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FEMS Immunology and Medical Microbiology 45 (2005) 369–381

MiniReview

Functional genome of the human pathogenic fungusParacoccidioides brasiliensis

Maria Sueli S. Felipe a,*, Fernando A.G. Torres a, Andrea Q. Maranhao a,Ildinete Silva-Pereira a, Marcio J. Pocas-Fonseca a, Elida G. Campos a,Lıdia M.P. Moraes a, Fabrıcio B.M. Arraes a, Maria Jose A. Carvalho a,

Rosangela V. Andrade a, Andre M. Nicola a, Marcus M. Teixeira a,Rosalia S.A. Jesuıno b, Maristela Pereira b, Celia M.A. Soares b, Marcelo M. Brıgido a

a Departamento de Biologia Celular, Laboratorio de Biologia Molecular, Instituto de Biologia, Universidade de Brasılia, Brasılia, DF 70910-900, Brazilb Departamento de Bioquımica, Universidade Federal de Goias, Goiania, GO 74001-970, Brazil

Received 2 May 2005; accepted 7 May 2005

First published online 19 July 2005

Abstract

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis,an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation processthat this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanismsinvolved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This reviewfocuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimor-phism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium andyeast phases.� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Transcriptome; Functional genomics; Metabolism; Cell differentiation; Host–pathogen interaction; Paracoccidioides brasiliensis

0928-8244/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2005.05.013

* Corresponding author. Tel.: +55 61 3072423; fax: +55 61 3498411.E-mail addresses: msueli@unb.br (Maria Sueli S. Felipe), ftorres@

unb.br (F.A.G. Torres), andreaqm@unb.br (A.Q. Maranhao), xocolau@unb.br (I. Silva-Pereira), mpossas@unb.br (M.J. Pocas-Fonseca),elida@unb.br (E.G. Campos), lmoraes@unb.br (L.M.P. Moraes),fabricio@unb.br (F.B.M. Arraes), mariajac@gmail.com (Maria JoseA. Carvalho), rvieira@unb.br (R.V. Andrade), amnicola@uol.com.br(A.M. Nicola), marcus.teixeira@gmail.com (M.M. Teixeira), rosalia@icb.ufg.br (R.S.A. Jesuıno), mani@icb.ufg.br (M. Pereira), celia@icb.ufg.br (C.M.A. Soares), brigido@unb.br (M.M. Brıgido).

1. Introduction

1.1. Paracoccidioides brasiliensis and the disease

Paracoccidioidomycosis (PCM) occurs in LatinAmerica where it is estimated that as many as 10 millionindividuals may be infected, with about 2% developingthe disease. PCM is the most important systemic myco-sis in Brazil with the most endemic area of this disease inLatin America. In those highly endemic areas the PCMannual incidence rate has been estimated in three casesper 100 thousand inhabitants and case lethality from2% to 23% [1–4]. The mycosis has also been reportedamong patients with AIDS in Latin America [5].

. Published by Elsevier B.V. All rights reserved.

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Paracoccidioides brasiliensis exists in two morpholog-ical forms: mycelium in the soil and yeast in the host.When the host inhales conidia or becomes in contactwith mycelial fragments, the pathogen converts to thebudding yeast form within hours [6]. It may remain con-fined to the lung or disseminate widely in the body, espe-cially to the components of the mononuclear phagocyticsystem [7]. PCM presents several clinical forms rangingfrom a localized and benign disease to a progressive andpotentially lethal systemic infection [8]. All patientsfrom whom the fungus is isolated should be treatedand pulmonary fibrosis is still the major sequel, despitethe availability of new antifungal drugs for therapy.The outcome of the infection depends on several factors,such as the host responses and the virulence of theinfecting isolate.

Experimental and clinical investigations have indi-cated the relevance of humoral and/or cellular immuneresponses in the pathogenesis and evolution of PCM[9,10]. The cell-mediated immunity has been regardedas the most important host defense mechanism againstthe fungus. Severe forms of the disease are associatedwith depressed immune cellular response and with highIgG levels [11,12]. P. brasiliensis is a facultative intracel-lular microorganism [13] and thus macrophages mayplay a pivotal role in the pathogenesis of the disease.GP43, the main antigenic glycoprotein secreted by P.

brasiliensis [14] down regulates phagocytosis and themicrobicidal activity of cells against P. brasiliensis [15].Another glycoprotein gp70 also down regulates mouseperitoneal macrophage functions in vitro and abolishesgranuloma formation in the lungs, suggesting itsinvolvement in the fungal establishment and progressionof lesions in the primary infection [16]. The glycoproteingp43 is a protective molecule against P. brasiliensis inexperimental models of PCM. A 15-amino-acid peptide(P10) present in gp43 is responsible for glycoprotein-mediated T-cell activation and protection against PCMin BALB/c mice [17]. DNA vaccination using the gp43gene could also elicit protective immunity against P. bra-siliensis [18]. Other reliable vaccine candidates have beenrecently described. Antigens F0 and FII obtained fromfractionation of P. brasiliensis antigens using anionicchromatography on fast protein liquid chromatography(FPLC) promoted significant decrease of organ colonyforming units (CFUs) in the lung after challenge infec-tion in animal models [19].

