related inhibition of the respective pathways [22,23,103,104].
Melanins are found in S. schenckii spores and yeast cells
and are produced in vitro and during infection using
hamsters as host model. Its detection has also been
confirmed by immunofluorescence with monoclonal
antibodies raised against S. schenckii melanin [21,22]. In
S. schenckii, melanin pigments can protect the fungus from
the mammalian host’s innate immune responses providing
resistance to killing by phagocytosis and oxidizing agents
[22,105]. Recently it was reported that S. schenckii and
S. brasiliensis also produce pyomelanin, a melanoid pigment derived from the degradation of L-tyrosine via a
4-hydroxyphenylpyruvate dioxygenase [103].
Genomic comparison showed that both S. schenckii
and S. brasiliensis possess enzymes with central roles in
melanin synthesis via DHN and DOPA pathways, and
also in pyomelanin synthesis. Sporothrix schenckii and
S. brasiliensis loci which are postulated to be involved in
the melanin biosynthesis pathway are described in
Additional file 2: Table S15. We found 19 loci related
to melanin biosynthesis in S. schenckii and 17 in S. brasiliensis. Homology with previously described melaninrelated enzymes found in the sequence analyses are:
pigment biosynthesis protein yellowish-green 1, polyketide
synthase I and III, tetrahydroxynaphthalene/trihydroxinaphtalene reductase, scytalone dehydratase, laccase,
tyrosinase and 4-hydroxyphenylpyruvate dioxygenase,
as illustrated in Additional file 13: Figure S11. The multiple functions of melanin in a cell and, especially, the
resistance to antifungal drugs and survival of the host
Page 15 of 22
immune system, are a strong motivation for the study of
the genetic characteristics of melanin biosythesis.
Conclusions
In this study we provide high quality genomic sequence
assemblies and annotations for S. schenckii and S. brasiliensis. Genomic analyses showed a convergent evolutionary fate compared to other dimorphic fungi, even
though Sporothrix is a close relative of plant-associated
Sordariomycetes. Similar to other dimorphic fungal pathogens we have observed a lack of polysaccharide lyase genes
which are associated with decay of plants, suggesting
evolutionary adaptations from a plant pathogenic or
saprobic to an animal pathogenic life style. In addition,
convergent branches linking Sporothrix and other pathogenic dimorphic fungi were also observed in genes involved in signal transduction and secondary metabolism
which suggest similar evolutionary traits. The recent
hypothesis of habitat shift from a saprobic life style in
fermented plant material to mammal transmission may
explain numerous plant/related atavisms. Comparative
genomics reveals a certain degree of specialization in
the Sporothrix lineage which may contribute to our
understanding of how fungal-environment-human interactions lead to the selection of pathogenic phenotypes of these species. The Sporothrix system may bring
new opportunities for functional studies in order to
understand the biology of fungi and infection.
Methods
Fungal strains and DNA extraction
Sporothrix schenckii strain 1099–18 (ATCC MYA-4821)
was originally obtained from the Mycology Section, Department of Dermatology, Columbia University, New
York, isolated from a patient manifesting subcutaneous
sporotrichosis, and has been widely used in experiments
of cell wall composition and virulence studies in mice
models [97,106]. Sporothrix brasiliensis strain 5110 (ATCC
MYA-4823) was isolated from a feline skin lesion in the
epidemic area of sporotrichosis in Rio de Janeiro, Brazil,
presenting high virulence in mouse model [77]. Mycelial
cells were cultivated in Sabouraud broth at 25°C, with
shaking (150 rpm) for 14 days, collected by centrifugation
and washed 3 times with Phosphate-buffered saline (PBS)
solution. Cells were disrupted using the Precellys®24Dual (Bioamerica) with help of CK28 hard tissue homogenizing tubes. DNA extraction was performed using
Qiagen DNeasy Plant Mini Kit, according to manufacturer’s protocols.
Genome sequencing and assembly
Sporothrix schenckii and S. brasiliensis genomes were
sequenced using next generation 454 pyrosequencing
(Roche). Shotgun and paired-end 3 kb inserts libraries
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were constructed and sequenced in the 454 GS FLX platform according to Roche’s protocols at the Computational
Genomics Unity of the National Laboratory for Scientific
Computing (LNCC, Petrópolis, RJ, Brazil). Genomic
assemblies were carried out using Newbler and Celera
Assembler. Sequence gap filling and the removal of
contigs corresponding to rDNA genes were manually
done, decreasing the numbers of scaffolds and contigs.
