203-212 203 Overview and Perspectives on the Transcriptome of Paracoccidioides Brasiliensis

Review Rev Iberoam Micol 2005; 22: 203-212 203 Overview and perspectives on the transcriptome of Paracoccidioides brasiliensis

Rosângela V. Andrade1, Silvana P. da Silva2, Fernando A. G. Torres1, Marcio José Poças-Fonseca1, Ildinete Silva-Pereira1, Andrea Q. Maranhão1, Élida G. Campos1, Lídia Maria P. Moraes1, Rosália S. A. Jesuíno3, Maristela Pereira3, Célia M. A. Soares3, Maria Emília M. T. Walter1, Maria José A. Carvalho1, Nalvo F. Almeida4, Marcelo M. Brígido1 and Maria Sueli S. Felipe1

1Departamento de Biologia Celular, Universidade de Brasília, Brasília; 2Departamento de Bioquímica, Universidade Estadual de Londrina, Londrina; 3Departamento de Bioquímica, Universidade Federal de Goiás, Goiânia; 4Departamento de Computação e Estatística, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil

Summary Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more com- prehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis. Key words Transcriptome, Expressed Sequence Tags, Dimorphic and thermo-regulated fungus, Paracoccidioides brasiliensis

Panorama y perspectivas del transcriptoma de Paracoccidioides brasiliensis

Resumen Paracoccidioides brasiliensis es un hongo de dimorfismo termorregulado que es el agente causal de la paracoccidioidomicosis, una enfermedad endémica muy extendida en América Latina que afecta a 10 millones de individuos. Se asume que la patogenicidad es una consecuencia de la transición dimorfa de micelio a levadura durante la infección humana. Esta revisión muestra los resultados del proyecto de transcriptoma de P. brasiliensis, que generó 6.022 grupos ensam- blados de las fases miceliar y levaduriforme. El análisis por ordenador utilizando herramientas bioinformáticas reveló varios aspectos del transcriptoma de este patógeno, como el metabolismo general y diferencial en el micelio y las levadu- ras, el ciclo celular, la replicación, reparación y recombinación del ADN, el apa- rato para la biogénesis del ARN, las maquinarias de traslación y destino de las proteínas, la pared celular, las enzimas hidrolíticas, las proteasas, las proteínas con anclaje GPI, los chaperones moleculares, el entendimiento de la resistencia a fármacos y los transportadores, la respuesta al estrés oxidativo y la virulencia. Este análisis ha proporcionado una visión más amplia de algunos aspectos específicos considerados relevantes para entender el conocimiento básico y aplicado de P. brasiliensis.

Palabras clave Transcriptoma, Etiquetas de secuencia, Hongo dimorfo y termorregulado, Paracoccidioides brasiliensis

Corresponding address: Dr. Maria Sueli S. Felipe Laboratório de Biologia Molecular Departamento de Biologia Celular Universidade de Brasília Brasília, DF, 70910-900 Brazil Tel.: +55 61 3072423 Fax: +55 61 3498411 E-mail: [email protected]

Paracoccidioidomycosis (PCM) is the most impor- activation of macrophages, the major defensive cell tant systemic mycosis in Brazil and widespread in Latin against P. brasiliensis [5,27,102]. In the absence of such America with about 10 million individuals infected being cytokines, such as in susceptible hosts, macrophages serve ~2% developing the disease [88]. Since 85% of PCM as a protected environment in which fungus can undergo cases occur in Brazil, this disease represents a major intracellular replication and disseminates from the lungs health problem, being classified as the first cause of death to other organs [17,18,19,20,50,75] as suggested in histo- among systemic mycoses, and the eighth, with respect to plasmosis [113]. All patients from whom the fungus is infectious and parasitic diseases [33,88]. PCM represents isolated must be treated, and despite new antifungal drugs, a serious public health challenge, with social and econo- pulmonary fibrosis is still the most frequent sequel. The mical importance. Infected individuals comprise rural outcome of infection depends on several factors, including workers living where forests and agriculture abound [16] host responses and the virulence of the infecting isolate. and immunocompromised patients [94] including indivi- PCM treatment lasts up to five years and uses basically duals with AIDS. PCM is