1.2. Genetics of P. brasiliensis

Phylogenetic analysis of members of the family Ony-genaceae demonstrated a close relationship of P. brasil-iensis with the pathogenic fungi Blastomyces

dermatitidis, Emmonsia parva and Histoplasma capsula-tum [20]. The ascomycete P. brasiliensis has never beenobserved to mate in the laboratory or in the nature.

The lack of a known sexual phase has hampered thestudy of its genetics, limiting our knowledge about themechanisms that contribute to its dimorphism, pathoge-nicity, and virulence. Some transcription factors relatedto ascomycete�s sexual reproduction and transcripts in-volved in meiotic recombination were recently describedin the P. brasiliensis transcriptome, representing a strongevidence for the occurrence of sexual cycle in this fungus[21].

The genetic composition of P. brasiliensis is poorlyknown and information about the genome size andchromosome organization is scarce. In P. brasiliensis,unicellular hyphae and fungal propagules or conidiaare uninucleate, while yeasts are multinucleate [22,23].Pulsed-field gel electrophoresis (PFGE) and confocalfluorescence microscopy have allowed the genomic char-acterization and chromosomal mapping of P. brasilien-sis [24,25]. These techniques were employed to estimatethe DNA content and to define the ploidy of P. brasili-ensis. The nuclear genome size estimated by PFGE wasaround 30 Mb and the results obtained by confocal fluo-rescence microscopy provided strong evidence to thehaploid/diploid or even aneuploid nature of P. brasilien-sis [24]. DNA sequencing of �50 kb showed a density ofone gene per 3.5–4.5 kb, suggesting a total of 7500–9000genes present in P. brasiliensis genome [26]. The electro-phoretic pattern revealed chromosomal polymorphismin the fungus, which presented 4–5 chromosomalDNA molecules according to the analyzed isolate, show-ing molecular sizes ranging from 2–10 Mb [24,25]. Ran-domly amplified polymorphic DNA (RAPD) analysishave delineated a strong variability among the fungusisolates, with genotypic differences showing some corre-lation with virulence, geographic distribution and sus-ceptibility or resistance to drugs [27–30].

1.3. Dimorphism and the biological model

P. brasiliensis is dimorphic; at room temperature itgrows in the mycelia form as branching septate hyphaefrom which chlamydospores or aleurio-conidia are pro-duced [31]. In the infected host the fungus presents mul-tiple-budding yeast form [32]. The morphologictransition is governed predominantly by temperatureand is correlated with host invasion. Its ability to revers-ibly switch from hyphae to yeast growth in response toenvironmental signals is not only inherent to its patho-genicity, but in addition provides an excellent modelto understand the cellular differentiation. Mammalianestrogens inhibit this transition, giving rise to a higherincidence of disease in males. Clinical disease is morecommon in adult males, with a male/female ratio of13:1 in some endemic areas. Receptors for 17b-estradiolwere detected in the cytosol of mycelial and yeast formsof P. brasiliensis, suggesting that this female hormoneinhibits mycelium-to-yeast-form transition [33].

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Association between morphogenesis and virulencehas been presumed, since isolates unable to transforminto yeasts are not virulent [34]. The morphogenetic con-version in P. brasiliensis is correlated with changes in thecell wall composition, organization and structure. Mostnotably, there is an increase in chitin content and achange in the glucan polymer from b-1,3-glucan to a-1,3-glucan when the fungus differentiates from myceliumto the yeast form [35]. Of particular interest is that mu-tant isolates of P. brasiliensis with decreased amounts ofa-1,3-glucan presents reduced virulence correlating wellto the induction of a greater inflammatory responsecompared to wild isolates [36]. It has been proposed thatthe presence of a-1,3-glucan in the yeast phase couldprotect the fungus against the enzymes of the host de-fense system [37]. Homologues of b-1,3-glucan-synthase,N-acetyl-b-D-glucosaminidase and mannosyltransferasegenes were cloned from P. brasiliensis and attempts todefine their functions are under progress [38–40].

Our group has been working for the last 8 years in or-der to identify the differentially expressed genes duringthe dimorphism of P. brasiliensis. Total proteins fromboth fungus phases were analyzed by 2-D gel electro-phoresis and/or Western blotting with serum of P. bra-siliensis infected patients [41,42]. Several genes upregulated in yeast cells have been identified: HSP70,HSP60, mannosyltransferase, ClpB, catalase, glyceral-dehyde-3-phosphate dehydrogenase, PbY20, malatedehydrogenase, fructose biphosphate aldolase and triosephosphate isomerase, were the most extensively charac-terized of those genes [43–51]. Most of those genes en-code proteins which are immunogenic in humansduring infection. Also, differential display reverse trans-criptase (DDRT)-PCR allowed the identification of dif-ferentially expressed genes of both fungus phases. M32,M51, M73 and two hydrophobins Pbhyd1 and Pbhyd2are up regulated genes in mycelium cells [52,53].

Fig. 1. Evolution of the number of groups as a function ofaccumulating reads. Experimental data points correspond to accumu-lation of reads throughout the project. Dashed line corresponds tovirtual transcriptome considering the same level of expression for eachtranscript. Continuous line corresponds to a more realistic modelconsidering a set of highly expressed genes and another of poorlyexpressed genes (see text). Both simulations have considered a totalnumber of 8000 genes.