The assembled scaffolds generated by the two species
were aligned and oriented using MAUVE [107]. Similarity scores and dot-plot graphs were generated using
LALING/PLALING (http://fasta.bioch.virginia.edu/fasta_
www2/fasta_www.cgi?rm=lalign).
Ab initio Gene prediction, annotation and protein family
classification
Gene predictions were performed using three different
approaches: SNAP [108], AUGUSTUS [109] and EXONERATE [110] using ORF’s identified in the G. clavigera
strain kw1407/UAMH 11150 [26] as reference and for
training and genomic comparisons. Proteins deduced for
G. clavigera proteome were aligned to the S. brasiliensis
and S. schenckii assembled genomes using Exonerate
(percent threshold equal 50) with the model protein2genome. Gene predictions (SNAP and AUGUSTUS) and
protein (EXONERATE) alignments were used as input
in order to identify consensus gene structures using EVidenceModeler [111]. Consensus ORF’s were subjected to
Blast searches against NCBI refseq_protein, KEGG and
SwissProt databases. Automatic annotations were performed using SABIA - upgraded for eukaryotic organisms [112] and validated ORF’s were considered with
minimum query/subject coverage of 60% and minimum
positive 50%. In addition, gene categories according
KEGG were inspected manually in order to re-assemble
the metabolic pathways of S. brasiliensis and S. schenckii.
Alignments were made by Blastp and the lowest e-value
was used to consider homologous sequences. Next, loci
identified in S. schenckii and S. brasiliensis genomes
were blasted against the genome libraries of 14 selected
fungi (Neurospora crassa, Aspergillus nidulans, A. fumigatus, Talaromyces marneffei, Paracoccidioides lutzii,
P. brasiliensis, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Fusarium graminearum,
Magnaporthe oryzae, Sordaria macrospora, Verticillium
dahliae, Grosmannia clavigera) to infer the putative
orthologues. Gene products were categorized according to
biological process, cellular component and molecular
function using GeneOntology (GO) using Blast2GO.
Secreted proteins were identified using SignalP3.0
(http://www.cbs.dtu.dk/services/SignalP/) using hidden
Markov model.
The prediction of mobile genetic entities was performed
by similarity searches using the following approaches and
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databases: a) Nucleotide Blast against Repbase version
17.10 (http://www.girinst.org/repbase/) [113], Dfam database version 1.1 [114] and Gypsy database version 2.0
(GyDB) [115]; b) PSI-BLAST (Position-Specific Iterated
BLAST) using profiles of proteins corresponding to major
clades/families of Transposable Elements (TEs) implemented with TransposonPSI tool (http://transposonpsi.
sourceforge.net/); c) reverse position-specific BLAST algorithm (RPSBLAST) against Conserved Domain Database
(CDD) version March 2013 [116]; and d) tblastn taking
specific protein subsets against the Sporothrix genome.
These subsets were built from NCBI Non Redundant (NR)
database version March 2011 using particular description
terms related to transposable elements including (apurinic/
apyrimidinic endonuclease, aspartic proteinase, ATPase,
endonuclease, envelope, GAG protein, helicase, integrase,
polymerase B, replication protein A, reverse transcriptase,
RNase, transposase, tyrosine transposase/recombinase). In
addition, the Tandem repeat finder (TRF) algorithm was
used for finding tandem repeats [117]. Transposable elements were classified accordingly [118]. All results were
obtained using locally compiled databases. Perl scripts were
built for automation of genome scans, report generation
and data integration. Artemis sequence visualization and
annotation tool [119] was used for manual curation and
annotation of transposable elements.
Whole genome gene families were identified using
InterproScan combined with Pfam domain assignments.
Annotation of carbohydrate-active enzymes was performed
in a two-step procedure where the translated protein
sequences were compared to the full length sequences
derived from the Carbohydrate-Active enZymes (CAZy)
database (www.cazy.org; [120]) using BLAST [121]. The
query sequences that had an e-value <0.1 were subjected to
a BLAST search against sequence fragments corresponding
to individual catalytic and carbohydrate-binding modules described in CAZy, along with a HMMer search
[122] using hidden Markov models corresponding to
each CAZy module family. A family assignment was
considered reliable when the two methods gave the
same result. Borderline cases were resolved by inspection of conserved features such as the presence of
known catalytic residues.
