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Biochemical characterization of potential virulence markers in the human fungalpathogen Pseudallescheria boydiiAndré L.S. Santos a; Vera C. B. Bittencourt b; Marcia R. Pinto c; Bianca A. Silva a; Eliana Barreto-Bergter b
a Laboratório de Estudos Integrados em Bioquímica Microbiana, b Laboratório de Química Biológica deMicrorganismos, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes(IMPPG), Centro de Ciências da Saúde (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilhado Fundão, Rio de Janeiro, RJ, Brazil c Departamento de Microbiologia, Instituto de Ciências Biológicas,Universidade de São Paulo, São Paulo, SP, Brazil
First Published on: 23 February 2009
To cite this Article Santos, André L.S., Bittencourt, Vera C. B., Pinto, Marcia R., Silva, Bianca A. and Barreto-Bergter,Eliana(2009)'Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheriaboydii',Medical Mycology,47:4,375 — 386
To link to this Article: DOI: 10.1080/13693780802610305
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Biochemical characterization of potential virulence
markers in the human fungal pathogen Pseudallescheria
boydii
ANDRE L.S. SANTOS*, VERA C.B. BITTENCOURT$, MARCIA R. PINTO%, BIANCA A. SILVA* &
ELIANA BARRETO-BERGTER$*Laboratorio de Estudos Integrados em Bioquımica Microbiana, $Laboratorio de Quımica Biologica de Microrganismos,Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes (IMPPG), Centro de Ciencias da Saude(CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundao, Rio de Janeiro, RJ, Brazil, and %Departamentode Microbiologia, Instituto de Ciencias Biologicas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a
saprophytic filamentous fungus recognized as a potent etiologic agent of a wide
variety of infections in immunocompromised as well as in immunocompetent
patients. Very little is known about the virulence factors expressed by this fungal
pathogen. The present review provides an overview of recent discoveries related to
the identification and biochemical characterization of potential virulence attributes
produced by P. boydii, with special emphasis on surface and released molecules.
These structures include polysaccharides (glucans), glycopeptides (peptidorham-
nomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases,
phosphatases and superoxide dismutase), which have been implicated in some
fundamental cellular processes in P. boydii including growth, differentiation and
interaction with host molecules. Elucidation of the structure of cell surface
components as well as the secreted molecules, especially those that function as
virulence determinants, is of great relevance to understand the pathogenic
mechanisms of P. boydii.
Keywords Pseudallescheria boydii, peptidorhamnomannans, glucans,
glucosylceramides, hydrolytic enzymes, virulence
Introduction
Pseudallescheria boydii (anamorph Scedosporium apios-
permum) is a ubiquitous ascomycetous fungus found
worldwide in soil, manure, decomposing vegetable
materials and multiple water sources including creeks
contaminated with sewage and standing water [1]. P.
boydii causes human infections by inhalation or after
traumatic subcutaneous implantation, allowing hyphal
fragments and conidial forms to penetrate the skin. The
clinical manifestations of P. boydii infections, collec-
tively termed pseudallescheriasis, are quite varied since
these infections can affect practically all the organs of
the body. The most common manifestation is myce-
toma, a chronic granulomatous infection of the skin
and subcutaneous tissue [2,3]. Following this subcuta-
neous infection, the lungs are the most common site of
P. boydii infection. Pulmonary infection ranges from
colonization of bronchiectatic and tuberculous cavities
to invasive necrotizing pneumonia [3]. For instance, P.
boydii is among the most common filamentous fungi
Andre L.S. Santos and Eliana Barreto-Bergter are members of the
ECMM/ISHAM Working Group on Pseudallescheria/Scedosporium
Infections. Part of this work was presented during the last meeting of
the working group that was held in Angers (France) on June 2007.
Correspondence: E. Barreto-Bergter, Departamento de
Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de
Goes (IMPPG), Centro de Ciencias da Saude (CCS), Bloco I,
Universidade Federal do Rio de Janeiro (UFRJ). Tel: � 55 2562
6741; fax: � 55 2560 8344; E-mail: eliana.bergter@micro.ufrj.br
Received 30 April 2008; Received in final revised form
29 October 2008; Accepted 9 November 2008
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colonizing the lungs of patients with cystic fibrosis with
a frequency ranging from 0.7�9% [4]. The lungs alsoappear to be the site of entry for most cases of
disseminated pseudallescheriasis. Nonmycetoma P.
boydii infections occur almost exclusively in compro-
mised hosts. These infections have been known for a
long time, but in recent years, a marked increase in
severe invasive infections has been noticed, mainly in
immunocompromised hosts [5]. The optimal treatment
for these infections is unknown, and the mortality rateis very high despite aggressive antifungal treatment [3].
The role of P. boydii in fatal infections may be
underestimated due to the present lack of detailed
diagnostics. Diagnosis of a Pseudallescheria/Scedospor-
ium infection is difficult, because clinical features and
histopathology are similar to those of aspergillosis,
fusariosis, and other relatively common hyalohyphomy-
cosis [6,7]. On the basis of nuclear DNA-DNA reasso-ciation, some studies have proved that important genetic
variation exists in P. boydii [4,8,9]. Other authors have
reported considerable differences with respect to growth
and sporulation [10�13]. In addition, a high variability
in antifungal susceptibility of the different isolates and
in their clinical response has been observed [14,15]. This
could be explained by the fact that P. boydii does not
represent a single species but instead is a complexcomprising at least six known species (P. boydii,
Pseudallescheria angusta, Pseudallescheria ellipsoidea,
Pseudallescheria fusoidea, Pseudallescheria minutispora
and Scedosporium aurantiacum) and two cryptic species
represented by clades 3 and 4 as described by Gilgado et
al. [16]. The discovery of new antifungal agents thus
remains an important challenge for the scientific com-
munity, which is further complicated by the similaritybetween fungal and mammalian cells. Like mammalian
cells, fungi are eukaryotic, so they share many structures
and metabolic pathways with mammalian cells, making
it more difficult to identify specific targets for the
development of new antifungals.
Despite the growing importance of Pseudallescheria/
Scedosporium infections, very little is known about the
physiology and biochemistry of this intriguing humanpathogenic fungus. A brief glimpse at the PubMed
database (www.pubmed.com) corroborates this state-
ment in that it reveals that around 70% of the works are
concerned with clinical case reports and/or epidemiol-
ogy studies, while papers on biochemistry and/or
physiology correspond to less than 4%. In addition,
few studies on innate and adaptive immune response
against the P. boydii complex have recently beenpublished [17�19]. Collectively, these data reinforce
the relevance of biochemical studies in Pseudal-
lescheria/Scedosporium. In this context, the character-
ization of cell wall and other surface components, as
well as secreted molecules are relevant to the develop-ment of new antifungal drugs and for an understanding
of the fungus’ pathogenic mechanisms. In the present
review, we summarize the current knowledge on the
biochemical markers expressed by P. boydii, including
molecules which have been implicated in some funda-
mental cellular processes including growth, differentia-
tion and interaction with host molecules.