2. The functional genome project of P. brasiliensis

In an attempt to accelerate the discovery of differen-tially expressed genes in both mycelium and yeast cells,the ‘‘Functional and Differential Genome Project of P.brasiliensis’’ was developed with the collaboration of11 academic/research institutions located in the Midwestregion of Brazil and financed by the Brazilian govern-ment (MCT/CNPq). The main goals of the project werethe identification of genes: (i) differentially expressed inmycelium and yeast cells; (ii) related to the generaland differential metabolism of this pathogen in myce-lium and yeast cells; (iii) related to signal transductionpathways involved in the dimorphic transition, prolifer-ation, cell wall construction, osmoregulation and hightemperature growth; (iv) related to host–pathogen inter-action; and (v) related to drug targets and essential genes

ortologues to Candida albicans. Ultimately, the func-tional analysis of these genes might lead to the identifi-cation of drug targets for disease control.

The project resulted in the sequencing of 25,511clones derived from non-normalized cDNA librariesfrom mycelium and yeast cells of P. brasiliensis. ESTswere generated either from PCR amplification of cDNAinserts, cloned in the k ZAPII phage vector, or from ex-cised phagemids. The sequences of the 5 0-ends of theamplicons (which ranged in size from 0.5–3.5 kb) weredetermined by single-pass sequencing. Only 19,718high-quality sequences (9777 from yeast and 9941 frommycelium cells) having more than 100 nucleotides with aPHRED P 20 were used for clusterization by CAP3analysis which yielded 2655 contigs and 3367 singlets[54]. The resulting 6022 groups represent genes ex-pressed in P. brasiliensis. Fig. 1 shows the evolution pat-tern of new groups, defined here as contigs plus singlets,when compared to two simulated transcriptome models.The first model assumes that every mRNA is equallyrepresented in the cell, while the second and more real-istic model assumes that half of the mRNA mass corre-sponds to 10% of transcripts and the remaining 90%transcripts participates with the other half. In both sim-ulated models a total number of 8000 genes were consid-ered. Our data fits very close to the second modelsupporting that the reported transcriptome cover about80% of the estimated P. brasiliensis gene content [26].

In order to analyze P. brasiliensis transcriptome, wedeveloped an automated pipeline including open sourceprograms to achieve base calling, clustering and annota-tion. Clustering was performed using CAP3 programdue to its more inclusive performance on overlapping se-quences. The resulting 6022 groups were automatically

Fig. 2. (a) Number of groups, composed of six or more reads, found ineach interval of percentage of yeast EST per group. The exclusivegroups for mycelium are denoted in the 0% of yeast EST interval andyeast exclusive groups are those in the 100% of yeast EST interval.(b) Representation of exclusive groups considering the number ofreads per group. The arrow indicates where the number of reads issignificant (95% confidence with n > 5 reads) to be considered asexclusive to mycelium or yeast cells.

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annotated using Blast and FASTA over protein dat-abases. The GenBank number, COG, GO, Saccharomy-

ces cerevisiae and S. pombe ORFomes were included inthis analysis. InterProScan were also used for detectingprotein domain and motifs. All the annotation datawere displayed in a web interface for human referee edi-tion and gene annotation. The annotation quality wassignificantly improved by such a human review step.An improvement of up to 2.6 times in the annotatedE.C. number was obtained by reviewer intervention. Al-most 40% of all of groups was assigned to an E.C. num-ber (data not shown) allowing a reasonable descriptionof the fungus metabolism.

Blast analysis revealed that 68.5% of these genes arerelated to other fungal genes and 52.9% are ortologuesto Aspergillus nidulans genes which reflects the closephylogenetic relationship of this filamentous fungi withP. brasiliensis. Nevertheless, 30.2% of the genes foundin P. brasiliensis represent new genes, which are uniqueto this organism. A total of 6022 clusters were submittedto categorization analysis: 2129 successfully annotated,1604 non-conclusively annotated and 2289 not identi-fied. We classified 3522 groups in 18 functional COGcategories as follows: cellular metabolism (29%); tran-scription (12%); protein synthesis (10%); energy produc-tion (9%); control of cellular organization (4%) andother categories [54]. Most important in this work wasthe fact that 4% of the total number of annotated genesbelonged to signal transduction and cellular communi-cation pathways which have been correlated to cell dif-ferentiation in dimorphic/pathogenic fungi.

3. Differentially expressed genes

The detection of differentially expressed genes in P.

brasiliensis was the main goal of this project. Yeastand mycelium ESTs were clusterized together and basedon the EST distribution per contig, the analyzed groupscould be categorized as: exclusive for a particular mor-photype, when the ESTs included came only from a par-ticular cDNA library; differentially expressed, whenmost ESTs came from one morphotype; and common,when the contigs were formed from equivalent amountof ESTs generated from each morphotype cDNA library(Fig. 2(a)).

Differentially expressed genes in mycelium and yeastcells were detected by statistical comparison of the num-ber of sequences in corresponding PbAESTs [54]. Infact, mycelium up-regulated genes M51, M32, hydro-phobins 1/2 and the highly expressed yeast PbY20 pro-tein have been previously described as differentiallyexpressed by DDRT-PCR, northern blot or proteomeanalysis in P. brasiliensis [41,52,53] and all them wereconfirmed as differentially expressed in the transcrip-tome analysis. Interestingly, we have found a yeast-

phase preferentially expressed gene that possibly en-codes a previously characterized P. brasiliensis estradiolbinding protein [55], also described in C. albicans and inother fungi [56]. A set of two cDNA microarrays, onefrom each phase, containing PCR products related to1152 ESTs were analyzed confirming that those se-quences correspond to differentially expressed genes[54].