Gene family expansion and contractions
Gene families were determined using OrthoMCL approach comparing with other 13 fungi (Additional file 2:
Table S1). Domains were annotated for each orthologous
cluster using Interpro [123], Pfam [124] and SMART
[125] databases. Significant enrichment or depletion
of domains in the Sporothrix lineage were calculated
based on hypergeometric comparisons (P < 0.05) and
the reported p-values are adjusted for multiple testing
using q-value [126,127]. The expanded families with
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highest discrepancies between Sporothrix and other compared fungi were individually analyzed. Protein sequences
of LysM, PKS enoyl reductase, Ras, Rab and Rho small
gtpases we individually aligned using ClustalW [128]
and the domains were manually checked. Uninformative
positions of the alignment were eliminated using trimal
[129] and the best-fit protein substitution model was
inferred based on likelihood values under AIC criteria,
implemented in ProtTest [130]. Phylogenetic analysis of
expanded gene families were carried out using Maximum likelihood methods implemented in PhyML 3.0
software and 1.000 of non-parametric bootstraps were
tested for branch support [131]. Gene duplications/losses
for the given family trees were inferred using Notung 2.6
software [132].
Blast reciprocal best hit and phylogenomic analysis
Bidirectinoal-best Blast Hit (BBH) were performed using
two different datasets: first we performed the comparisons between S. schenckii, S. brasiliensis and G. clavigera
genomes in order to identify the unique genes in Sporothrix lineage. In addition, Blast reciprocal best hits were
performed to identify common orthologues in 25 fungal
genomes (Additional file 2: Table S1) using minimum
query/subject coverage of 50% and e-value of E ≤ 1×10−20.
A total of 395 orthologs were found in all species analyzed
and were aligned using MAFFT [133] and retrieved
alignments were trimmed using Trimal [129] in order
to exclude spurious sequences or poorly aligned regions.
Phylogenomic analysis was performed using RAxML
[134] and the Dayhoff aminoacid substitution model
was selected according ProtTest [130]. Divergence time
between species was calculated with help of r8s v 1.8
[135] program using Langley-Fitch model [136] considering the origin of the Ascomycota at 500 to 650 MYA
(Millions Years Ago) [137].
Cell wall protein/adhesin analysis
Two programs were used for the prediction of cell wall
proteins/adhesins: ProFASTA [138] and FungalRV [139].
Analysis using the fasta files of the complete genomes of
S. schenckii and S. brasiliensis were performed for prediction of GPI-anchor secretion signal and transmembrane
domain identification. The SignalP 4.0 server (http://www.
cbs.dtu.dk/services/SignalP/) was applied for prediction
of the presence and location of signal peptide cleavage
sites in amino acid sequences, using the method of
Input sequences, which do not include TM regions.
Then, the TMHMM Server v. 2.0 (http://www.cbs.dtu.
dk/services/TMHMM/) was used for prediction of transmembrane helices in proteins, and finally the Big-PI fungal
predictor [140] was used for GPI modification sites. The
ProFASTA requires the combination of these three analyses to prospect cell wall proteins and adhesins, with the
Page 17 of 22
following parameters: SignalIP 4.0 positive; TMHMM
2.0 < 1 helices and number of AA to exclude as 45 from
N-terminus and 35 for C-terminus; Big-PI positive. The
FungalRV validates only proteins with score up to 0.5
for adhesin or adhesin-like features.
Autophagy, peroxisome and endocytosis
The initial tool used for this annotation was the KEGG
automatic classification, which adequately identified
genes involved in peroxisome biogenesis. However, the
automatic annotation algorithms only picked up a few
genes involved in autophagosome biogenesis and endocytosis, so different approaches were necessary. For the
genes involved in autophagy, we started by collecting on
the SGD all protein sequences annotated as involved in
autophagy and autophagosome biogenesis in Saccharomyces cerevisiae. All of these sequences were blasted against
the S. schenckii and S. brasiliensis databases in order to
correctly identify homologous protein sequences. To
narrow down the list, we focused the analysis on 17
genes that are necessary for autophagosome biogenesis
in yeast [141] plus those that are shown on KEGG.
Regarding endocytosis, the KEGG table only showed
two genes involved in the process itself and several
genes involved in vacuolar degradation. To overcome
this limitation, the genes that were used for annotation
were those listed in a review article as being involved in
clathrin-mediated endocytosis in S. cerevisiae [142]. All
protein sequences encoded by these genes were blasted
against the S. schenckii and S. brasiliensis databases.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.
Additional files
Additional file 1: Core genes for general and secondary metabolism.
