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Abadio et al. BMC Genomics 2011, 12:75
http://www.biomedcentral.com/1471-2164/12/75

RESEARCH ARTICLE

Open Access

Comparative genomics allowed the identification
of drug targets against human fungal pathogens
Ana Karina R Abadio1,3, Erika S Kioshima1, Marcus M Teixeira1, Natalia F Martins2, Bernard Maigret3,
Maria Sueli S Felipe1*

Abstract
Background: The prevalence of invasive fungal infections (IFIs) has increased steadily worldwide in the last few
decades. Particularly, there has been a global rise in the number of infections among immunosuppressed people.
These patients present severe clinical forms of the infections, which are commonly fatal, and they are more
susceptible to opportunistic fungal infections than non-immunocompromised people. IFIs have historically been
associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies,
including side effects, toxicities, drug interactions and antifungal resistance. Thus, the search for alternative
therapies and/or the development of more specific drugs is a challenge that needs to be met. Genomics has
created new ways of examining genes, which open new strategies for drug development and control of human
diseases.
Results: In silico analyses and manual mining selected initially 57 potential drug targets, based on 55 genes
experimentally confirmed as essential for Candida albicans or Aspergillus fumigatus and other 2 genes (kre2 and
erg6) relevant for fungal survival within the host. Orthologs for those 57 potential targets were also identified in
eight human fungal pathogens (C. albicans, A. fumigatus, Blastomyces dermatitidis, Paracoccidioides brasiliensis,
Paracoccidioides lutzii, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum). Of those, 10
genes were present in all pathogenic fungi analyzed and absent in the human genome. We focused on four
candidates: trr1 that encodes for thioredoxin reductase, rim8 that encodes for a protein involved in the proteolytic
activation of a transcriptional factor in response to alkaline pH, kre2 that encodes for a-1,2-mannosyltransferase and
erg6 that encodes for Δ(24)-sterol C-methyltransferase.
Conclusions: Our data show that the comparative genomics analysis of eight fungal pathogens enabled the
identification of four new potential drug targets. The preferred profile for fungal targets includes proteins
conserved among fungi, but absent in the human genome. These characteristics potentially minimize toxic side
effects exerted by pharmacological inhibition of the cellular targets. From this first step of post-genomic analysis,
we obtained information relevant to future new drug development.

Background
The frequency and diversity of invasive fungal infections
have changed over the last 25 years. The emergence of
less common, but medically important, fungi has
increased, especially in the large populations of immunocompromised patients and of those hospitalized with
serious underlying diseases [1,2]. These patients develop
more severe clinical forms of mycoses, which are commonly fatal, and they are more susceptible to infections
* Correspondence: msueli@unb.br
1
Department of Cellular Biology, University of Brasília, Brasília, Brazil
Full list of author information is available at the end of the article

by opportunistic fungi than non-immunocompromised
people [3]. The antifungal agents currently available for
the treatment of systemic mycoses include four groups
of drugs: polyenes (amphotericin B), azoles (fluconazole,
itraconazole, ketoconazole, posaconazole and voriconazole), echinocandins (caspofungin, anidulafungin, and
micafungin) and flucytosines [4]. Conventional amphotericin B, despite being a broad-spectrum fungicidal
agent with little intrinsic or acquired resistance, is limited by its serious toxicities and lack of an oral formulation for systemic therapy. In recent years, three lipid
formulations of amphotericin B (amphotericin B lipid