Peptidorhamnomannan: a potential antigeninvolved in adhesion
Polysaccharides and peptidopolysaccharides are espe-
cially relevant for the architecture of the fungal cell
wall, but several of them are immunologically active
compounds with great potential as regulators of
pathogenesis and the immune response of the host. Inaddition, some of these molecules can be specifically
recognized by antibodies in patients’ sera, suggesting
that they can be also useful in the diagnosis of fungal
infections [20].
In the search for structures that could be helpful in
the diagnosis of pseudallescheriasis, much attention has
been paid to the study of P. boydii cell wall antigens.
Peptidopolysaccharides have been isolated from itsmycelial form and characterized using chemical and
immunological methods. Hot aqueous extraction, fol-
lowed by treatment with Cetavlon in the presence of
sodium borate, provided a precipitate of peptidorham-
nomannan (PRM) [21]. Sugar component analysis of
the PRM molecule showed the presence of rhamnose
(Rha), mannose (Man), galactose (Gal) and glucose
(Glc) in a ratio of 29.5:60.5:5.5:4.5, respectively.Methylation analysis and 1H- and 13C-nuclear magnetic
resonance (NMR) spectra indicated the presence of a
rhamnomannan with a structure distinct from that of
similar components isolated from Sporothrix schenckii
[22]. Specifically the former consists in a-rhamnopyr-
anosyl-(103)a-rhamnopyranose side-chain epitopes
linked (103) to a (106) linked a-mannopyranosyl
core [21]. This PRM reacted poorly with an antiserumraised against whole cells of S. schenckii and strongly
with one against P. boydii hyphae. These characteristics
and immunological differences suggest that this major
rhamnose-containing antigen of P. boydii may be useful
for the specific diagnosis of infections attributable to
this fungus [21].
PRM from P. boydii mycelia is a complex glycocon-
jugate consisting of a peptide chain substituted withboth O- and N-linked glycans. O-linked oligosacchar-
ides were released by b-elimination under mild alka-
line reducing conditions [23]. Three oligosaccharide
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fractions were obtained and the major oligosaccharide,
a hexasaccharide, was characterized by methylation
analysis, 1H- and 13C-NMR spectroscopy, matrix-
assisted laser desorption ionization mass spectrometry
(MALDI MS) and eletrospray ionization quadrupole
time-of-flight tandem mass spectrometry (ESI MS/
MS). It was a branched structure, with a main chain
of a-rhamnopyranosyl-(103)a-rhamnopyranosyl-(103)-a-mannopyranosyl-(102)-mannitol substituted at
O-6 of mannitol with an a-glucopyranosyl-(104)-b-
galactopyranosyl group [23]. Immunofluorescence ana-
lysis using a polyclonal anti-PRM antibody demon-
strated that the PRM molecule is expressed in both
conidia and mycelia of P. boydii (Fig. 1A).
The O-linked oligosaccharides (Fig. 1B) may account
for a significant part of the antigenicity of PRM,
because de-O-glycosylation decreased by around 80%
its activity. The immunodominance of the O-linked
oligosaccharide chains was evaluated by testing their
ability to inhibit the reactivity between PRM and anti-
P. boydii rabbit antiserum in an enzyme-linked immu-
nosorbent assay (ELISA) hapten system. Up to 75%
inhibition was obtained with the hexasaccharide frac-
tion [23]. Similar results were obtained with the
peptidogalactomannan from Aspergillus fumigatus [24]
and PRM from S. schenckii [25]. Besides the contribu-
tion of O-linked oligosaccharides to the antigenicity of
PRM, O-glycosylation is critical for fungal adhesion to
host cells, because de-O-glycosylation of PRM effi-
ciently inhibits the adhesion of P. boydii conida to
epithelial cells, as well as preventing their endocytosis
[26]. Corroborating this result, the treatment of conidia
with a polyclonal anti-PRM antibody reduced both
adhesion and endocytosis by epithelial cells [26] (Fig.
1C). In a similar way, the pre-incubation of epithelial
cells with soluble PRM drastically diminished the
interaction process [26] (Fig. 1C). Interestingly,
P. boydii PRM binds to a polypeptide of 25 kDa on
Fig. 1 (A) Interferential contrast images
(a, c) and the corresponding immunofluor-
escence labeling (b, d) by anti-peptidorham-
nomannan (PRM) antibody in conidia (a,b)
and mycelia (c,d) of Pseudallescheria boydii.
(A) Major O-linked oligosaccharides from
P. boydii PRM. (C) Inhibition assay of the
interaction between P. boydii conidia and
HEp2 cells by soluble PRM (� PRM) and
by anti-PRM antibody (� anti-PRM), in
contrast to non-treated conidia (control).
(D) Light micrographs of different phases
of the interaction between P. boydii conidia
and A549 lung epithelial cells after 1, 2 and
4 h. Note the germ tube projection from the
conidial cell after 2 and 4 h of interaction
(arrows).
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the HEp2 cell surface, suggesting its participation as an
adhesin molecule [26].Studies on the interaction between P. boydii and
HEp2 cells demonstrated that conidia attached to, and
were ingested by, epithelial cells in a time-dependent
process [26]. Similarly, the same interaction pattern was
observed during the interaction with A549 cells, an
epithelial lung lineage (Fig. 1D). In P. boydii, conidia
differentiation into mycelia is a crucial event during its
life cycle. In this context, after 2�4 h of interaction, theconidia produced a germ-tube like projection, which
was able to penetrate the epithelial cell membrane,
leading to the cell’s death [26] (Fig. 1D). Interestingly,
when conidia were incubated alone in culture medium
(e.g., DMEM or Sabouraud), the germ-tube formation
was observed only after 6 h of incubation [26]. The
differentiation of conidia into mycelia is an important
step observed in other opportunistic fungi during theinteraction, invasion and dissemination processes [27].
Glucosylceramides: an antigenic moleculelinked to fungal differentiation
Glycosphingolipids are amphiphatic molecules consist-
ing of a ceramide lipid moiety linked to a glycan chain
of variable length and structure. The ceramide mono-hexosides (CMHs) gluco- and galactosylceramides are
the main neutral glycosphingolipids expressed in al-
most all fungal species [28]. A few exceptions include
Torulaspora delbrueckii, Candida glabrata, Saccharo-
myces cerevisiae, Kluyveromyces polysporus and
Kluyveromyces yarrowii [29]. Fungal cerebrosides have
conserved structures in which modifications include
different sites of unsaturation, as well as the varyinglength of fatty acid residues in the ceramide moiety.
These molecules have been related with sorting of
molecules to cell surface, cell differentiation, growth
and pathogenicity [30�37].
CMHs were purified from lipidic extracts of P. boydii
and analyzed by high-performance thin-layer chroma-
tography (HPTLC), gas chromatography coupled to
mass spectrometry (GC-MS), fast atom bombardment-mass spectrometry (FAB-MS), and NMR. This combi-
nation of techniques allowed the identification of
CMHs from P. boydii as molecules containing a glucose
residue attached to 9-methyl-4,8-sphingadienine in
amidic linkage to 2-hydroxyoctadecanoic or 2-hydro-
xyhexadecanoic acids [34] (Fig. 2A).