Depending on the relative expression level of eachgene, contigs could be formed from two ESTs (the mostcommon contigs) up to 657 ESTs as found for the abun-dant expressed M51 gene, a possible non-coding RNA.Among the most abundant ESTs identified in the tran-scriptome are those encoding for ribosomal proteins(60S acidic proteins P1-a and P2, S2, S3, S10, S14 andL24) and the highly expressed mycelium genes M51,M32 previously described [52], hydrophobin 1 [53] andalso the Y20 protein highly expressed in yeast cells[41]. A transcript for a putative formamidase is the sec-ond most abundant EST. Formamide is a by-product oforganic matter decay, which is readily available for theassimilation of saprophytic microorganisms such as P.

brasiliensis.

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Fig. 2(b) shows a plot of the number of exclusivegroups found as the minimum number of ESTs pergroup increases. The number of exclusive groups fallssharply with the increment of the cut-off number ofEST per contig. The statistical significance of theseexclusive groups was estimated by Audic–Claverie test[57]. Therefore, we consider significantly exclusivegroups of P. brasiliensis those containing more than 6EST, yielding 22 exclusive groups for mycelium and 24for yeast cells (Table 1).

Among the up-regulated genes possibly involved indimorphism in P. brasiliensis, we highlight those encod-ing for heat shock proteins (HSPs) which may help toprotect the pathogen from the host environment [54].The P. brasiliensis HSP70 is preferentially expressed inthe yeast phase [43]. The differential expression seemsto be controlled by the differential splicing of thehsp70 introns, which are effectively removed only inthe yeast parasitic phase [44]. Studies that address HSPshost interaction have been carried out in P. brasiliensis

[46]. Recombinant P. brasiliensis HSP60 was recognizedby 75 serum samples of infected patients, showing highsensitivity and specificity in immunoblot assays. Thosefindings allowed us to suggest the usefulness of HSP60

Table 1Genes found in P. brasiliensis that are formed by six or more reads and thconfidence)

Mycelium-exclusive genes Yea

No. of reads Annotation No

75 Lactoylglutathione lyase 27

56 Pbhyd1 1551 Hypothetical protein 1413 Hypothetical protein 1112 Hypothetical protein 1010 Nid 109 Pbhyd2 98 Amino acid permease 88 Nid 87 Hypothetical protein 87 Hypothetical protein 8

7 Hypothetical protein 77 PbM32 77 Peroxisomal membrane protein PEX16 (peroxin-16) 77 Nid 67 Hexose transporter 66 Involved in cell-type-specific transcription and

pheromone response; Mcm1p6

6 RNA splicing factor Pad-1 66 Carboxypeptidase y 6

6 Related to myo-inositol transport protein ITR1 66 Hypothetical protein 66 Glutamyl-tRNA synthetase; mitochondrial 6

66

Nid: not identified.

singly or in association with other recombinants anti-gens in the PCM diagnosis.

In this context, the expression of several different heatshock proteins (HSPs) detected in P. brasiliensis tran-scriptome is compatible to this fungus necessity to over-come the temperature stress in order to differentiate,infect and colonize the human host. Forty-eight distinctchaperones or co-chaperones transcripts were identified[54]. Calnexin, the cytoplasmic hsp60 (cct7) and the sba1co-chaperone genes were over expressed in myceliumcells, while the co-chaperone cpr1, hsp42, hsp60, hsp70and hsp90 sequences were up regulated in the yeastform. The increased expression (about 38% higher) ofheat shock genes observed in the yeast phase was al-ready expected, since yeast form differentiation bothin vitro as in vivo depends on the shift from the environ-mental temperature to around 37 �C.

4. General and differential metabolism of P. brasiliensis

The transcriptome of P. brasiliensis generated anoverview of the metabolic pathways, which occur inmycelium and yeast cells and will be summarized herein

at are exclusively expressed either in mycelium or yeast cells (95% of

st-exclusive genes

. of reads Annotation

Involved in secretion of proteins that lack classicalsecretory signal sequencesAccumulation of Dyads; predicted membrane protein60S ribosomal protein L19Accumulation of Dyads; predicted membrane proteinPyridoxamine-50-phosphate oxidaseHypothetical proteinHypothetical proteinConserved hypothetical proteinCu–Zn superoxide dismutaseNidAcyl carrier protein, mitochondrial precursor (ACP)(NADH-ubiquinone oxidoreductase 9.6 kDa subunit)Dihydrolipoamide S-succinyltransferasePeptidyl-prolyl cis–trans isomerase b precursorHypothetical proteinConserved hypothetical proteinDan4pArgininosuccinate synthase

Hypothetical proteinCatalyzes synthesis of immediate precursor to riboflavin;Rib4pHypothetical proteinNidNADH-cytochrome b5 reductasePutative sterol transporter60S acidic ribosomal protein P2