© 2011 Abadio et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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Abadio et al. BMC Genomics 2011, 12:75
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complex, amphotericin B cholesteryl sulfate and liposomal amphotericin B) have been developed and approved
by the Food and Drug Administration (FDA). Although
less nephrotoxic than deoxycholate amphotericin B,
lipid amphotericin B nephrotoxicity still limits treatment
compared to the newer triazoles and echinocandins [5].
The triazoles are the most widely used antifungal agents
and have activity against many fungal pathogens, with
less serious nephrotoxic effects observed than with
amphotericin B. However, the azoles antifungals have
many drug-drug interactions with multiple drug classes
owing to their interference with hepatic cytochrome P450 enzymes [6]. Another problem with azoles therapy
is the acquired resistance of many pathogens to these
drugs, which is the most common cause of refractory
infection. Thus, the search for alternative therapies and/
or the development of more specific drugs is a challenge. Recently, efforts have been devoted to the chemistry side of discovering new antifungal agents, including
the development of third-generation azoles or a new
therapeutic class of antifungal drugs, such as echinocandins [7]. Additionally, nanotechnology approaches have
improved the development of innovative products that
reduce side effects by lowering dose administration of
already available drugs, such as amphotericin B nanoencapsulated [8-10]. Many advances have been made in
antifungal drug development in the past decade. However, the search for more specific drugs, in an effort to
overcome the global problem of resistance to antifungal
agents and minimize the serious side effects, is increasingly relevant and necessary.
Currently, drug research and development are expensive and time consuming. An estimated 14 years and an
average of $1.8 billion is the investment required to
develop a new drug that will reach the market [11].
Selecting new molecular targets by comparative genomics, homology modeling and virtual screening of compounds is promising in the process of new drug
discovery. In fact, technological advances over the past
two decades have led to the accumulation of genomewide sequence data for many different fungal species. As
the number of sequenced genomes rapidly increases,
searching and comparing sequence features within and
between species has become a part of most biological
inquires [12]. Currently, 183 fungi genomes have been
sequenced, either completely or are in the process of
sequencing, and 40 human pathogenic fungi genomes
have been sequenced. (Data collected on 09/07/2010 in
the following databases: Fungal Genomes, TIGR, Sanger,
Broad Institute and NCBI). Seven of the human pathogens are of great importance in systemic mycosis: Candida albicans, Aspergillus fumigatus, Blastomyces
dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Paracoccidioides brasiliensis and Histoplasma

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capsulatum, which are strong candidates for post-genomic studies.
Comparative genomics strategy is a useful tool in
identifying potential new drug targets, such as putative
essential genes and/or those affecting the cell viability
that are conserved in pathogenic organisms [13-16]. By
this methodology, ten genes conserved in three bacteria
species (Staphylococcus aureus, Mycobacterium tuberculosis and Escherichia coli 0157: H7) were selected as
candidates for an antibacterial drug [14]. Since the
publication of the nematode Brugia malayi complete
genome, Kumar and colleagues [15] conducted a comparison analysis between the genomes of B. malayi and
Caenorhabditis elegans and were able to identify 7,435
orthologs genes, from which 589 were identified as
essential, as well as absent in the human genome, resulting in a list of candidate target genes for new drug
development. Recently, Caffrey and colleagues [16] identified new drug targets in the metazoan pathogen Schistosoma mansoni, the causative agent of Schistosomiasis.
The authors identified 35 orthologs essential genes and
potential drug targets against this human pathogen.
Here we identified potential drug targets applied to
human fungal pathogens using comparative genomics
strategy. Ten genes were present in all pathogenic fungi
analyzed and absent in the human genome. Among
them, four genes (trr1, rim8, kre2 and erg6) were
selected for future research and new drug development.
Two of those genes codify for proteins (TRR1 and
KRE2) that showed significant identity when compared
to templates already deposited in the databank PDB
(Protein Database Bank), which were used to perform
homology modeling of both enzymes. These results
will be used to virtually screen combinatorial libraries,
offering new perspectives on technological development
and innovation of antifungal agents against human
pathogens.

Results and Discussion
Drug target selection

Direct demonstration of A. fumigatus and C. albicans
gene essentiality was achieved using conditional promoter replacement (CPR) [17] and gene replacement and
conditional expression (GRACE) strategies [18], respectively. Therefore, the initial ensemble of genes, experimentally described as essential in C. albicans and/or
A. fumigatus were used to identify 55 orthologs. In addition, two non-essential genes (kre2 and erg6), but which
are important to cell viability within the host [19,20],
were added to the list of possible drug targets. The
alignments of those 57 sequences against the genome of
the 8 pathogenic fungi P. lutzii, P. brasiliensis isolates
(Pb18 e Pb3), A. fumigatus, B. dermatitidis, C. albicans,
C. immitis, C. neoformans, H. capsulatum confirmed the