P. boydii CMHs are antigens recognized by anti-
bodies from a rabbit infected with this fungus. Theseantibodies were purified and used in indirect immuno-
fluorescence, which revealed that CMHs are detectable
on the surface of mycelia and pseudohyphae but not
conidial forms of P. boydii, suggesting a differential
expression of glucosylceramides according to themorphological phase of the fungus [34]. Biosynthesis,
expression or chemical structures of CMHs seem to be
modified during the conidium to mycelium transition,
which suggests a role for CMHs in fungal differentia-
tion. In agreement with this hypothesis is the observa-
tion that antibodies against CMH were able to inhibit
the differentiation of conidia into mycelia in P. boydii
(Fig. 2B), but did not influence mycelial growth.Similarly, cellular differentiation in C. albicans was
affected by antibodies against GlcCer [34]. The me-
chanisms by which anti-CMH antibodies inhibit fungal
growth and/or differentiation remain to be established,
but there is a possibility that CMHs are associated with
enzymes involved in the hydrolysis and synthesis of the
cell wall and/or with glycosylphosphatidylinositol-an-
chor precursors during cell differentiation and division.In this context, binding of antibodies to CMHs could
impair the action of CMH-associated functional pro-
teins, and inhibit cell wall synthesis.
Anti-GlcCer antibodies are not exclusive molecules
that can bind to GlcCer and impair the fungal growth.
Thevissen et al. [38] showed that the antifungal peptide
RsAFP2, a defensin purified from radish seeds, pre-
sented a potent fungicidal action. A series of experi-ments revealed that the cell target of the RsAFP2
defensin was fungal GlcCer. Interestingly, mammalian
GlcCer was not recognized by this defensin. In addi-
tion, the GlcCer-lacking yeast, S. cerevisiae, was
resistant to the antifungal effects of the RsAFP2
defensin. These results suggest that, most probably,
association of RsAFP2 with fungal GlcCer activates
signaling pathways leading to cell death rather thancausing direct membrane permeabilization. Further
experiments in fact revealed that the RsAFP2-GlcCer
association activates the production of endogenous
reactive oxygen species (ROS) by C. albicans [39].
Glycosphingolipid synthesis has also been proposed
as an attractive target for development of new anti-
fungal drugs [40]. The administration of a fungal
glucosylceramide synthase (GCS) inhibitor could beeffective in prophylaxis and/or therapy. However,
compounds that inhibit the mammalian GCS enzyme
[41] do not inhibit the fungal enzyme perhaps because
of a different substrate specificity between the fungal
enzyme and the mammalian one [42].
Glucans: structural molecules that bind toToll-like receptor
In a recent study, Bittencourt et al. [19] determined the
chemical structure of an a-glucan extracted from the
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P. boydii cell wall employing a combination of techni-
ques including gas chromatography, 1H-TOCSY, 1H-
and 13C-NMR spectroscopy and methylation analysis.
This polysaccharide consists of a glycogen-like struc-
ture with linear 4-linked a-D-glucopyranosyl residues
substituted at O-6 with a-D-glucopyranosyl units. The1H-NMR spectrum of the purified a-glucan also
confirmed the similarity of the glucan of P. boydii
with glycogen from other species including A. fumigatus
[43], Mycobacterium bovis [44] and rabbit liver [45].
The host immune response to fungi is in part
dependent on activation of evolutionarily conserved
receptors able to recognize pathogen-associated mole-
cular patterns (PAMPs), among them, the phagocytic
receptors and the toll-like receptors (TLR). TLRs are
present in a variety of human cells, including the
gastrointestinal tract and cell lineages involved in the
immune response [reviewed in 46]. In fungi, the (103)-
b-glucans have a well-characterized role as a ligand for
these receptors [47,48] and as an activator of immune
response [49,50]. Although cell wall a-glucans are also
of interest because of their role in the virulence of
several important fungal pathogens [51�53], the sig-
nificance of these polysaccharides in the processes of
interaction with host immunological cells is poorly
known. In order to investigate whether the a-glucan of
P. boydii could be involved in the phagocytic process,
macrophages were incubated with conidia in the
Fig. 2 (A) Chemical structure of the cer-
amide monohexosides (CMHs) extracted
from Pseudallescheria boydii mycelia. (B)
The incubation of conidial cells for 48 h in
the presence of anti-CMH antibodies (�anti-CMH) blocked the conidia to hyphae
transition in Sabouraud medium (� anti-
CMH).
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absence or in the presence of increasing concentrations
of the polysaccharide [19]. Phagocytosis of conidia was
inhibited in a dose-dependent way, where an a-glucan
concentration of 100 mg/ml caused approximately 50%
inhibition of phagocytosis [19] (Fig. 3A). The role of a-
glucan in the internalization of conidia was further
characterized comparing the phagocytic index of con-
idia submitted to treatment with a-amyloglucosidase
with that from untreated conidia. Removing of a-
glucan from conidial surface by enzymatic treatment
caused a significant decrease in the phagocytic index,
suggesting that a-glucan present in the P. boydii surface
plays an essential role in the internalization of conidia
by macrophages [19].
Absence of intracellular signaling upon TLR engage-
ment by PAMPs, in a MyD88-dependent pathway,
results in increased susceptibility to a wide variety of
microorganisms including fungal pathogens, such as C.
albicans [54]. Despite the progress in understanding the
interaction of some fungal PAMPs with TLR receptors,
the molecular nature of these fungal ligands responsible
for cell activation is still in its beginning.
Distinct from the glycogen and others a-glucans
from lichens and oysters, a-glucan of P. boydii induced
TNF-a secretion by mice peritoneal macrophages in
vitro. The secretion of inflammatory cytokines, like
TNF-a and IL-12, by mice macrophages and dendritic
cells stimulated by a-glucan of P. boydii, is a mechanism
that involves TLR2, CD14 and MyD88 adapters [19]
(Fig. 3B). Recognition of a-glucan might have rele-
vance in the immunomodulation during fungal infec-
tion favoring the host resistance through IL-12 secre-
tion and consequent induction of a Th1 phenotype, or,
alternatively, contributing to the pathology through
TNF-a release provoking tissue injury.The presence of (103)-b-D-glucans in P. boydii cell
wall have yet to be described. These polysaccharides are
components of the cell wall of a wide variety of fungi
[55] and the enzyme b(103)-glucan synthase, a well-
conserved component of fungal morphogenetic ma-
chinery, is a target for human antifungal therapy.