Additional file 2: Table S1. Information about the 25 genomes
retrieved for phylogenomic inference. Table S2. Pfam domain
expansions/contractions of Sporothrix schenckii and S. brasiliensis. Table S3.
SMART domain expansions/contractions of Sporothrix schenckii and
S. brasiliensis. Table S4. INTERPRO domain expansions/contractions of
Sporothrix schenckii and S. brasiliensis. Table S5. Comparative genomic
analysis of carbohydrate active enzymes (CAZy) of Sporothrix schenckii
and S. brasiliensis. Table S6. Putative adhesins of S. schenckii. Table S7.
Putative adhesins of S. brasiliensis. Table S8. Cell wall and adhesin-related
genes characterized in Sporothrix schenckii and S. brasiliensis. Table S9.
Glucan synthase and glucanase genes identified in Sporothrix schenckii
and S. brasiliensis. Table S10. Chitin synthase and chitinase genes identified
in Sporothrix schenckii and S. brasiliensis. Table S11. Genes involved in
N-linked glycosylation in Aspergillus nidulans and Neurospora crassa, and
their putative orthologs in Sporothrix schenckii and S. brasiliensis. Table S12.
Enzymes activities related with protein glycosylation in Aspergillus nidulans
and Neurospora crassa, and their putative orthologs in Sporothrix schenckii
and S. brasiliensis. Table S13. Genes involved in O-linked glycosylation in
Aspergillus nidulans and Neurospora crassa, and their putative orthologs in
Sporothrix schenckiia and S. brasiliensis. Table S14. Genes involved in
Teixeira et al. BMC Genomics 2014, 15:943
http://www.biomedcentral.com/1471-2164/15/943
Page 18 of 22
GPI-anchor elaboration in Aspergillus nidulans and Neurospora crassa,
and their putative orthologs in Sporothrix schenckii and S. brasiliensis.
Table S15. Melanin biosynthesis pathway and putative orthologs in
Sporothrix schenckii and S. brasiliensis. Table S16. Comparative analysis
of core genes related to amino acids, Secondary, Energy, Cofactor and
Vitamin metabolisms of Sporothrix schenckii and S. brasiliensis. Table S17.
Genomic identification and classification of phospholipases A, C, and D
enzyme families in Sporothrix schenckii and S. brasiliensis genomes.
Table S18. Homologs of vitamin and cofactor genes presented analyzed
by FUNGIpath in both Sporothrix species. Table S19. Orthologous genes
related to catabolism and transport in Sporothrix schenckii and S. brasiliensis.
Authors’ contributions
Designed the experiments: MMT LGPA RCS JCR JFS BH HMMM SRA JES GAN
PSC LMLB ATRV MSSF; Sequencing, assembling and annotation: MMT LGPA
PK FLA ESK AKRA LF LSD KSF RCS JCR NCA HCP AMN PA ALG VPM LDFP AV
CBC PAS OLC FFMO TCS ALNB MAS LMO MMM HV MMLC BH HMMM JES;
Comparative genomics analysis: MMT LGPA PK FLA ESK AKRA LF LSD KSF
RCS JCR NCA HCP AMN PA ALG VPM LDFP AV CBC PAS OLC FFMO TCS
ALNB MAS LMO MMM HV MMLC SH BH HMMM JES; Contributed to analysis
tools: MMT BH GAN PSC HMMM SRA JES LMLB ATRV MSSF; Wrote the
manuscript: MMT MSSF. All authors read and approved the final manuscript.
Additional file 3: Figure S2. Phylogenetic distribution of LysM domains
containing proteins in Sporothrix. The LysM domains are displayed along
the taxa (red bars), chitin binding module type 1 (CB1) (blue bars) plus
catalytic sites identified (glycoside hydrolase - GH or Pectin Lyase – PL).
Gene paralogous duplications are highlighted by red boxes.
Acknowledgments
We are grateful to FAP-DF, CNPq and Capes for the financial support and
fellowships of the projects Pronex (grant number 193000569/2009) and
Genoprot (grant number 559572/2009-3). HMMM is supported by CONACyT,
México (grant number CB2011-166860). BH is an Honorary Professor of
Glycomics at the Faculty of Health and Medical Sciences, University of
Copenhagen, Denmark.
Additional file 4: Figure S3. Unrooted maximum likelihood tree of Ras
Small GTPase proteins (IPR020849) family shows high diversification in
the Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi
are highlighted in red.