Abadio et al. BMC Genomics 2011, 12:75
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presence of all the genes (Additional file 1). As a result,
ten conserved genes were selected as drug targets
because they were present in all species analyzed and
were also absent in the human genome, as shown in
Table 1.
Six criteria were used to select the potential targets: 1)
be essential or relevant for fungi survival; 2) be present
in all analyzed pathogens, therefore allowing a broad
spectrum of drug action; 3) be absent in the human
genome, therefore avoiding unwanted side effects; 4) be
preferentially an enzyme and have the potential for
assayability; 5) not be auxotrophic, thereby avoiding
host provision of the necessary substrate for the blocked
pathway; and 6) have a cellular localization potentially
accessible to the drug activity. Applying these criteria,
four potential drug targets were identified: trr1, rim8,
kre2 and erg6 genes. Only trr1 and rim8 are essential
genes, but kre2 and erg6 are involved in cell viability
and survival within the host. In addition, those genes
were also identified as potential drug targets in P. lutzii
isolate Pb01 transcriptome, as described by Felipe and
colleagues [21].
The trr1 is an essential gene that encodes for the cytoplasmatic enzyme thioredoxin reductase [22]. This protein plays a critical role in maintaining the cell redox
status [22] and is part of the complex so-called thioredoxin system, which contains thioredoxin (Trx), thioredoxin reductase (Trr) and NADPH, protecting cells
against oxidative stress [23]. Thioredoxin reductase is
necessary for the viability of C. neoformans [24] and is
essential for erythrocytic stages in Plasmodium falciparum [25]; it also appears to be essential for growth in
S. aureus [26]. S. cerevisiae strain deleted for trr1 gene

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is hypersensitive to hydrogen peroxide and high temperatures [27,28].
The rim8 is also an essential gene that encodes for a
protein involved in the proteolytic activation of a transcriptional factor in response to alkaline pH and is
located near the plasma membrane [29]. RIM8 (for
yeasts) or PalF (for filamentous fungi) protein binds
strongly to the C-terminal cytoplasmic tail of the seven
transmembrane domains, the putative pH sensor PalH.
Alignment of protein sequences suggests structural similarity of RIM8 to mammalian arrestins, but the sequence
similarity was restricted to short stretches of amino acid
sequences, mostly corresponding to b-strands in arrestin
crystal structures [30]. The RIM8 protein performs an
essential step in the signaling pathway activating
RIM101, which, in turn, regulates alkaline pH-response.
This pathway is also involved in the activation of the
yeast-to-hyphal transition required for host-pathogen
interaction [31].
The kre2 gene encodes for the enzyme a-1,2-mannosyltransferase that is located in the Golgi complex. It has a
short amino-terminal cytoplasmic domain, a hydrophobic
membrane-spanning domain and a large carboxy-terminal
catalytic domain [32]. This enzyme is responsible for the
addition of the a-1,2-linked mannose residues to O-linked
carbohydrates and is also involved in N-linked glycosylation [33-36]. Cell wall-associated proteins are commonly
glycosylated and defects in this process may result in protein misfolding, instability, and/or reduced enzymatic
activity [36]. Absence of MNT1p in S. cerevisiae resulted
in the synthesis of truncated O-linked oligosaccharides
and this interfered with the functioning and/or synthesis
of cell wall compounds [33,34]. Mutants of C. albicans

Table 1 Potential target genes selected for new antifungal drug development
PDB
template

Organism

Evalue

PDB sequence identidy
(%)

1ITJ

Saccharomyces cerevisiae

3e115

65

1VDC

Arabidopsis thaliana

1e-94

57

*
1HV8

*
Methanocaldococcus
jannaschii

*
7e-42

*
30

*

*

*

*

Mitochondrion membrane
Golgi apparatus Membrane

2QK9
2VY0

Homo sapiens
Pyrococcus furiosus

0.8
6e-4

34
32

Membrane

1R1M

Neisseria meningitidis

0.3

32

Gene

Biological process

Cytolocalization

trr1

Cell redox homeostasis

Cytoplasm

aur1
mak5

Cellular metabolism
Ribosome biogenesis

Golgi and membrane
Nucleolus

chs1

Cell wall biogenesis/
degradation

Membrane

tom40 Protein transport
kre6
Cell wall biogenesis/
degradation
fks1
Cell wall organization/
biogenesis
kre2
Protein mannosilation

Golgi membrane

1S4N

Saccharomyces cerevisiae

6e-96

50

erg6

Ergosterol biosynthesis

Endoplasmatic reticulum
membrane

3BUS

Lechevalieria
aerocolonigenes

5e-18

32

rim8

pH-response regulator

Cytoplasm

3G3L

Bacteroides fragilis

3,9

38

*Structure absent in PDB (http://www.rcsb.org/pdb/home/home.do).