Measurement of b-glucan has emerged as an adjunct
diagnostic strategy for invasive fungal infections
[47,56]. Recent data indicate that b-glucans are released
from fungal cell walls into the systemic circulation of
patients with invasive fungal infections who were or
where not receiving antifungal medication. Its presence
in blood or other body fluids could be a serological
marker of fungal sepsis in patients with fungaemia,
aspergillosis, candidiasis, fusariosis, trichosporonosis
or infections caused by Pneumocystis jiroveci (carinii),
Acremonium spp. or Saccharomyces spp. [reviewed in
48]. Interestingly, Scedosporium spp. and P. boydii,
produced and extracellularly released low b-glucan
levels [57]. In addition, Kahn et al. [58] demonstrated
that caspofungin inhibits b-glucan synthesis and re-
duces in vitro growth of clinical members of the genera
Alternaria, Curvularia, Acremonium, Bipolaris, Tricho-
derma and Scedosporium. Among the new antifungal
drugs, inhibitors of b-glucan synthesis, as well as
Fig. 3 (A) Ingestion of Pseudallescheria
boydii conidia by mice peritoneal macro-
phages in the absence (upper panel) or in
the presence (lower panel) of a-glucan. Note
that macrophages treated with the polysac-
charide presented a reduction in the number
of intracellular conidia in comparison with
the non-treated ones. The arrows show the
conidia ingested by macrophage cells. (B)
Schematic representation of cytokine pro-
duction by cells of the innate immune system
by the a-glucan of P. boydii, in a mechanism
involving TLR2, CD14 and MyD88.
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second-generation azole and triazole derivatives, have
characteristics that render them potentially suitable foruse against some resistant fungi [59]. Fungal b-glucans
exist both in soluble and in particulate forms and are
believed to have several functions. Cytoplasmic and
exocellular b-glucans probably act as carbon storage
materials, which may be re-utilized by the fungus under
conditions of carbon limitation, suggesting an impor-
tant survival function [60]. Soluble forms of b-glucans
may inhibit phagocytosis by monocytes, by blockingthe receptors for b-glucans present on phagocytes;
according to Miyazaki et al. [47], this could explain
the development of a fungal infection.
Proteolytic enzymes: cleavage of key hostcomponents
The pathogenic filamentous fungi use extracellularenzymes to degrade the structural barriers of the
host. In animal tissues, these barriers are mostly
composed of proteins, therefore the fungus requires
proteolytic enzymes to invade them. For this reason, it
is logical to assume that this class of hydrolytic enzymes
could act by making this tissue invasion easier, but they
could also participate in the infection by eliminating
some mechanisms of the immune defense and/or help-ing in the obtaining of nutrients [61�63]. Despite the
importance of proteases in fungus-host interaction,
little is known about these molecules in P. boydii.
Evidence about the expression of protease in this
fungus was first reported by Larcher et al. [64], who
had purified and characterized an extracellular pro-
tease produced by a clinical isolate of P. boydii. The
highest yield of enzyme production was obtained whenthe fungus was cultivated in modified Czapek-Dox
liquid medium supplemented with 0.1% bacteriological
peptone and 1% glucose as nitrogen and carbon
sources respectively. Analysis of the purified enzyme
by SDS-PAGE revealed a single polypeptide chain with
an apparent molecular mass of 33 kDa. The inhibition
profile and N-terminal amino acid sequencing con-
firmed that this enzyme belongs to the subtilisin familyof serine protease that has numerous biochemical and
physical similarities with those of the previously
described serine protease of A. fumigatus [65]. Interest-
ingly, the 33-kDa serine protease is able to degrade
human fibrinogen, suggesting an action as a mediator
of the severe chronic bronchopulmonary inflammation
from which cystic fibrosis patients suffer [64].
Recently, our group described the secretion of newproteolytic enzymes related to the metalloprotease
class. By means of SDS-PAGE containing bovine
serum albumin (BSA) as co-polymerized substrate,
Silva et al. [66] identified a 28-kDa proteolytic enzyme
released to the extracellular environment by mycelia ofP. boydii. This protease was detected during the growth
of this fungus in Sabouraud dextrose medium for 13
days and reached its maximal production on day 7. The
28 kDa protease was active in acidic pH and had its
activity completely blocked by 1,10-phenanthroline, a
potent zinc-metalloprotease inhibitor [66]. In addition,
in an effort to know more about some biochemical
properties of this enzyme and possible other proteolyticenzymes induced after growth on Sabouraud medium
for 7 days, mycelia of P. boydii were incubated for
additional 20 h in PBS�glucose and then analyzed for
proteolytic activity. The cell-free PBS-glucose super-
natant was submitted to SDS-PAGE and 12 secreted
polypeptides were observed. Two of them of 28 and 35
kDa presented proteolytic activity when BSA was used
as a copolymerized substrate. These extracellularproteases were also most active in acidic pH (5.5) and
fully inhibited by 1,10-phenanthroline. Other metallo,
cysteine, serine and aspartic proteolytic inhibitors did
not significantly alter the proteolytic activities. To
confirm that these enzymes belong to the metallo-
type proteases, the apoenzymes were obtained by
dialysis against chelating agents, and supplementation
with different cations, especially Cu2� and Zn2�,restored their activities. Except for gelatin, both me-
talloproteases hydrolyzed various copolymerized sub-
strates, including human serum albumin, casein,
hemoglobin and immunoglobulin G. Additionally, the
metalloproteases were able to cleave different soluble
proteinaceous substrates such as extracellular matrix
components (laminin and fibronectin) and sialylated
proteins (mucin and fetuin). Collectively, these proper-ties could help the fungus to escape from natural
human barriers and defenses [67]. Interestingly, the
major 28 kDa secreted metalloprotease was also
detected in cellular extracts from mycelia and conidia
[68]. However, quantitative protease assay, using solu-
ble albumin, showed a higher metalloprotease produc-
tion in mycelial cells in comparison with conidia. In
this sense, conidia synthesized a single protease of 28kDa, while mycelia yielded 6 distinct metalloproteases
ranging from 28 to 90 kDa [68]. The regulated
expression of proteases in the different morphological
stages of P. boydii represents potential target for
isolation of stage-specific proteolytic enzymes and their
biochemical and immunological analysis.
Ecto-phosphatases
Fungal pathogens have developed a diversity of strate-
gies to interact with host cells, manipulate their
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Potential virulence markers in Pseudallescheria boydii 381
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behaviors, and thus survive and propagate. During the
process of pathogenesis, phosphorylation of proteinson hydroxyl amino acids (serine, threonine and tyr-
osine) occurs at different stages, including cell-cell
interaction, as well as adherence and changes in host
cellular structure and function induced by infection.
The phosphorylation reactions are catalyzed in a
reversible fashion by specific protein kinases and
phosphatases that belong to either the invading fungal
cells or the infected host cells [69,70].Ecto-phosphatase activities were characterized in
intact mycelial forms of P. boydii, which are able to
hydrolyze, with different specificities the artificial
substrates p-nitrophenylphosphate (p-NPP), b-glycero-
phosphate and phosphoaminoacids such as phospho-
serine, phosphotyrosine and phosphothreonine [71].