Additional file 5: Figure S4. Unrooted maximum likelihood tree of Rho
Small GTPase proteins (IPR003578) family shows high diversification in
the Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi
are highlighted in red.
Additional file 6: Figure S1. Gene tree and species tree reconciliation
of LysM domain-containing genes showing specific Sporothrix duplications
(blue boxes).
Additional file 7: Figure S5. Unrooted maximum likelihood tree of Rab
Small GTPase proteins (IPR003579) family shows high diversification in
the Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi
are highlighted in red.
Additional file 8: Figure S6. Gene tree and species tree reconciliation
of small GTPase Ras gene family showing independent gene losses in
other species and the increased copy number in Sporothrix (blue boxes).
Additional file 9: Figure S7. Gene tree and species tree reconciliation
of small GTPase Rho gene family showing independent gene losses in
other species and the increased copy number in Sporothrix (blue boxes).
Additional file 10: Figure S8. Gene tree and species tree reconciliation
of small GTPase Rab gene family showing independent gene losses in
other species and the increased copy number in Sporothrix (blue boxes).
Additional file 11: Figure S9. Chart pies showing the efficiency of
the algorithms used to predict the putative adhesins and/or cell wall
GPI-anchored proteins of (A) S. schenckii (n = 118) and (B) S. brasiliensis
(n = 106). The relative percentage of putative adhesins and/or
GPI- anchored proteins, predicted by either ProFASTA or Fungal RV, is
shown as well as the proteins in common by both predictors.
Additional file 12: Figure S10. Phylogenetic tree of relatedness of
Sporothrix spp. chitin synthases. The Mega 4 software package was
employed, using ClustalW for sequence alignment. Construction of the
phylogenetic tree was done by the neighbor-joining method using 1000
replications. The seven chitin synthases identified for both, Sporothrix
brasiliensis and S. schenckii, cluster within the seven chitin synthase classes
(I to VII) previously reported [83]. GenBank accession numbers of sequences,
and names of fungal species used for construction of the tree are displayed
in Additional file 12: Table S10.
Additional file 13: Figure S11. Melanin biosynthesis pathways for
DHN-Melanin, DOPA-melanin and pyomelanin proposed for S. schenckii
and S. brasiliensis based on melanin biosynthetic pathways described in
other pathogenic fungi. The putative enzymes identified in the genomes
of S. schenckii and S. brasiliensis are indicated in circles. Locus tags, Gene
products, numbers of exons, size of transcripts, estimated protein sizes
and current annotations are listed.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF,
Brazil. 2Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brazil.
3
Departamento de Biologia Celular, Instituto de Biologia Roberto Alcântara
Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
4
Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo
Horizonte, MG, Brazil. 5Grupo Informática de Biossistemas, Centro de
Pesquisas René Rachou, FIOCRUZ, Minas, Belo Horizonte, MG, Brazil.
6
Departamento de Análises Clínicas, Universidade Estadual de Maringá,
Maringá, PR, Brazil. 7Programa de Pós-Graduação em Ciências e Tecnologias
em Saúde, Universidade de Brasília, Ceilândia, Brasília, DF, Brazil. 8Instituto de
Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São
Paulo, Campus Diadema, São Paulo, SP, Brazil. 9Pós-Graduação em Ciências
Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF,
Brazil. 10Programa de pós-graduação em Medicina Tropical, Universidade de
Brasília, Brasília, DF, Brazil. 11Programa de pós-graduação em Bioinformática,
Universidade Federal de Minas Gerais, Minas Gerais, Brazil. 12Departamento
de Microbiologia Imunobiologia e Parasitologia, Universidade Federal de São
Paulo, São Paulo, SP, Brazil. 13Centro de Microbiología y Biología Celular,
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela.
14
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. 15Centre
National de la Recherche Scientifique, Aix-Marseille, Université, CNRS,
Marseille, France. 16Departamento de Biología, Universidad de Guanajuato,
Guanajuato, Mexico. 17Departamento de Análises Clínicas e Toxicológicas,
Universidade de São Paulo, São Paulo, SP, Brazil. 18Department of Plant
Pathology & Microbiology, University of California, Riverside, CA, USA.
Received: 11 February 2014 Accepted: 25 September 2014
Published: 29 October 2014
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doi:10.1186/1471-2164-15-943
Cite this article as: Teixeira et al.: Comparative genomics of the major
fungal agents of human and animal Sporotrichosis: Sporothrix schenckii
and Sporothrix brasiliensis. BMC Genomics 2014 15:943.
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