characterised by granulomatous sulphonamides, azoles and amphotericin B, with success- inflammation, suppression of cellular immunity and high ful rates [52]. antibody titres [40,90]. The disease may develop as a Information on the genetic composition of benign and localized form to severe and disseminated [49] P. brasiliensis is scarce. The nuclear genome size estima- affecting the skin, lymph nodes and various internal ted by PFGE (Pulsed Field Gel Eletrophoresis) is around organs, including the lungs and the central nervous system 30 Mb and confocal fluorescence microscopy studies sug- [89]. All patients from whom the fungus is isolated should gest that P. brasiliensis is haploid/diploid or even aneu- be treated, and pulmonary fibrosis is still the major sequel ploid [42]. The electrophoretic pattern revealed despite the availability of new antifungal drugs for chromosomal polymorphism in the fungus, which presen- therapy. ted 4-5 chromosomal DNA molecules according to the The etiological agent of PCM is the fungus analyzed isolate, showing molecular sizes ranging from 2- Paracoccidioides brasiliensis, an ascomycete closely 10 Mb [42,71]. Based on the DNA sequencing of ~50 Kb related to other pathogenic fungi such as B l a s t o m y c e s of a P. brasiliensis genome fragment, it is estimated that de r m a t i t i d i s and Histoplasma capsulatum [7]. P. brasiliensis is a total of 7,500-9,000 genes [86]. exists as mycelium in the soil environment and as yeast in Our group has been working for the last 10 years in the host. The unicellular hyphae and fungal propagules or order to identify the differentially expressed genes invol- conidia are uninucleate, while yeasts are multinucleate. ved in the dimorphism of P. brasiliensis and also genes Infection typically occurs by inhalation of dry airborne encoding proteins which are immunogenic in humans spores, fungal propagules or mycelium fragments, which during infection. Several genes up-regulated in mycelium settle in the airways following the thermally regulated and yeast cells have been previously identified by DDRT- transition to the parasitic yeast phase [67,96]. The yeast PCR (Diferencial Display Reverse Transcriptase PCR), form has a well-established habitat as a parasite of ani- proteome analysis and immunoscreening of cDNA libra- mals, including humans, armadillos and penguins [6,25]. ries from mycelium and yeast cells using patient’s sera Many aspects of P. brasiliensis ecology remain unclear, [1,8,34-37,43,46,58,73,82,92,93,109]. especially those that concern its environmental niche An important and efficient approach for the com- [68,88]. The morphological switch from mycelium to parative study of various genomes is the sequencing of yeast is the most important biological feature that enables expressed sequence tags (ESTs) which reflects the expres- P. brasiliensis to colonize, invade and survive in the sion profile of different tissues, cell types or developmen- warm-blooded host [96]; strains unable to differentiate tal stages [84]. EST data is useful for the discovery of into yeast do not cause disease [14]. In vitro, this process homologous genes, detection of polymorphism and alter- can be reversibly driven mainly by a temperature switch native splicing of mRNAs; identification of vaccine candi- from 26 °C to 36 °C. The morphogenetic changes are date molecules, new drug targets, gene prediction and directly associated to the life cycle of this fungus; it expression studies. This review will focus on some biolo- undergoes molecular alterations during the morphogenetic gical aspects derived from P. brasiliensis t r a n s c r i p t o m e switch from hypha to the yeast phase [36]. project which was carried out by the PbGenome Network, The degree of pathogenesis varies according with a consortium of 11 public and private institutions from the host features and infecting lineage virulence. The immune Midwest region of Brazil [43,44]. response of the hosts against P. brasiliensis depends on factors such as, sex, age, nutritional state and genetic inheritance. PCM can be restricted to respiratory tract or The transcriptome project of P. brasiliensis disseminated all over the organism, and so, being lethal – an overview [49]. Thus, the