MgCl2, MnCl2 and ZnCl2 were able to increase the p-
NPP hydrolysis while CdCl2 and CuCl2 inhibited it.High sensitivity to specific inhibitors of alkaline and
acid phosphatases suggests the presence of both acid
and alkaline phosphatase activities at the surface of P.
boydii mycelia. Cytochemical localization of the acid
and alkaline phosphatase showed electron-dense cer-
ium phosphate deposits on the whole cell wall, as
visualized by transmission electron microscopy. How-
ever, the alkaline conditions favored the detection,suggesting the predominance of phosphatases with
alkaline characteristics on the cell surface of this
fungus. The enhancement of p-NPP hydrolysis by
increasing pH values (2.5�8.5) over an approximately
5-fold range corroborate this finding. For some cells,
the electron-dense precipitate indicative of the alkaline
phosphatase activity was also found apart from the
cells; however, under the experimental conditionsemployed in that work, no enzymatic activity was
biochemically detected in culture supernatant [71].
Several biological roles for extracytoplasmic phos-
phatases have been proposed. In Candida parapsilosis, a
surface phosphatase activity was described to be
involved with fungal adhesion to host cells [72] and in
C. albicans, the endocytosis by vascular endothelial
cells is associated with tyrosine phosphorylation ofspecific host cell proteins [73]. These ecto-enzymes have
also been associated with cell differentiation [74,75] and
may also have a role as ‘safeguard’ enzymes to protect
the cells from acidic conditions by buffering the
periplasmic space with phosphate released from poly-
phosphates [76]. From a general standpoint, the
demonstration of a direct relationship between protein
phosphorylation on serine/threonine/tyrosine and fun-gal virulence represents a novel concept of great
importance in deciphering the molecular and cellular
mechanisms that underlie pathogenesis. However,
knowledge of the relevance of these enzymes in P.
boydii biology or pathogenesis is still very limited.
Superoxide dismutase
In spite of their diversity, all the primary or opportu-
nistic pathogenic fungi have to face the first line of host
defense against fungal infection: oxidative response of
phagocytes. Evolution of antioxidant systems could
therefore be a key process determining fungal adapta-tion and resistance in the host environment, contribut-
ing to the emergence of pathogenicity among fungi.
Many enzymatic, e.g., catalase, glutathione peroxidase
and superoxide dismutase (SOD), and non-enzymatic
systems, e.g., melanin and mannitol, participate to
reactive oxygen species elimination [77]. SODs are
ubiquitous metalloenzymes, catalyzing the dismutation
of toxic superoxide anions into hydrogen peroxide.Three main isoforms are described, depending on their
metal cofactor: manganese- (MnSOD), iron- (FeSOD),
or copper/zinc- (Cu/ZnSOD) SODs. Mn- and Fe-
dependent SODs are found in bacteria, whereas
eukaryotic cells usually present both Mn-SODs in
mitochondria and Cu,Zn-SODs located in the cyto-
plasm [78]. SODs have already been characterized from
various organisms, including pathogenic fungi such asC. albicans, C. neoformans, Trichophyton mentagro-
phytes var. interdigitale, A. fumigatus, Aspergillus flavus,
Aspergillus nidulans and Aspergillus terreus [79�83].
Recently, a Cu,Zn-SOD was characterized from P.
boydii mycelial form [84]. The purified enzyme pre-
sented a relative molecular mass of 16.4 kDa under
reducing conditions and was inhibited by potassium
cyanide and diethyldithiocarbamate, which are twowell-known inhibitors of Cu,Zn-SODs. The encoding
gene was sequenced and a database search for sequence
homology revealed for the deduced amino acid se-
quence 72 and 83% identity rate with Cu,Zn-SODs
from A. fumigatus and Neurospora crassa, respectively
[84]. Iron availability has been suggested as a significant
controlling factor on expression levels of antioxidant
enzymes in various microorganisms such as A. fumiga-
tus and A. nidulans [85]. Similarly, iron starvation leads
to an increased production of the SOD enzyme in P.
boydii [84]. Interestingly, under conditions of low iron
availability, most fungi excrete siderophores in order to
mobilize extracellular iron and stimulate SOD levels to
a protective antioxidant role [86].
Conclusion
Pseudallescheria/Scedosporium species are a well recog-
nized cause of serious fungal infections that are
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382 Santos et al.
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difficult to treat. Infections due to these opportunistic
fungi are usually marked by a poor response toantifungal therapy, in agreement with the in vitro
resistance of the fungus to most available antifungal
agents. In addition, several key steps of fungal patho-
genesis are still unknown, which motivates new studies
on the characterization of fungal molecules with key
roles in cell growth, differentiation and in the interac-
tion with the host.
In this context, several studies have focused onunderstanding how the fungal cell wall is assembled.
The cell wall is an excellent target for the action of
antifungal agents, since most of its components are
absent in mammalian cells. Over recent years, poly-
saccharides, peptidopolysaccharides, O-linked oligo-
saccharides, glycosphingolipids and several hydrolytic
enzymes (e.g., proteases and phosphatases) have been
identified in Pseudallescheria/Scedosporium. The role ofeach molecule on fungal physiology and pathogenesis is
only beginning to be elucidated. In this context, P.
boydii PRM molecules have been shown to be useful for
diagnostic purposes and also to influence the interac-
tion of this fungus with the host cells. An a-glucan,
resembling a glycogen-like polysaccharide, is accessible
at the surface of conidia and mediates their interaction
with receptors of cells of the host innate immunesystem. However, the partial effect of a-glucan on
inhibition of phagocytosis indicates that other putative
phagocytic ligands may be present on P. boydii conidia.
Besides its involvement in phagocytosis of conidia, a-
glucan also stimulates the secretion of inflamatory
cytokines by macrophages and dendritic cells by a
mechanism involving TLR-2, CD14 and MyD88. So, it
is possible that this polysaccharide is a TLR2-activat-ing molecule representing typical PAMP in P. boydii.
Glucosylceramides having highly conserved structures
are involved in fungal development. In this context,
specific antibodies against P. boydii CMHs were able to
arrest the fungal growth and differentiation. Assuming
the concept that sphingolipid ordered membrane
microdomains contribute to the differential trafficking
of cell surface components [87], it is reasonable thatantibodies to CMHs could impair the transport to the
cell wall of molecules involved in conidia to hyphae
transition. Extracellular proteases described in P. boydii
were able to cleave human serum proteins, extracellular
matrix components as well as sialylated proteins. These
hydrolytic properties could help the fungus to obtain
amino acids for its nutrition, to escape from human
defenses, and to disseminate through host barriers.Ecto-phosphatase activities were also characterized in
intact mycelial forms of P. boydii, but the relevance of
these enzymes in biology or pathogenesis of this fungus
is still unclear. In addition, the Cu,Zn-SOD enzyme can
help the fungus during the oxidative response generatedby the host phagocytic cells, minimizing the toxic effect
of superoxide anions.
Determination of structural and functional aspects
of the abovementioned molecules could contribute to
the design of new agents capable of inhibiting fungal
growth and/or differentiation and will provide critical
information in understanding the nature of host-fungus
interactions.
