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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

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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.

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