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in this topic [54]. The fungus has the basic enzymaticapparatus to carry out the biosynthesis of several carbo-hydrates including glycogen, starch, maltose and sugarnucleotide (UDP-glucose and UDP-galactose) glucan,chitosan, chitobiose, chitin, glycosaminoglycans, lipo-polysaccharides, peptideoglycans, mucopolysaccharidesand lipoglycoproteins, but not pectin. In addition,P. brasiliensis is able to produce N-glycans such as theglycosylphosphatidyl-inositol (GPI) anchor. Severalmono- and disaccharides can be taken up (D-glucose,D-fructose, D-galactose, D-mannose, D-sorbitol, D-man-nitol, D-inositol, a-trehalose, sucrose), except L-sorbose,L-fucose and L-lactose. In order to validate the transcrip-tome data, two isolates of P. brasiliensis, Pb01 andPb18, were grown in McVeigh-Morton minimum med-ium with adaptations [58] and containing different car-bon sources, including ethanol and glycogen [54]. Theexperiment confirmed the data of carbon sources uptakeobtained in the transcriptome analysis, except for L-lac-tose, which is also consumed by P. brasiliensis [54].These isolates showed different behaviors; Pb18 was un-able to grow in the presence of fructose, galactose andglycerol.

Pyruvate cannot be converted to L-lactate, since nolactate dehydrogenase was found. P. brasiliensis is ableto perform alcohol fermentation, gluconeogenesis andboth oxydative and non-oxydative branches of pentosephosphate pathway. Fatty acid biosynthesis and b-oxi-dation pathway have also been predicted and also iscapable of synthesizing many phospholipids and cardio-lipin. Purines and pyrimidines can be synthesized bothby the de novo and salvage pathways. The glyoxylate cy-cle is active allowing the fungus to grow in two-carbonsubstrates such as ethanol and acetate. P. brasiliensis iscapable to synthesize all amino acids, except asparagine.Arginine can be degraded by arginase yielding ornithineand urea, which is degraded by urease to ammonia andCO2. Riboflavin, FMN, pyridoxine, pantothenate, fo-late, FAD, NAD, NADP and porphyrin can be synthe-sized. Thiamine and biotin are probably not producedby the fungal metabolism.

Nitrate and ammonium assimilation pathways arepresent in P. brasiliensis transcriptome. It is also ableto utilize formamide, which is degraded to formate bythe action of the differentially and highly expressed for-mamidase present in the mycelium saprophytic form ofP. brasiliensis found in the environment. It can producesulfate from sulfite and the former can be incorporatedinto adenylylsulfate (APS) and 3-phosphoadenylylsul-fate (PAPS). Sulfite can be regenerated from PAPS,via PAPS reductase. Furthermore, sulfite can be trans-formed into H2S, which reacts with O-acetyl-L-serineyielding L-cysteine and acetate for the glyoxylate cycle.

Genes encoding enzymes participating in secondarymetabolic pathways were detected in P. brasiliensis tran-scriptome such as terpenoid and melanin biosynthesis.

In P. brasiliensis all enzymes necessary for the terpe-noids biosynthesis were detected, including geranylgera-nyl pyrophosphate synthase. It is known that melanin isa virulence factor especially for pathogenic fungus. InCryptococcus neoformans melanin protects the fungusfrom the host immune response [59], and melanin-defi-cient mutants are indeed avirulent in animal models ofcryptococcosis [60]. P. brasiliensis encodes a gene fortyrosinase whose product is the key enzyme requiredfor melanin biosynthesis.

Differentially expressed genes encoding for metabolicenzymes, detected in the differential transcriptome byEST analysis, indicate that mycelium saprophytic cellspossess an aerobic metabolism [54]. The mycelium up-regulated enzymes correspond to the main regulatorypoints of the citrate cycle, such as isocitrate dehydroge-nase and succinyl-CoA synthetase; this strongly suggestsa metabolic shunt to oxidative phosphorylation. Also,glucokinase, adenylate kinase, uridine kinase and trans-aldolase genes are induced in mycelium cells [54]. In con-trast, P. brasiliensis yeast cells overexpress the genesencoding alcohol dehydrogenase I and pyruvate dehy-drogenase, producing ethanol and reflecting a probableanaerobic behavior of yeast form [54]. Also, severalpathways that provide substrates for the glyoxylate cycleare up-regulated in the yeast cells, mainly isocitratelyase, aminotransferase, acetamidase, acyl-CoA synthe-tase and PAPS reductase generating acetyl-CoA andacetate.

5. Signal transduction pathways

The Ca2+/calmodulin and cAMP-PKA signal trans-duction pathways in particular must play an importantrole in cell differentiation in P. brasiliensis. Functionalanalysis of calmodulin from P. brasiliensis revealed thatdrugs that block the Ca2+/calmodulin-dependent ki-nases inhibited the mycelium-to-yeast transition [61].Likewise, the observed increase in cAMP concentrationduring the mycelium-to-yeast transition suggests thatthe cAMP-PKA pathway is involved in the dimorphicprocess in this fungus [62].