forms of PCM can be divided into two groups: PCM infection, which is generally self-limited The Pb Genome project aimed to identify the trans- and restricted to the site of contact with fungi fragments cribed genes of mycelium and yeast cell-types of or to a single organ, affecting both sexes indistinctly; and P. brasiliensis separately, being of particular interest those PCM disease, which preferentially attacks males and can related to the dimorphic process, which could be key to a evolve benignly to a PCM infection or disseminate syste- better comprehension of the pathobiology of this fungus. mically imputing severe damage to the host. Ultimately, we sought the understanding of the cellular The fungus-host interaction will depend mainly on differentiation process at the molecular level, by means of the host immunological response and fungal virulence. the massive identification and annotation of genes. Clinical and experimental data indicate that cell-mediated The strategy employed to study the P. brasiliensis immune response is the main mechanism of defense (Pb01 isolate) transcriptome was to sequence ESTs deri- against P. brasiliensis infection, whereas specific antibo- ved from non-normalized cDNA libraries from mycelium dies produced in large amounts do not confer protection and yeast cells cultivated at 23 °C and 36 °C, respectively [11,24,47,60,66]. Protective cell-mediated immune res- (Figure). A total of 25,511 clones were sequenced corres- ponse in PCM is characterized by the production of cyto- ponding to 6,022 groups which represents the repertoire of kines (TNF-α, IL-12 and IFN-γ) that are required for the P. brasiliensis expressed genes [15]. This covers about Transcriptome of P. brasiliensis Andrade RV, et al. 205

Figure. Strategy for EST generation employed in the P. brasiliensis transcriptome project. Briefly, mRNA purified from mycelium and yeast cells were used as template for cDNA synthesis and then cloned into the multiple cloning site (MCS) of the λZAP expression vector (Stratagene). cDNA were sequenced from the 5´-end generating an EST (expressed sequence tags) collection which was deposited in the P. brasiliensis ESTs data bank (PbdbESTs) acces- sed through https://www.biomol.unb.br/Pb.

80% of the total number of predicted genes since it is esti- cycle), most of them experimentally confirmed by cDNA mated that P. brasiliensis contains ~8,000 genes. The microarray and confirmed by Northern blot analysis [43]. 6,022 clusters were categorized as follows: 2,129 success- The overall analysis indicates that alcohol fermentation fully annotated, 1,604 non-conclusively annotated and occurs preferentially in yeast cells while mycelium 2,289 not identified. The PbAESTs (P. brasiliensis saprophytic cells possess an aerobic metabolism. The Assembled ESTs) were classified in 18 functional COG putative capacity of the pathogen to also grow in anaero- categories: cellular metabolism (29%), transcription biosis was evidenced by the alternative conversion of (12%), protein synthesis (10%), energy production (9%), pyruvate to ethanol. Lastly, it may be able to utilize two- control of cellular organization (4%) and other categories. carbon sources in the form of acetate and ethanol through the glyoxylate cycle, since several pathways that provide Metabolic features. Annotation of the of P. brasi - substrates for the glyoxylate cycle are up-regulated in the l i e n s i s transcriptome has set the grounds for a global yeast cells. understanding of its metabolism in both mycelia and yeast forms [4,43]. This fungus is able to use several carbohy- Cell cycle, DNA replication, repair and recombi - drate sources including starch; and it can store reduced nation. Data derived from transcriptome analysis revealed carbons in the form of glycogen and trehalose; these pro- that cell cycle, DNA replication, DNA repair, and the vide energy reserves that are relevant for metabolic adap- recombination machineries are highly similar to that of tation, protection against stress and infectivity the yeast Saccharomyces cerevisiae [87]. Among ortho- mechanisms. The glyoxylate cycle, which is also involved logs detected in both species there are genes related to in pathogenicity, is present in this