Acknowledgements
This study was supported by grants from Conselho
Nacional de Desenvolvimento Cientıfico e Tecnologico
(CNPq), Financiadora de Estudos e Projetos (FINEP),Fundacao de Amparo a Pesquisa do Estado do Rio de
Janeiro (FAPERJ), Fundacao Universitaria Jose Boni-
facio (FUJB), Programa de Apoio a Nucleos de
Excelencia (PRONEX) and Fundacao de Amparo a
Pesquisa do Estado de Sao Paulo (FAPESP, grant 05/
02776-0). Marcia Ribeiro Pinto is supported by a
FAPESP fellowship (grant 05/56161-6) and Vera Car-
olina Bordallo Bittencourt is supported by a FAPERJ(grant 152945/05).
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the paper.
References
1 Kwon-Chung KJ, Bennett JE. Pseudallescheriasis and Scedospor-
ium. In: Kwon-Chung KJ, Bennett JE (eds). Medical Mycology.
Philadelphia: Lea and Febiger, 1992: 678�694.
2 De Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of Clinical
Fungi, 2nd. ed. Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands, and University Rovira i Virgili, Reus, Spain,
2000
3 Steinbach WJ, Perfect JR. Scedosporium species infections and
treatments. J Chemother 2003; 15: 16�27.
4 Defontaine A, Zouhair R, Cimon B, et al. Genotyping study of
Scedosporium apiospermum isolates from patients with cystic
fibrosis. J Clin Microbiol 2002; 40: 2108�2114.
5 Guarro J, Kantarcioglu AS, Horre R, et al. Scedosporium
apiospermum: changing clinical spectrum of a therapy-refractory
opportunist. Med Mycol 2006; 44: 295�327.
6 Lopez FA, Crowley RS, Wastila L, Valantine HA, Remington JS.
Scedosporium apiospermum (Pseudallescheria boydii) infection in a
heart transplant recipient: a case of mistaken identity. J Heart
Lung Transpl 1998; 17: 321�324.
7 Kleinschmidt-de-Masters BK. Central nervous system aspergillo-
sis: a 20-year retrospective series. Hum Pathol 2002; 33: 116�124.
8 Gueho E, De Hoog GS. Taxonomy of the medical species of
Pseudallescheria and Scedosporium. J Mycol Med 1991; 1: 3�9.
9 Rainer J, De Hoog GS, Wedde M, Graser I, Gilges S. Molecular
variability of Pseudallescheria boydii, a neurotropic opportunist. J
Clin Microbiol 2000; 38: 3267�3273.
– 2009 ISHAM, Medical Mycology, 47, 375�386
Potential virulence markers in Pseudallescheria boydii 383
Downloaded By: [International Society for Human and Animal Mycology] At: 15:43 19 April 2009
10 Zeng JS, Fukushima K, Takizawa K, et al. Intraspecific diversity
of species of the Pseudallescheria boydii complex. Med Mycol
2007; 45: 547�558.
11 Gordon MA. Nutrition and sporulation of Allescheria boydii. J
Bacteriol 1957; 73: 199�205.
12 Cazin J, Decker DW. Carbohydrate nutrition and sporulation of
Allescheria boydii. J Bacteriol 1964; 88: 1624�1628.
13 Cazin J, Decker DW. Growth of Allescheria boydii in antibiotic-
containing media. J Bacteriol 1965; 90: 1308�1313.
14 Carrillo A, Guarro J. In vitro activities of four novel triazoles
against Scedosporium spp. Antimicrob Agents Chemother 2001; 45:
2151�2153.
15 Capilla J, Serena C, Pastor FJ, Ortoneda M, Guarro J. Efficacy of
voriconazole in treatment of systemic scedosporiosis in neutro-
penic mice. Antimicrob Agents Chemother 2004; 48: 4009�4011.
16 Gilgado F, Cano J, Gene J, Guarro J. Molecular phylogeny of the
Pseudallescheria boydii species complex: proposal of two new
species. J Clin Microbiol 2005; 43: 4930�4942.
17 Gil-Lamaignere C, Maloukou A, Rodriguez-Tudela JL, Roilides
E. Human phagocytic cell responses to Scedosporium prolificans.
Med Mycol 2001; 39: 169�175.
18 Gil-Lamaignere C, Roilides E, Lyman CA, et al. Human
phagocyte cell responses to Scedosporium apiospermum (Pseudal-
lescheria boydii): variable susceptibility to oxidative injury. Infect
Immun 2003; 71: 6472�6478.
19 Bittencourt VCB, Figueiredo RT, da Silva RB, et al. An alpha-
glucan of Pseudallescheria boydii is involved in fungal phagocy-
tosis and Toll-like receptor activation. J Biol Chem 2006; 281:
22614�22623.
20 Barreto-Bergter E, Pinto MR, Rodrigues ML, Bittencourt VBC,
Gorin PAJ. (Ed) Hugo Verli. Structural and functional aspects of
fungal polysaccharides, peptidopolysaccharides and ceramide
monohexosides. In: Insights into Carbohydrate Structure and
Biological Function 2006; 147�173. Transworld Research Network:
Kerala, India.
21 Pinto MR, Mulloy B, Haido RM, Travassos LR, Barreto-Bergter
E. A peptidorhamnomannan from the mycelium of Pseudal-
lescheria boydii is a potential diagnostic antigen of this emerging
human pathogen. Microbiology 2001; 147: 1499�1506.
22 Lopes-Alves L, Travassos LR, Previato JO, Mendonca-Previato
L. Novel antigenic determinants from peptidorhamnomannans of
Sporothrix schenckii. Glycobiology 1994; 4: 281�288.
23 Pinto MR, Gorin PA, Wait R, Mulloy B, Barreto-Bergter E.
Structures of the O-linked oligosaccharides of a complex glyco-
conjugate from Pseudallescheria boydii. Glycobiology 2005; 15:
895�904.
24 Leitao EA, Bittencourt VC, Haido RM, et al. Beta-galactofur-
anose-containing O-linked oligosaccharides present in the cell
wall peptidogalactomannan of Aspergillus fumigatus contain
immunodominant epitopes. Glycobiology 2003; 13: 681�692.
25 Penha CV, Bezerra LM. Concanavalin A-binding cell wall
antigens of Sporothrix schenckii: a serological study. Med Mycol
2000; 38: 1�7.
26 Pinto MR, de Sa AC, Limongi CL, et al. Involvement of
peptidorhamnomannan in the interaction of Pseudallescheria
boydii and HEp2 cells. Microbes Infect 2004; 6: 1259�1267.
27 Osherov N, May GS. The molecular mechanisms of conidial
germination. FEMS Microbiol Lett 2001; 199: 153�160.
28 Barreto-Bergter E, Pinto MR, Rodrigues ML. Structure and
biological functions of fungal cerebrosides. An Acad Bras Cienc
2004; 76: 67�84.