Abadio et al. BMC Genomics 2011, 12:75
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that lack CaMNT1 and CaMNT2 have truncated O-mannan, marked reduction in adherence and attenuated virulence [34]. Although CaMNT1p is not essential for
viability, MNT1p-mediated O-glycosylation of proteins of
C. albicans is essential for normal host-fungus interactions
[37].
The erg6 gene encodes for the enzyme Δ-(24)-Sterol
C-methyltransferase that is located in the endoplasmic
reticulum. It shows a transmembrane portion and an
active site positioned toward the cytoplasm [38,39]. This
enzyme catalyzes the attachment of a methyl group acting in a bifurcation point of the ergosterol/cholesterol
biosynthesis pathway [40]. In S. cerevisiae, erg6 mutants
showed alteration in membrane fluidity and permeability
[41,42]. In C. albicans, mutants that do not synthesize
Δ-(24)-Sterol C-methyltransferase showed an increase in
the plasma membrane permeability, resulting in cells
with severely compromised phenotypes [20]. erg6
mutants, in Candida lusitaniae, showed a severe growth
defect and decreased ergosterol content [43].
Conserved domains in protein sequences and
phylogenetic analysis

A multiple protein sequence alignment showed the presence of conserved domains mainly in the catalytic site of
the four selected candidates (Additional files 2, 3, 4, 5).
The catalytic site of the protein TRR1 contains the fouramino acid-residue sequence Cys-Ala-Thr-Cys
[44,22,45], and these two highly conserved cysteine residues (Cys142 and Cys145 in C. albicans) are essential for
its redox activity (Additional file 2). RIM8 protein alignments showed that in the C-terminal domain, the residue
Ile-331 of A. nidulans, involved in PalF-PalH receptor
binding, was conserved in all the fungi sequences (it is
located in position Ile-320 in P. brasiliensis). The amino
acid residue Ser-86 of A. nidulans, present in the
N-terminal domain and responsible for PalF-PalH interaction and pH signaling, was replaced by the conserved
Cys-75 in P. brasiliensis, B. dermatitidis, H. capsulatum,
Cys-76 in C. immitis and Cys-77 in A. fumigatus (Additional file 3).
The catalytic site of KRE2 proteins contains the conserved amino acid residues His312, His377, Asp350 and
Glu318 in C. albicans. If those residues are individually
replaced, in C. albicans, the enzyme activities are fully
abolished [46,47]. Our analysis showed highly conserved
catalytic domains of all the sequences of the analyzed
proteins for all the pathogenic fungi. The domain
YNLCHFWSNFEI, previously described as important to
the catalysis mechanism, was also conserved in all fungi
analyzed (Additional file 4).
The ERG6 protein showed four conserved regions in
several sterol methyltransferase (SMT) proteins, including Regions II, III and IV, generally present in AdoMet-

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dependent enzymes and, Region I, observed in all SMT
enzymes [48]. Region I, a highly conserved region rich
in aromatic amino acids, contains a signature motif
YEXGWG [49]. The mutation of the amino acid residues situated in Region I altered the catalytic behavior
of the fungal SMT [50-52]. In addition, specific-site
mutation in Region II and Region III of ERG6 protein
showed that certain residues (Cys128, Gly129, Pro133
and Ala193 in S. cerevisiae) were important to C-methylation activity [52]. All important amino acid residues
for ERG6 protein activity were conserved in all fungi
genomes analyzed (Additional file 5).
The alignments of protein sequences were also used in
phylogenetic studies performed by Bayesian analysis to
construct phylogenetic trees relating TRR1, RIM8, KRE2
and ERG6 orthologs. The phylogenetic trees showed the
evolutionary relationships between the different species
used in this work and separated them in different
groups (Figure 1). P. brasiliensis, B. dermatitidis, H. capsulatum, A. fumigatus and C. immitis were clustered
apart from C. albicans and C. neoformans. The posterior
probability values were added to the phylogenetic
branches and received values near 1, showing the consistency and reliability of these branches. In the four
phylogenetic trees, the P. brasiliensis isolate Pb01 was
separated from the other isolates (Pb3 and Pb18). These
findings are in agreement with Teixeira et al. 2009 [53],
in which 13 single-locus topologies showed that the
genus Paracoccidioides contains two highly divergent
groups. As proposed by Teixeira and colleagues [53],
these results reinforce the existence of two species for
Paracoccidioides genus: P. brasiliensis (Pb18 and Pb3)
and P. lutzii (Pb01).
Homology modeling of TRR1 and KRE2