fungus. Classical path- cytoskeleton structure and assembly, chromosome segre- ways of lipid biosynthesis and degradation, including gation and cell cycle control genes (CDCs). We identified those of ketone body and sterol production, are well repre- at least one representative gene from each step of ini- sented in the database of P. brasiliensis. It is able to synt- tiation of DNA replication. hesize de novo all nucleotides and amino acids, with the We also identified the major players in DNA sole exception of asparagine, which was confirmed by the damage repair in P. brasiliensis, with the exception of fungus growth in minimal medium. Sulphur metabolism, photo-reactivation repair mechanism. Oxidative DNA as well as the accessory synthetic pathways of vitamins damage is mainly repaired by BER (base excision repair), and cofactors, is likely to exist in this fungus so this kind of repair may play an important role in host We have identified up-regulated genes in myce- defense resistance. P. brasiliensis had shown a large resis- lium and yeast cells encoding enzymes of central metabo- tance to high concentrations of H2O2 (Dantas, personal lic pathways (glycolysis, citrate cycle, sulphur and amino communication) that can induce oxidative DNA damage. acid metabolism, alcohol fermentation and glyoxylate So, maybe BER could be an important mechanism in the 206 Rev Iberoam Micol 2005; 22: 203-212 response of P. brasiliensis against oxidative stress caused Of the mitochondrial ribosomal proteins, we found by H2O2. In a growth-restricting environment, genetic orthologs to S. cerevisiae large (20) and small (18) riboso- adaptation of a microbial population involves mechanisms mal subunit proteins. Although orthologs to S. cerevisiae (e.g. error-prone polymerases) that lead to an elevated mitochondrial EF-Tu, EF-G and RF1 were detected, no mutation rate. Evidence for the presence of mutagenic sequences corresponding to functional EF-Ts were identified. pathways in P. brasiliensis may account for the high As for the post-translational apparatus, 64 trans- variability observed in karyotypes of different isolates and cripts associated to protein modifications and 28 to pro- the difficult to determine if P. brasiliensis isolates are one tein degradation were identified. These results suggest single species. that in P. brasiliensis these machineries are well conserved, when compared to other organisms. RNA biogenesis apparatus. The P. brasiliensis RNA biogenesis apparatus is very similar to those of other Cell wall. Besides being the first barrier against eukaryotes, such as S. cerevisiae [2]. PbAESTs related to host defences, the cell wall of human pathogenic fungus almost all categories of S . c e r e v i s i a e RNA biogenesis displays important antigens, thus presenting an active role were found. Two out of 12 S. cerevisiae RNA pol II core during infection [64]. Cell wall is a dynamic structure, subunits, Rbp3 and Rpb7, were found, probably reflecting whose components are continuously modified and rea- the growth phase from where the cDNA libraries, used in rranged during fungal life cycle [83]. A striking difference ESTs generation, were constructed. We have also found in the composition of the cell wall of P. brasiliensis is that orthologs to TBP, and at least one subunit of each TFII in α–glucan is the main yeast cell wall polysaccharide, while P. brasiliensis transcriptome, except TFIIB. β–glucan and galactomannan predominate in mycelium. With respect to the pre-mRNA processing, 65 Morphogenetic shift from hypha to the yeast form PbAESTs orthologs to S. cerevisiae basal splicing machi- is essential to P. brasiliensis pathogenicity and cell wall nery and 21 orthologs of 5’and 3’-ends formation proces- α-1,3-glucan and β-1,3-glucan seem to contribute to this ses were found. Components involved in RNA process [95]. In this view, studies on the enzymes invol- interference were detected, suggesting that this gene ved in cell wall biosynthesis and recycling, as well as on expression regulation mechanism is probably used by the cell-wall-associated molecules, should provide impor- P. brasiliensis. Twelve PbAESTs related to Pol I and Pol tant information for the design of drugs that affect selecti- III