Transcriptome analysis and reverse annotation in-deed revealed several components of MAP-kinase path-way signaling for cell integrity, cell wall construction,pheromone/mating and osmotic regulation; cAMP/PKA, regulating fungal development and virulence;Ca+2-calmodulin-calcineurin, controlling growth at hightemperature; and a ras homologue sequence, openingthe possibility of cross-talk among them [54]. In severalpathogenic and non-pathogenic fungi, RAS is involvedin filamentation, pseudohyphal/hyphal growth and mat-ing [63]. Further studies are required to elucidate RASfunction in mycelium-to-yeast transition and in themechanism of pathogenicity of P. brasiliensis. Rho1p,

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a small GTP binding protein of the Rho subfamily andalso found in P. brasiliensis transcriptome, is requiredfor cell growth and coordinated regulation of cell wallconstruction [64] through the synthesis of b-1,3 glucan.RHO1 is an essential gene in C. albicans [65] and hasbeen pointed out as a therapeutic target for treatinginfection by this pathogen.

6. Host–pathogen interaction

In the last few years, molecular approaches havedemonstrated the importance of hydrolytic enzymes inthe pathogenesis of infection, causing damage to hostcells and providing nutrients to the pathogen in a restrictenvironment [66,67]. Probably, the best-characterizedset of enzymes involved in pathogenesis of fungi is theproteases. An extracellular SH-dependent serine pro-teinase has been characterized from the yeast phase ofP. brasiliensis; it cleaves the main components of the ba-sal membrane in vitro, thus being potentially relevant infungal dissemination [68,69]. Our group had character-ized the kex2 gene of P. brasiliensis encoding a putativeGolgi localized protein and potentially associated to thefungus dimorphism [70] and a ClpB homologue, a heat-inducible protease, differentially expressed in the P. bra-siliensis [48].

Adhesion of pathogenic microorganism to host tis-sues has been regarded as the first and major step in col-onization and dissemination of the parasite. Fungaladhesion on the host tissues plays a critical role in infec-tion. P. brasiliensis produces an antigen, the glycopro-tein gp43, which presents a high mannose content, andis described with the capacity of promoting binding tolaminin [71]. This antigen is stored in large vesicles be-fore secretion into the extracellular space as a mem-brane-free material [72]. Gp43 is involved in adhesionto the basal membrane or to other components of theextracellular matrix, playing a major role in the dissem-ination of the fungus [71]. P. brasiliensis yeast adheresin vitro to epithelia cells and gp43 antiserum abolishedmost of the fungus binding activity [73]. The analysisof P. brasiliensis transcriptome allowed the identifica-tion of putative additional homologues genes involvedin the pathogen–host interaction (Table 2).

Human response to invading microorganisms needsthe production of reactive oxygen species (ROS) by leu-kocytes. The enzyme that catalyzes the production ofROS is a multicomponent enzyme called NADPH oxi-dase. Genes coding classical reactive oxygen speciesdetoxification enzymes are present in P. brasiliensis,such as catalase, superoxide dismutases, glutathione per-oxidase and cytochrome c peroxidase [54]. Surprisingly,we found one gene (contig 492) homologous to themammalian gp67phox in P. brasiliensis (Table 3), butno other genes similar to neutrophil NADPH oxidase

subunits. This gene was 80% identical to genes codinghypothetical proteins in Aspergillus nidulans and Neuros-

pora crassa and 53% identical with a gene coding ahypothetical protein in Magnaporthe grisea. It will beimportant to research the role of the protein coded bythe gene in contig 492 and examine if it interacts withany of the human NADPH subunits.

Reactive nitrogen species (RNI) refers to oxidationstates and adducts of the nitrogenous products of nitricoxide synthases, ranging from nitric oxide (NO�) to ni-trate ðNO�

3 Þ. Nitric oxide (NO�) participates in P. brasil-

iensis killing [74–76]. NO� is produced from the aminoacid L-arginine by a family of enzymes called nitric oxidesynthases (NOS). Because RNI are essential for the con-trol of murine parococcidioidomycosis, the yeast formof P. brasiliensis may have mechanisms for RNI resis-tance. Candidates for RNI resistance gene products bypresumptive mechanism of action have been reported[77]. Among the described candidates we identified thefollowing in P. brasiliensis transcriptome (Table 3):superoxide dismutases (SODs), peroxiredoxins, flavohe-moglobin and glucose-6-phosphate dehydrogenase.SODs may confer resistance to RNI by avoidingOONO� to be formed from superoxide ðO��

2 Þ [78]. Per-oxiredoxins have been identified in most genomes se-quenced, but their functions are only partlyunderstood. Peroxiredoxins purified from Salmonella

typhimurium, Mycobacterium tuberculosis, and Helico-

bacter pylori convert peroxynitrite to nitrate [79]. Athiol-specific antioxidant gene (TSA1) expresses prefer-entially in the yeast phase of P. brasiliensis and may pro-tect against ROS and RNIs during yeast growth. It isalso highly expressed in hyphae of C. albicans [80]. Glu-cose-6-phosphate dehydrogenase is the first enzyme ofthe pentose phosphate pathway and produces NADPH.S. typhimurium having a disrupted gene for glucose-6-phosphate dehydrogenase are hypersusceptible to S-nitrosoglutathione reflecting the role of NADPH inthe redox cycling of glutathione and other detoxificationmechanisms [81]. Therefore, P. brasiliensis have genesreported as candidate genes for RNI resistance andinhibitors for the proteins coded by these genes mayimprove immunity.