In the absence of experimentally solved structures, computational methods were used to predict 3D protein
models and provide information regarding protein functions and structures [54]. Homology modeling is efficient in new drug design, from the biological target
conception through new drug discovery [55]. Of the
four selected potential targets obtained from our comparative genomic analyses, only TRR1 and KRE2 showed
a reasonable sequence identity to the templates found in
PDB (Table 1). Consequently, we performed the homology modeling only for these two proteins.
According to the BLAST search performed on the entire
PDB database, the thioredoxin reductase (TRR1) of P. brasiliensis showed good sequence identity with two templates, specifically 3ITJ (PDB ID) of S. cerevisiae (65%
sequence identity) and 1VDC (PDB ID) of Arabidopsis
thaliana (57% sequence identity). In the case of a-1,2mannosyltransferase (KRE2) of P. brasiliensis, only the
PDB template 1S4N (PDB ID) of S. cerevisiae showed a

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Figure 1 Phylogenetic analysis between human pathogenic fungi performed by Bayesian analysis. Phylogenetic trees generated from
Bayesian analysis of the genes trr1 (a), rim8 (b), kre2 (c) and erg6 (d). The length of the vertical lines linking one protein is proportional to the
estimated distance between their sequences. The posterior probability values were added to the phylogenetic branches. Af: Aspergillus fumigatus,
Bd: Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis, Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum,
Pl: Paracoccidioides lutzii, Pb3: P. brasiliensis isolate 3, Pb18: P. brasiliensis isolate 18.

reasonable sequence identity (50%). Starting from the
BLAST alignment between P. brasiliensis TRR1 and KRE2
proteins with the PDB templates as found above, we
manually modified them in order to preserve the secondary structures and the correspondence between cysteine
residues (Additional file 6a and 6b).
In these alignments between the target sequences and
template structures, a fragment of the C-terminus
region of TRR1 (Glu325-Leu358) and of the N-terminus
region of KRE2 (Met1-Phe70) did not align. Therefore, a
BLAST search with the fragment sequences was performed to verify if these regions had similarity with proteins deposited in the PDB. No confirmation was found,
so these fragment terminus regions of TRR1 and KRE2

were removed from the models. This was legitimate,
since these fragments are not involved in the active site
of the proteins and should not interfere with the virtual
screening that we intend to perform using these models.
The refinement of the homology models was obtained
through molecular mechanics optimization; the stable
structures of TRR1 and KRE2 are displayed in Figure 2.
Figure 2a shows that the enzyme TRR1 has 9 helices
and 17 sheets. Figure 2b shows that the enzyme KRE2
has 18 helices and 11 sheets.
The TRR1 monomeric protein of S. cerevisiae is composed of two domains that form the binding sites of
NADPH and FAD similar to plants. The FAD molecule
is bound to the S. cerevisiae TRR1 protein and is

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Figure 2 The predicted tridimensional structure of TRR1 and KRE2 proteins obtained by homology modeling. The structures of TRR1
(a) and KRE2 (b) proteins. The a-helix is represented by the color red, the b-sheet is represented by yellow and loops are represented by green.
(a - left panel) The KRE2 active site presents the conserved residues His292, His357, Asp330 and Glu298. (Mutation of these residues abolished
the protein activity in C. albicans.) Additional residues found in the KRE2 active site include Glu216, which interacts with the metal ion Mn2+ and
creates the reactive nucleophylic center for the glycosyltransferase reaction, and Tyr189, which coordinates the donor and acceptor binding that
allows the transfer of the mannose to the growing oligosaccharide. (b - right panel) The TRR1 protein is composed of two domains that
comprise the binding sites of NADPH and FAD. The NADPH binding domain contains the active Cys145 and Cys148 residues. Other important
residues of the TRR1 active site are Ala151, Val152, Pro153 and Ile154 that form a hydrophobic region in the NADPH binding domain.