machineries were assigned as S. cerevisiae orthologs. vely the pathogen, and for new preventive or therapeutic Finally, 25 and 10 PbAESTs associated to rRNA and approaches. The P. brasiliensis transcriptome project has tRNA processing were detected. Altogether, our results revealed several data concerning the gene products that enable us to depict, for the first time, a global view of could be involved in this fungus cell wall metabolism transcription and RNA processing in P. brasiliensis. [107]. Even though P. brasiliensis is classified as an Chitosan was not yet identified in P. brasiliensis ascomycete, no sexual cycle has ever been observed. cell wall; nevertheless, the gene encoding chitin deacety- However we found transcripts encoding transcription fac- lase (cda), the enzyme that converts chitin to chitosan, is tors possibly related to sexual development such as over expressed in the yeast phase, according to transcrip- MCM1, a MAD box protein, which interacts with cofac- tome and cDNA microarray analyses [43,44]. tors that regulate mating in yeast, homologues to the Hydrophobins are small proteins that form an amphipathic mating type MAT-1 and NsdD, from A s p e r g i l l u s film at hydrophilic/hydrophobic interfaces covering fun- n i d u l a n s , and a putative MAT-2 encoding sequence. gal structures [114]. Two hydrophobin single-copy genes In addition to the genes described above, the description were detected in P. brasiliensis genome; Northern blot of other transcription factors regulated by signal transduc- analysis revealed that both mRNAs are mycelium-specific tion pathways strongly suggest, a not yet experimentally and highly expressed during the onset of the mycelium-to- observed, sexual cycle in P. brasiliensis. yeast transition [1]. Although yeast cells present a higher chitin content Translation and protein fate machineries. than mycelium, the chitin synthase genes Pbrc h s1 , P . b r a s i l i e n s i s translational and post-translational machi- Pbrchs2, Pbrchs3, Pbrchs4 and Pbrchs5 transcripts levels neries are very similar to those of other eukaryotes, such were higher in the former [79], suggesting an involvement as S. cerevisiae [103]. In the P. brasiliensis transcriptome of these multigenic family in morphogenesis. Our group we were able to find 78 of the 79 predicted cytosolic ribo- identified a new P. brasiliensis chitin synthase gene somal proteins, corresponding to the whole small subunit ( P b rc h s6) whose expression is restrict to the mycelium and almost complete large subunit set of proteins at the phase. exception of that encoding L23. Transcripts encoding pro- Genes whose products are involved in the synthesis teins of the large subunit (L24, P1 and P2) and of the small of N-linked outer chain mannans, such as d p m1, p m t1 , subunit (S2, S3, S10 and S14) are highly expressed. Also, mnn2 and mnn9 were represented in P. brasiliensis trans- the L19 cytosolic ribosomal protein of P. brasiliensis criptome. Their transcripts were exclusive of the yeast revealed to be differentially expressed in yeast cells. phase, except for the mnn2 mRNA which was present in From the analysis of 27 genes related to translation both phases. initiation in S. cerevisiae, 19 orthologs were identified in Some transglycosidase genes ( b g l2, g a s1, c r h1 , the P. brasiliensis transcriptome. All eukaryotic elonga- gel1, gel2 and gel3), possibly related to the cell wall syn- tion factors were detected being eEF1A one of the most thesis and fungal morphogenesis [10] were identified in expressed genes. A transcript for the putative initiation the P. brasiliensis transcriptome. In this context, myce- factor eIF5A was found in our analysis, which is not lium and yeast cells were tested for several glycohydrola- essential to the general protein synthesis but is essential to ses activities: high levels of β-glucanases, low amounts of yeast viability. We also found eEF3 which is required for α-glucanase, chitinase and maltase were detected. ribosome translocation in fungi and the translation termi- β–1,3–glucanase increasing levels correlated to the shift nation factor eRF3, but not eRF1. Sixteen PbAESTs, sho- to the mycelial phase (unpublished data). wing aminoacyl-tRNA synthetases