7. Drug targets and essential genes

Novel, non-traditional drug targets have been foundthrough the analysis of genome sequences. Also, com-parative genomics can be used to define fungi specificmolecules allowing the identification of a global set ofbroad-spectrum antifungal drug targets absent in hu-mans. Genes involved on cell wall metabolism in P. bra-

siliensis have been isolated: 1,3-b-glucan synthase [38],chitin synthases [82,83], RHO [84], mannosyltransferase[40] and 1,3-a-glucan synthase [85]. In P. brasiliensis

Table 2Genes involved in the pathogen–host interaction in microorganisms with orthologues in P. brasiliensis

Function Gene Product Putative role in the host–pathogen interaction Accession No. or PbAEST

Antioxidant response catP Catalase P Protection against the host; required for growth inprimary macrophages

AF428076

cat A Catalase A Protection against the host AY494834

HSPs hsp60-Mitochondrial HSP60 HSP; adhesion to macrophages AF059523hsp70-Mitochondrial HSP70 HSP, diagnostic antigen AY254044

Protein modification mnt Mannosyl transferases Reduced adherence of C. albicans AF374353AY462125

dph Dolichol-phosphate mannose synthase Required for adhesion and infection ofStreptomyces coelicolor

AY425945

Protein destination sap9 Aspartic protease Up-regulated in C. albicans in human blood 4362pigA,C,P First steps of GPI anchors synthesis Complete GPI anchors are required for resistance

to macrophages in C. albicans

(A) 4926(P) 2368(C) 2893

Amino acid metabolism leu2 3-Isopropyl-malate dehydrogenase C. albicans up regulation during infection 1346

Glucose metabolism tps1 Trehalose-6-phosphate synthase Hypha production in C. albicans 442eno Enolase Immunosupressive protein in Streptococcus

sobrinus

1539

tip Triosephosphate isomerase Cell wall protein of C. albicans AY250089fba1 Fructose, 1,6-biphosphate aldolase Cell wall protein of C. albicans; plasminogen

binding proteinAY233454

gapdh Glyceraldehyde-3-phosphate dehydrogenase Cell wall protein of C. albicans; plasminogenbinding protein

AY061958

Nucleotide synthesis ade2 Adenine phosphoribosyltransferase 1 C. albicans unable to colonize the host 495ura3 Orotidine-50-phosphate decarboxylase C. albicans mutant unable to colonize the host 2396

Lipid synthesis fas2 Fatty acid synthase subunit C. albicans with less ability to colonize the host 3750

Cell wall associated proteins gel Glucanosyltransferase homologues – GPI anchored Antigens, vaccine candidates; modulation of thehost immune system in Coccidioides immitis

AY340235-Gel2AY307855-DFG5AY324033-Gel3AY380566-Gel1AY495673

Metabolism of ammonia urease Urease Ammonia production can be linked to thecapacity to resist to strong acidity by Helicobacter

2456amiE Amidase E 377, 323amiF Amidase F 2585, 687

Secreted factors vps34 Phosphatidylinositol-3-kinase Adherence – of C. albicans 5012

376M.S.S.Felip

eet

al./FEMSIm

munologyandMedica

lMicro

biology45(2005)369–381

Table 4Genes found in P. brasiliensis transcriptome by BBH analyses that were considered essential in C. albicans by GRACE and current methods

Gene Product PbAEST E-value

alg7 UDP-N-acetylglucosamine-1-phosphate transferase 3461 4e � 51cct8a Component of chaperonin 3796 5e � 36cdc28 Cell division control protein 28 1093 9e � 34dpb2 DNA-directed DNA polymerase epsilon subunit 4564 1e � 11gna1 Acetyltransferase 4968 2e � 15gsp1 GTP-binding protein 422 5e � 97hem3 Porphobilinogen deaminase 1219 2e � 58his1 ATP phosphoribosyltransferase 5609 6e � 42nmt1 N-myristoyltransferase 668 1e � 59prsb Ribose-phosphate pyrophosphokinase 1732 4e � 50psa1 GDP-mannose pyrophosphorylase 1090 4e � 75rho1 GTP-binding protein of the rho subfamily 265 6e � 82sec14 Phosphatidylinositol (PI)/phosphatidylcholine transfer 1808 6e � 85sec4a GTP-binding protein 3363 6e � 05ypt1 GTP-binding protein of the rab family (by homology) 1768 6e � 83

a PbAESTs that matched C. albicans genes, but not the opposite.b C. albicans genes that matched PbAESTs, but not the opposite.

Table 3Candidate P. brasiliensis genes likely to interfere with ROS production and RNI resistance

PbAEST Annotation Number of EST Organism/Accession No./E-value

Mechanism likely to involve interference with NADPH oxidase activity

4362 Aspartic protease 1 Aspergillus oryzae/BAC00848.1/3e � 425557 Gpi-anchored aspartic protease 1 Botryotinia fuckeliana/AAR87747.1/2e � 34492 Neutrophil cytosol factor 2 NAPDH oxidase 8 Bos taurus/NP_776545.1/7e � 28

Mechanism likely to involve conversion of RNI to less toxic forms

2509 [Cu–Zn] Superoxide dismutase 19 Aspergillus fumigatus/Q9Y8D9/1e�68190 Putative thiol-specific antioxidant protein TSA1 7 Ajellomyces capsulatus/AAG31645.1/3e � 961811 Thioredoxin peroxidase type VI 4 Homo sapiens/AAF04856.1/2e � 122492 Flavohemoglobin 2 Dictyostelium discoideum/BAA83811.1/5e � 065023 Glucose-6-phosphate dehydrogenase 1 Neurospora crassa/XP_331503.1/7e � 41