stabilized by interactions with the residues Pro13, Glu33,
Gln45, Asn54, Gln136, Asp288 and Gln296. The
NADPH binding domain contains active cysteine residues and is linked to the FAD domain by a short
b-sheet [56,57]. Figure 2a shows the two domains in the
P. brasiliensis TRR1 model.
In KRE2 protein, the catalytic mechanism of the active
site involves nucleophylic substitutions mediated by
acidic amino acid residues and an essential Mn2+ cofactor. Heterologous expression of site-specific mutants of
C. albicans MNT1 protein in Pichia pastoris confirmed

the nature of a nucleophilic reaction center, where the
two conserved histidines (His292 and His357 in P. brasiliensis) that coordinated the metal ion cofactor Mn 2+
and created the reactive nucleophylic center required
the nonprocessing, GDP-mannose-dependent, retaining
glycosyltransferase reaction [46]. However, Lobsanov
and colleagues [47] examined the structure and catalysis
mechanism of S. cerevisiae KRE2 enzyme (1S4N template) by crystallography and proposed a novel mechanism for this interaction, and the precise function for the
conserved amino acids was determined by site-direct

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mutagenesis by Thomson and colleagues [46]. The proposed mechanism of retaining glycosyltransferases such
as CaMNT1p involves a two-step displacement. The
first step involves attack on the sugar anomeric center
by one of the carboxylates, and then a second carboxylate acts as the active site nucleophilic to displace the
GDP from the sugar nucleotide, leading to formation of
a glycosyl-enzyme intermediate. The metal ion Mn2+ is
coordinated by a direct interaction of the residue glutamate (Glu216 in KRE2 protein of P. brasiliensis) as
shown in Figure 2b, right panel. Transfer of the mannose to the growing oligosaccharide is completed by displacement of the enzyme from the intermediate by the
hydroxyl group of the acceptor [46]; in C. albicans
KRE2 protein, tyrosine (Tyr209) coordinates the donor
and acceptor binding from the N-terminal domain and
plays a role in the catalysis [47].

Conclusions
We reported a comparative genomic strategy to provide
a list of potential antifungal drug targets for the human
pathogenic fungi P. brasiliensis, P. lutzii, A. fumigatus,
B. dermatitidis, C. albicans, C. immitis, C. neoformans
and H. capsulatum. The preferred profile for fungal targets was proteins conserved among these fungi, but
absent in the human genome, aiming to minimize the
potential toxic side effects exerted by pharmacological
inhibition of the cellular targets. In general, the potential
drug targets were selected following the criteria of
essentiality, presence in all human pathogenic fungi considered here, absence in humans, be preferentially an
enzyme, not be auxotrophic and have accessible cell
localization.
In silico and manual mining provided four genes as
potential drug targets: trr1 that encodes for thioredoxin
reductase, rim8 that encodes for a protein involved in
the proteolytic activation of a transcriptional factor in
response to alkaline pH, kre2 that encodes for a-1,2mannosyltransferase and erg6 that encodes for Δ-(24)Sterol C-methyltransferase. The increase in structural
databases allows the satisfactory prediction of structures
by theoretical methods, with advantages over more
costly experimental methods. We performed the homology modeling for the potential targets that were identified to have a known 3D structure and that showed
good sequence identity to the templates found in PDB,
TRR1 and KRE2. In the absence of structures solved
experimentally, the available homology modeling tools
were extremely useful for the structural prediction of
the TRR1 and KRE2 proteins. From this first step of
post-genomic analysis, we obtained relevant information
for future technological development. Moreover, these
results are being used to virtually screen chemical

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libraries, which are under progress, generating new perspectives on technological development and innovation
of antifungal agents to these human pathogens.

Methods
Comparative analysis of human pathogenic fungi
genomes and drug target selection

The identification of potential drug targets was based on
55 genes experimentally confirmed as essential for Candida albicans [17] or Aspergillus fumigatus [18]. In
these cases, the genes were experimentally confirmed as
essential and represent a large spectrum of biological
functions, such as cellular metabolism, cell wall organization and biogenesis, ergosterol biosynthesis, ribosomal
biogenesis and post-translational modification of protein
[17,18]. Other 2 genes (kre2 and erg6) were added to the
initial screening since they were described as potential
drug targets [21].
The 57 gene sequences of were retrieved from the
GenBank databases (http://www.ncbi.nlm.nih.gov/) and
were used to screen the P. brasiliensis Pb01 transcriptome database (https://helix.biomol.unb.br/Pb/) using
blastn. The sequences of Paracoccidioides lutzii isolate
Pb01 were not applied as a filter since all 57 genes were
present and expressed in its genome/transcriptome.
Subsequently, the presence of these genes in the 2 isolates of P. brasiliensis (Pb3 and Pb18) was confirmed.
Using the isolate Pb01 sequence, released by Broad
Institute (http://www.broad.mit.edu/), the orthologs
search in other pathogenic fungi (A. fumigatus Af293,
B. dermatitidis ER3, C. albicans WO1, C. immitis
H538.4, C. neoformans serotype B, H. capsulatum
NAm1) and human genome was completed using blastx
because we have focused in the development of a new
antifungal not only for the Paracoccidioides species but
for all medically important fungi. The cut-off established for determining the presence of an ortholog was
a maximum E-value of 0.00001 (1e-5). A manual curation was performed to select the potential drug targets
following the criteria of essentiality, be present in
pathogenic fungi, be absent in humans, be preferentially
an enzyme, not be auxotrophic and have accessible cell
localization.
Multiple alignments of the orthologs genes and
phylogenetic analysis