predicted activities Taken together, these data can shed some light on were found in our analyses. the P. brasiliensis poorly understood cell wall metabo- Transcriptome of P. brasiliensis Andrade RV, et al. 207 lism, whose comprehension is essential to the develop- ved from Archaea to mammalian mitochondria, which not ment of more effective prophylactic and therapeutic pro- only degrades protein substrates but also binds DNA. Lon cedures against this pathogen. protease was previously described in P. brasiliensis [9]. The endopeptidase Clp/Hsp100 is a cytoplasmic Hydrolytic enzymes. Saprophytic fungi display a protease that plays an important role in many cellular pro- broad spectrum of protein- and polysaccharide-hydroly- cesses, including regulation of stress responses and pro- sing enzymes which allow the utilization of important tein quality control. Clp/Hsp100 proteins, such as ClpA available nutrients. This versatility is required since in the and ClpX, associate with the ClpP protease and direct the natural environment a large diversity of substrates, inclu- degradation of substrate proteins bearing specific sequen- ding animal, vegetal and fungal sources is expected to be ces. The first Clp protein described in the P. brasiliensis found. Since the mycelium form of P. brasiliensis is belie- was the ClpB [58]. ClpB is also a heat shock protein ved to be a saprobe, it should also be expected to have the which is induced upon the mycelium to yeast transition in enzymatic machinery necessary for the conversion of P. brasiliensis. Other members of the Clp protease family complex substrates [104]. Analysis of the P. brasiliensis were identified by analysis of the P. brasiliensis transcrip- transcriptome has revealed this to be true and these data tome: ClpA and ClpP2 was also further confirmed by enzymatic tests with the The 26S proteasome is a member of a family of supernatants of P. brasiliensis cultures grown with diffe- 'chambered proteases' and consists of the 20S proteasome, rent carbon sources [12]. which forms the proteolytically active core and a regula- The most important polysaccharides available in tory 19S complex. The 20S proteasome of higher eukar- the natural habitats of P. brasiliensis are hemicellulose, yotes is composed of seven distinct α and β subunits [63]. cellulose, and starch which can be hydrolysed to simple ESTs encoding all the α subunits (1 to 7) and six homolo- sugar for fungal metabolism. Xylanases are key enzymes gues to the 20S b subunit (1 to 6) are present in in the hydrolysis of hemicellulose. Although xylanase P . b r a s i l i e n s i s transcriptome. Of special note is the activity was detected in both fungal forms, it is six times presence of the β subunits 1, 2 and 5 suggesting that the higher in mycelium than in yeast, which is expected since complex is proteolytically active in P. brasiliensis. T h e hemicellulose is an abundant carbon source in the envi- eukaryotic 26S proteasome recognizes substrates that are ronment. P. brasiliensis also secretes cellulases confir- tagged with a “polyubiquitin chain” - a polymer assem- ming the transcriptome data which indicated the presence bled from the small (8 kDa), conserved protein ubiquitin. of β-1,4-glycosidase and endoglucanase homologues. ESTs coding deubiquitinating enzymes are present in this Another important plant polysaccharide is starch. transcriptome. In addition, components of the regulatory As expected, amylolitic activity was detected in 19S complex were found in the P. brasiliensis transcripto- P. brasiliensis mycelium cells and an α-amylase ortholog me in a total of nine different subunits. was found in P. brasiliensis transcriptome. Likewise, chitinolytic activity was only observed in mycelium cells. GPI-anchored proteins. A chitinase ortholog gene that contains motifs for extra- Detection of glycosylp- cellular localization was found. hosphatidylinositol (GPI) membrane anchors in proteins of P. brasiliensis was described for the first time in 1995 P r o t e a s e s . Proteases can be classified as aspartyl [56]. The P. brasiliensis transcriptome analysis allowed proteases, cysteine proteases, metalloproteases, serine an efficient retrieval of novel parasite GPI proteins [30]. proteases and