29 Saito K, Takakuwa N, Ohnishi M, Oda Y. Presence of gluco-
sylceramide in yeast and its relation to alkali tolerance of yeast.
Appl Microbiol Biotechnol 2006; 71: 515�521.
30 Rodrigues ML, Travassos LR, Miranda KR, et al. Human
antibodies against a purified glucosylceramide from Cryptococcus
neoformans inhibit cell budding and fungal growth. Infect Immun
2000; 8: 7049�7060.
31 Rodrigues ML, Shi L, Barreto-Bergter E, et al. A monoclonal
antibody to fungal glucosylceramide protects mice against lethal
Cryptococcus neoformans infection. Clin Vaccine Immunol 2007;
14:1372�1376.
32 Toledo MS, Levery SB, Suzuki E, Straus AH, Takahashi HK.
Characterization of cerebrosides from the thermally dimorphic
mycopathogen Histoplasma capsulatum expression of 2-hydroxy
fatty N-acyl(E)-D3-unsaturation correlates with the yeast-myce-
lium phase transition. Glycobiology 2001; 11: 113�124.
33 Levery SB, Momany M, Lindsey R, et al. Disruption of the
glucosylceramide biosynthetic pathway in Aspergillus nidulans and
Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide gluco-
syltransferase strongly affects spore germination, cell cycle, and
hyphal growth. FEBS Lett 2002; 525: 59�64.
34 Pinto MR, Rodrigues ML, Travassos LR, et al. Characterization
of glucosylceramides in Pseudallescheria boydii and their involve-
ment in fungal differentiation. Glycobiology 2002; 12: 251�260.
35 Silva AF, Rodrigues ML, Farias SE, et al. Glucosylceramides in
Colletotrichum gloeosporioides are involved in the differentiation
of conidia into mycelial cells. FEBS Lett 2004; 561: 137�143.
36 Nimrichter L, Cerqueira MD, Leitao EA, et al. Structure, cellular
distribution, antigenicity, and biological functions of Fonsecaea
pedrosoi ceramide monohexosides. Infect Immun 2005; 73: 7860�7868.
37 Ritterhaus PC, Kechichian TB, Allegood JC, et al. Glucosylcer-
amide synthase is an essential regulator of pathogenicity of
Cryptococcus neoformans. J Clin Invest 2006; 116: 1651�1659.
38 Thevissen K, Warnecke DC, Francois IE, et al. Defensins from
insects and plants interact with fungal glucosylceramides. J Biol
Chem 2004; 279: 3900�3905.
39 Aerts AM, Francois IE, Meert EM, et al. The antifungal activity
of RsAFP2, a plant defensin from Raphanus sativus, involves the
induction of reactive oxygen species in Candida albicans. J Mol
Microbiol Biotechnol 2007; 13: 243�247.
40 Dickson RCC, Sumanasekera C, Lester RL. Functions and
metabolism of sphingolipids in Saccharomyces cerevisiae. Prog
Lipid Res 2006; 45: 447�465.
41 Luberto C, Toffaletti DL, Wills EA, et al. Roles for inositol-
phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of
Cryptococcus neoformans. Gene Dev 2001; 15: 201�212.
42 Leipelt M, Warnecke D, Zahringer U, et al. Glucosylceramide
synthases, a gene family responsible for the biosynthesis of
glycosphingolipids in animals, plants, and fungi. J Biol Chem
2001; 276: 33621�33629.
43 Bahia MCFS, Haido RMT, Figueiredo MHG, et al. Humoral
immune response in aspergillosis: an immunodominant glycopro-
tein of 35 kDa from Aspergillus flavus. Curr Microbiol 2003; 47:
163�168.
44 Dinadayala P, Lemassu A, Granovski P, et al. Revisiting the
structure of the anti-neoplastic glucans of Mycobacterium bovis
Bacille Calmette-Guerin. Structural analysis of the extracellular
and boiling water extract-derived glucans of the vaccine sub-
strains. J Biol Chem 2004; 279: 12369�12378.
45 Zang LH, Howseman AM, Shulman RG. Assignment of the 1H
chemical shifts of glycogen. Carbohydr Res 1991; 220: 1�9.
– 2009 ISHAM, Medical Mycology, 47, 375�386
384 Santos et al.
Downloaded By: [International Society for Human and Animal Mycology] At: 15:43 19 April 2009
46 Carpenter S, O’Neill LA. How important are Toll-like receptors
for antimicrobial responses? Cell Microbiol 2007; 9: 1891�1901.
47 Miyazaki T, Kohno S, Mitsutake K, et al. Plasma (103)-b-D-
glucan and fungal antigenemia in patients with candidemia,
aspergillosis, and cryptococcosis. J Clin Microbiol 1995; 33:
3115�3118.
48 Kedzierska A, Kochan P, Pietrzyk A, Kedzierska J. Current status
of fungal cell wall components in the immunodiagnostics of
invasive fungal infections in humans: galactomannan, mannan
and (103)b-D-glucan antigens. Eur J Clin Microbiol Infect Dis
2007; 26: 755�766.
49 Hohl TM, van Epps HL, Rivera A, et al. Aspergillus fumigatus
triggers inflammatory responses by stage-specific beta-glucan
display. PLoS Pathog 2005; 1: e30.
50 Brown GD, Herre J, Williams DL, et al. Dectin-1 mediates the
biological effects of beta-glucans. J Exp Med 2003; 197: 1119�1124.
51 Reese AJ, Yoneda A, Breger JA, et al. Loss of cell wall alpha(1-3)
glucan affects Cryptococcus neoformans from ultrastructure to
virulence. Mol Microbiol 2007; 63: 1385�1398.
52 Borges-Walmsley MI, Chen D, Shu X, Walmsley AR. The
pathobiology of Paracoccidioides brasiliensis. Trends Microbiol
2002; 10: 80�87.
53 Rappleye CA, Engle JT, Goldman WE. RNA interference in
Histoplasma capsulatum demonstrates a role for alpha-(1,3)-
glucan in virulence. Mol Microbiol 2004; 53: 153�165.
54 Bellocchio S, Montagnoli C, Bozza S, et al. The contribution of
Toll-like/IL-1 receptor superfamily to innate and adaptive im-
munity to fungal pathogens in vivo. J Immunol 2004; 172: 3059�3069.
55 Douglas CM. Fungal b(103)-D-glucan synthesis. Med Mycol
2001; 39(Suppl. 1):55�66.
56 Pazos C, Ponton J, Palacio AD. Contribution of (103)-b-D-
glucan chromogenic assay to diagnosis and therapeutic monitor-
ing of invasive aspergillosis in neutropenic adult patients: a
comparison with serial screening for circulating galactomannan.
J Clin Microbiol 2005; 43: 299�305.
57 Odabasi Z, Paetznick VL, Rodriguez JR, et al. Differences in
beta-glucan levels in culture supernatants of a variety of fungi.
Med Mycol 2006; 44: 267�72.