Sequences were aligned by the ClustalW using dynamic
programming and hierarchical methods [58] available in
the BioEdit software [59]. The program identified conserved regions in the protein sequences between orthologs target genes by multiple sequence alignments.
The sequences were also used for phylogenetic analysis by Bayesian inference using Mr. Bayes software,

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version 3.1.2 [60]. Detected gaps in sequence alignments
were considered missing data and coded in terms of
presence or absence. The amino acid substitution model
selected was JTT [61]. The Markov Chain Monte Carlo
(MCMC) was initiated from a random tree and processed for 1000000 generations; sample trees were
retrieved every 1000 generations. Log-likelihood values
were plotted against the generation number, and the
first 25% of samples were discarded (”burn-in“). The
remaining samples were used to determine the distribution of posterior probability values. Phylogenetic trees
were produced with the help of the Treeview and Figtree 1.0 software.
Protein structure prediction

There is no crystallographic structure presently available for TRR1 and KRE2 of P. brasiliensis and also for
the other pathogenic fungi. Therefore, the 3D structures of TRR1 and KRE2 of P. brasiliensis were constructed by homology modeling based on known
structures with high percentage of identity in amino
acid sequences. We have initially modeled P. brasiliensis proteins but it will be similar for the other pathogenic fungi since the sequences of the proteins are
highly conserved. The known template structures were
searched in the PDB. There were two templates for
TRR1 protein: 3ITJ (PDB ID) of S. cerevisiae and
1VDC (PDB ID) of A. thaliana. There was one template for KRE2 protein: 1S4N (PDB ID) of S. cerevisiae.
The templates structures for ERG6 and RIM8 showed
low sequence identity, then not allowed the construction of 3D structures for these proteins by molecular
modeling. The amino acid residue sequences of TRR1
and KRE2 were compared with the primary sequences
of the structures deposited in the PDB using the
BLAST program. The homologous sequences allowed
the construction of a 3D model of TRR1 and KRE2
using the homology module of the Insight II software
package (Biosym/MSI, San Diego, Accelrys Inc. 2001).
Briefly, the target sequences were aligned with the
template structures, and coordinates from the templates were transferred to the targets TRR1 and KRE2.
For model optimization, the backbone atoms of the
structures were initially frozen and only the side chains
were allowed to move for a selective minimization by
conjugate gradient method. A second selective minimization, also by conjugate gradient method, was performed with only atoms of the complementary
determining region (CDR) loops moving. The last
minimization was performed by Steepest-descent
method with all atoms of the structure relaxed, resulting in whole, refined 3D structures. The molecular
visualization was performed by PyMOL open-source
software version 0.99rc6 (Delano Scientific LLC, 2006).