M.S.S. Felipe et al. / FEMS Immunology and Medical Microbiology 45 (2005) 369–381 377

transcriptome all these mentioned genes were confirmedand also new genes were identified as chitin synthaseasmA; chitin deacetylase; bud neck; malate synthase;isocitrate lyase, a glyoxylate pathway regulator gene;aur1 involved in sphingolipid biosynthesis; C-24 sterolmethyltransferase (ERG6); elongation factor 3 (EF-3)that is unique in fungi and essential for translationmachinery; urate oxidase and urease which plays a rolein both sporulation and pathogenesis [86]. Genes encod-ing for mannosyltransferase [40], chitin deacetylase, iso-citrate lyase, a glyoxylate pathway regulator gene arepreferentially expressed in the yeast phase [54].

Pathogenic microorganisms display several complexmetabolic processes that allow them to resist to thera-peutic drugs. In this view, the comprehension of themolecular mechanism of drug resistance is a crucial stepto overcome this problem, as well as to the developmentof more effective treatments. Twenty multiple drug resis-tance (MDR)-related sequences were present in P. brasil-

iensis transcriptome [54]. These genes represent membersof the Major Facilitator Superfamily (MSF), Pleiotropi-cal Drug Resistance (PDR) and other ABC transporters

and they include homologues to S. cerevisiae, C. albicansand Trichophyton rubrum MDR components.

Large-scale essential gene identification in C. albicans

has been performed in the last years in order to discovernew drug targets to combat fungal infections. Table 4shows the P. brasiliensis PbAESTs orthologues (identi-fied by Biderectional Best Hit analysis – BBH) to essen-tial genes for C. albicans as determined by GRACE, afunctional genomics approach, and other current meth-ods [65]. Among them, three essential genes (RHO1,ALG7 and YBR070c) were tested in mouse model ofC. albicans systemic infection for target validation andtwo of them (rho1 and alg7) are also present in the tran-scriptome of P. brasiliensis, which makes them possiblecommon drug targets in these two human pathogens.

8. Concluding remarks

The functional genome project described for P. bra-siliensis has provided us for the first time with a broadview of the pathogen�s metabolism and also new insights

378 M.S.S. Felipe et al. / FEMS Immunology and Medical Microbiology 45 (2005) 369–381

into biological features, which had been elusive to inves-tigation so far. The expression profiles of some genesthat were shown to be up-regulated or exclusively ex-pressed in each other form (22 mycelium-specific and24 yeast-specific) were confirmed by cDNA microarrayand northern blot analysis [54]. Also, differentially ex-pressed genes in P. brasiliensis have been recently de-scribed using similar approaches [87,88]. Expressionanalysis will be extended to the genes proposed to be in-volved in host–pathogen interaction. Experiments arecurrently being carried out in order to confirm thein vivo expression profile of these genes in macrophagesand human pulmonary epithelial cells infected by P. bra-siliensis. It is also of great interest the analysis of thenine pathogen-specific genes identified in this work,which represents major targets for drug therapy.

A mating process which had never been described inP. brasiliensis can now be subjected to investigation withthe discovery of S. cerevisiae orthologs related to mei-otic sister chromatids recombination (MSC1 andNAM8), meiosis regulatory genes (MSH4, MSH5,RIM101 and RMD1), synaptonemal complex genes(ZIP1 and ZIP2) and mating process-involved se-quences (such as MID2). Although mating has not yetbeen described in P. brasiliensis, the complete MAPKpathway for activation by mating pheromone wasunequivocally identified in the P. brasiliensis transcrip-tome [54]. These findings suggest that P. brasiliensis

could undergo sexual reproduction in some stage of itslife cycle.

Together, the genetic analysis of genes described inthis project will result in important information aboutexpression, cellular differentiation, pathogenicity and/or virulence of P. brasiliensis. These issues can only befurther addressed when genetic tools become availablefor this organism since genetic analysis in P. brasiliensis

has been hampered by the fact that cloning vectors andtransformation protocols are still being developed[89,90]. More important, since P. brasiliensis isolateshave different number of chromosomes [24] and cellsare normally multinucleated it seems unlikely that genedisruption by homologous recombination will be feasi-ble. In order to overcome these difficulties, other geneticapproaches may be employed. Trans-complementationanalysis in S. cerevisiae can be a useful genetic tool forgene functional analysis but it is limited by the availabil-ity of yeast mutants with clearly defined phenotypes forthe tester gene. RNA interference (RNAi), a naturalprocess, which relies in the ability of the cell to targetand destroy foreign genetic material, may represent anattractive alternative for gene analysis in P. brasiliensis.RNAi has been successfully used in other fungi such asC. albicans, C. neoformans and N. crassa [91] and wehave identified in P. brasiliensis five N. crassa orthologs(rrp-3, qde-2, sms-2, dcl-2 and recQ-2) which are relatedto this process. This observation is the first evidence that

RNAi may also occur in P. brasiliensis. Since RNA-mediated gene silencing occurs in the cytoplasm, RNAirepresents so far the most promising approach to over-come all the barriers encountered in P. brasiliensis forfunctional gene analysis.

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