58 Kahn JN, Hsu MJ, Racine F, Giacobbe R, Motyl M. Caspofungin
susceptibility in Aspergillus and non-Aspergillus molds: inhibition
of glucan synthase and reduction of beta-D-1,3 glucan levels in
culture. Antimicrob Agents Chemother 2006; 50: 2214�2216.
59 Canuto MM, Rodero FG. Antifungal drug resistance to azoles
and polyenes. Lancet Infect Dis 2002; 2: 550�563.
60 Pitson SM, Seviour RJ, McDougall BM. Noncellulolytic fungal b-
glucanases: their physiology and regulation. Enz Microb Technol
1993; 15: 178�190.
61 Hube B. Extracellular peptidases of human pathogenic fungi.
Contrib Microbiol 2000; 5: 126�137.
62 Monod M, Capoccia S, Lechenne B, et al. Secreted proteases from
pathogenic fungi. Int J Med Microbiol 2002; 292: 405�419.
63 Naglik JR, Challacombe SJ, Hube B. Candida albicans secreted
aspartyl peptidases in virulence and pathogenesis. Microbiol Mol
Biol Rev 2003; 67: 400�428.
64 Larcher G, Cimon B, Symoens F, et al. A 33 kDa serine
proteinase from Scedosporium apiospermum. Biochem J 1996;
315: 119�126.
65 Larcher G, Bouchara JP, Annaix V, et al. Purification and
characterization of a fibrinogenolytic serine proteinase from
Aspergillus fumigatus culture filtrate. FEBS Lett 1992; 308: 65�69.
66 Silva BA, Santos ALS, Barreto-Bergter E, Pinto MR. Extra-
cellular peptidase in the fungal pathogen Pseudallescheria boydii.
Curr Microbiol 2006; 53: 18�22.
67 Silva BA, Pinto MR, Soares RMA, Barreto-Bergter E, Santos
ALS. Pseudallescheria boydii releases metallopeptidases capable of
cleaving several proteinaceous compounds. Res Microbiol 2006;
157: 425�432.
68 Pereira MM, Silva BA, Pinto MR, Barreto-Bergter E, Santos
ALS. Proteins and peptidases from conidia and mycelia of
Scedosporium apiospermum strain HLPB. Mycopathologia 2009;
167: 25�30.
69 Cohen PTW. The structure and regulation of protein phospha-
tases. Annu Rev Biochem 1989; 58: 453�508.
70 Zhan XL, Hong Y, Zhu T, et al. Essential functions of protein
tyrosine phosphatase Ptp2 and Ptp3 and Rim 11 tyrosine
phosphorylation in Sascharomyces cerevisiae meiosis and sporula-
tion. Mol Biol Cell 2000; 11: 663�676.
71 Kiffer-Moreira T, Pinheiro AA, Pinto MR, et al. Mycelial forms
of Pseudallescheria boydii present ectophosphatase activities. Arch
Microbiol 2007; 188: 159�166.
72 Fernanado PHP, Panagoda GJ, Samaranayare LP. The relation
between the acid and alkaline phosphatase activity and the
adherence of clinical isolates of Candida parapsilosis to human
epithelial cell. APMIS 1999; 107: 1034�1042.
73 Belanger PH, Johnston DA, Fratti RA, Zhang M, Filler SG.
Endocytosis of Candida albicans by vascular endothelial cells is
associated with tyrosine phosphorylation of specific host cells
proteins. Cell Microbiol 2002; 4: 805�812.
74 Nakagura KH, Tachibana H, Kaneda Y. Alteration of the cell
surface acid phosphatase concomitant with morphological trans-
formation in Trypanosoma cruzi. Comp Biochem Physiol 1985; 81:
815�817.
75 Bakalara N, Santarelli X, Davis C, Baltz T. Purification, cloning
and characterization of an acid ectoprotein phosphatase differ-
entially expressed in the infectious bloodstream form of Trypa-
nosoma brucei. J Biol Chem 2000; 275: 8863�8871.
76 Touati E, Dassa E, Dassa J, Boquet PL. Acid phosphatase
(pH2.5) of Escherichia coli: regulatory characteristics. In: Tor-
riani-Gorini A, Rothman FG, Silver S, Wright A, Yagil E
(eds) Microorganisms. Washington, DC: ASM Press, 1987; 31�40.
77 Hamilton AJ, Holdom MD. Antioxidant systems in the patho-
genic fungi of man and their role in virulence. Med Mycol 1999;
37: 375�389.
78 Fridovich I. Superoxide radical and superoxide dismutases. Annu
Rev Biochem 1995; 64: 97�112.
79 Hamilton AJ, Holdom MD, Jeavons L. Expression of the Cu,Zn-
superoxide dismutase of Aspergillus fumigatus as determined by
immunohistochemistry and immunoelectron microscopy. FEMS
Immunol Med Microbiol 1996; 14: 95�102.
80 Holdom MD, Hay RJ, Hamilton AJ. The Cu,Zn-superoxide
dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus
nidulans and Aspergillus terreus: purification and biochemical
comparison with the Aspergillus fumigatus Cu,Zn-superoxide
dismutase. Infect Immun 1996; 64: 3326�3332.
81 Hwang CS, Rhie GE, Oh JH, et al. Copper- and zinc-contain-
ing superoxide dismutase (Cu/Zn-SOD) is required for the
protection of Candida albicans against oxidative stresses and the
expression of its full virulence. Microbiology 2002; 148: 3705�3713.
– 2009 ISHAM, Medical Mycology, 47, 375�386
Potential virulence markers in Pseudallescheria boydii 385
Downloaded By: [International Society for Human and Animal Mycology] At: 15:43 19 April 2009
82 Tesfa-Selase F, Hay RJ. Superoxide dismutase of Cryptococcus
neoformans: purification and characterization. J Med Vet Mycol
1995; 33: 253�259.
83 Ungpakorn R, Holdom MD, Hamilton AJ, Hay RJ. Purification
and partial characterization of the Cu,Zn-superoxide dismutase
from the dermatophyte Trichophyton mentagrophytes var. inter-
digitale. Clin Exp Dermatol 1996; 21: 190�196.
84 Lima OC, Larcher G, Vandeputte P, et al. Molecular cloning and
biochemical characterization of a Cu,Zn-superoxide dismutase
from Scedosporium apiospermum. Microbes Infect 2007; 9:
558�565.
85 Oberegger H, Zadra I, Schoeser M, Haas H. Iron starvation leads
to increased expression of Cu/Zn-superoxide dismutase in Asper-
gillus. FEBS Lett 2000; 485: 113�116.
86 Haas H, Zada I, Stoffler G, Angermayr K. The Aspergillus
nidulans GATA factor SREA is involved in regulation of side-
rophore biosynthesis and control of iron uptake. J Biol Chem
1999; 8: 4613�4619.
87 Kasahara K, Sanai Y. Possible roles of glycosphingolipids in lipid
rafts. Biophys Chem 1999; 82: 121�127.
This paper was first published online on iFirst on 23 February 2009.
– 2009 ISHAM, Medical Mycology, 47, 375�386
386 Santos et al.
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