Page 8 of 10

Additional material
Additional file 1: Essential genes found in C. albicans and/or
A. fumigatus and orthologs in other human pathogenic fungi.
Additional file 2: Amino acid alignment between conserved protein
residues of TRR1, in the human pathogenic fungi. Amino acid
sequence analysis of TRR1 protein. Af: Aspergillus fumigatus, Bd:
Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis,
Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum, Pb01:
Paracoccidioides brasiliensis isolate 01, Pb3: P. brasiliensis isolate 3, Pb18:
P. brasiliensis isolate 18. Positions of identity are indicated with asterisks, a
semicolon indicates conserved substitutions, and a dot shows a semiconservative substitution.
Additional file 3: Amino acid alignment between conserved protein
residues of RIM8, in the human pathogenic fungi. Amino acid
sequence analysis of RIM8 protein. Af: Aspergillus fumigatus, Bd:
Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis,
Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum, Pb01:
Paracoccidioides brasiliensis isolate 01, Pb3: P. brasiliensis isolate 3, Pb18:
P. brasiliensis isolate 18. Positions of identity are indicated with asterisks, a
semicolon indicates conserved substitutions, and a dot shows a semiconservative substitution.
Additional file 4: Amino acid alignment between conserved protein
residues of KRE2, in the human pathogenic fungi. Amino acid
sequence analysis of KRE2 protein. Af: Aspergillus fumigatus, Bd:
Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis,
Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum, Pb01:
Paracoccidioides brasiliensis isolate 01, Pb3: P. brasiliensis isolate 3, Pb18:
P. brasiliensis isolate 18. Positions of identity are indicated with asterisks, a
semicolon indicates conserved substitutions, and a dot shows a semiconservative substitution.
Additional file 5: Amino acid alignment between conserved protein
residues of ERG6, in the human pathogenic fungi. Amino acid
sequence analysis of ERG6 protein. Af: Aspergillus fumigatus, Bd:
Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis,
Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum, Pb01:
Paracoccidioides brasiliensis isolate 01, Pb3: P. brasiliensis isolate 3, Pb18:
P. brasiliensis isolate 18. Positions of identity are indicated with asterisks, a
semicolon indicates conserved substitutions, and a dot shows a semiconservative substitution.
Additional file 6: Manual alignments performed between P.
brasiliensis proteins and the PDB templates. (a) TRR1 protein and
templates 3ITJand 1VDC. The boxes represent the template regions that
were used as references for the homology modeling of TRR1 protein. In
the alignment between TRR1 protein and the templates, the big boxes
indicate that the 1VDC template was used as reference and the small
box indicates that the reference was the 3ITJ template. Considering the
global alignment, 3ITJ is the best template to use as a reference to
perform the homology modeling of P. brasiliensis TRR1 protein. However,
some regions of the 1VDC template present identical amino acids to P.
brasiliensis TRR1 protein, and those are different in the 3ITJ template. The
following colors represent amino acids: white (identical amino acids
between TRR1 protein and the two templates), green (identical amino
acids between TRR1 protein and 1VDC template), red (identical amino
acids between TRR1 protein and 3ITJ template), orange (unique amino
acids in 3ITJ template), dark blue (unique amino acids in 1VDC template),
and light blue (unique amino acids in TRR1 protein). The cysteine
residues that form the disulfide bonds are conserved between TRR1
protein and the two templates. (b) KRE2 protein and templateIS4N. The
boxes represent the template regions that were used as references for
the homology modeling of KRE2 protein. In the alignment between KRE2
protein and the 1S4N template, the boxes indicate the regions that were
used as references for 3D structure construction of KRE2. The following
colors represent amino acids: white (identical amino acids between KRE2
protein and the template), purple (similar amino acids between KRE2
protein and the template), red (unique amino acids in KRE2 protein), and
light blue (unique amino acids in 1S4N template). The cysteine residues
that form the disulfide bonds are conserved between KRE2 protein and
the template.

Abadio et al. BMC Genomics 2011, 12:75
http://www.biomedcentral.com/1471-2164/12/75

Acknowledgements
The authors gratefully acknowledge the Brazilian Agencies CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico) and FAP-DF
(Fundação de Apoio a Pesquisa do Distrito Federal) for financial support.
Author details
1
Department of Cellular Biology, University of Brasília, Brasília, Brazil.
2
Embrapa - Genetic Resources and Biotechnology, Brasília, Brazil. 3Lorrain
Laboratory of Computing Research and its Applications, University Henri
Poincaré-Nancy I, Nancy, France.

Page 9 of 10

17.

18.

19.
Authors’ contributions
AA, NM, MF and EK planned and designed the study, developed the
experiments and completed the data analysis, wrote the main draft of the
paper and supported the preparation of the figures and tables. BM
participated in the homology modeling experiments and helped in the
manuscript editing. MT participated in the phylogenetic analysis. All authors
read and approved the final manuscript.

20.

21.

Received: 26 August 2010 Accepted: 27 January 2011
Published: 27 January 2011
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doi:10.1186/1471-2164-12-75
Cite this article as: Abadio et al.: Comparative genomics allowed the
identification of drug targets against human fungal pathogens. BMC
Genomics 2011 12:75.

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