threonine proteases, depending on the natu- Several studies have now established that GPI-anchored re of the active site. Many of these enzymes are of medi- proteins represent a large class of functionally diverse cal importance, including exogenous proteases encoded in proteins. They can be enzymes, surface antigens, adhesion the genomes of disease-causing organisms. A significant molecules, and surface receptors [31]. GPI-anchored pro- number (53) of protease genes were identified in the teins are leading vaccine candidates, though to be of P. brasiliensis transcriptome database [80]. A total of 15 major importance for infection [39]. These proteins have cDNAs, which encode energy independent protease certain characteristics which allow their recognition, homologs in the fungus transcriptome were annotated mainly N-terminal signal peptides and C-terminal features (3 aspartyl, 2 cysteine, 8 metallo and 2 serine proteases). that mediate GPI anchor addition at amino acid residue Among those, 2 are exopeptidases and the remaining 13 designated the omega (ω) site [54]; a serine-threonine rich are endopeptidases. sequence which provides sites for glycosylation; and the A gene coding an aspartyl protease of the A1 presence of basic or hydrophobic residues in the ω-region family (clan AA) is present in P. brasiliensis transcripto- that partially determine cellular localization me. Zinc-containing metalloproteases are widely distribu- [28,111,54,55]. By searching some of these characteristics ted in prokaryotic and eukaryotic organisms and are in the translated sequences derived from P . b r a s i l i e n s i s classified into four groups comprehending DD-carboxy- ESTs, 20 predicted GPI-anchored proteins were identi- peptidases, carboxypeptidases, zincins and inverzincins fied: nine enzymes (α-amylase, aspartic proteinase, Cu- [70]. From the eight identified energy independent zinc and Zn-containing superoxide dismutase 1, ECM33 and metalloproteases in the P. brasiliensis transcriptome, four DFG5-like, β-1,3-glucanosyltransferases 1, 2 and 3, and present the consensus motif HEXXH which define those phospholipase B); two structural proteins (Crh-like and proteases as members of the zincins family. Among the GPI-anchored cell wall protein); one adhesion molecule cysteine proteases, a caspase homolog was detected, sug- (Extracellular Matrix Protein, EMP); three surface anti- gesting that the programmed cell death in P. brasiliensis gens (expression library immunization antigen 1, proline- could be proteolytically regulated by this class of mole- rich antigen, β-1,3-glucanosyltransferase 1); and six cules [98]. In addition, a Kex2 endoprotease was identi- hypothetical proteins. Predicted localization in plasma fied among the serine proteases. The Kex2 endoprotease membrane or cell wall was reported for most of them. presented the highest identity to the previously described Metabolism of GPI-proteins involves different pro- kex2 gene of P. brasiliensis [108]. cesses: GPI biosynthesis, attachment of the GPI anchor to As for genes coding energy dependent proteases, the protein carboxyl-terminus in the endoplasmic reticulum, 12 ESTs were detected. Among these, one corresponds to GPI protein trafficking and sorting, and protein release by a Lon protease, a multi-functional enzyme that is conser- the action of phospholipases. 208 Rev Iberoam Micol 2005; 22: 203-212

In mammals, around 20 genes participate in the hsp42, hsp60, hsp70 and hsp90 sequences were up regula- GPI biosynthesis pathway and are called PIG (phospha- ted in the yeast form. The increased expression (about tidylinositol-glycan) genes. The glycoslyltransferase com- 38% higher) of heat shock genes observed in the yeast plex composed by the proteins Pig-A, Pig-C, Pig-H, GPI1, phase was expected, since the differentiation of Pig-P and DPM2 (Dolichol-Phosphate-Mannose 2) cataly- P. brasiliensis cells relies on a thermal shift from the envi- ses the first step in the GPI synthesis [112]. Genes enco- ronmental temperature to around 37 °C both in vitro as in ding Pig-A, Pig-C, Pig-P and DPM2 of the vivo. Transcriptome data greatly improved our knowledge glycoslyltransferase complex were detected in of P. brasiliensis molecular chaperones, since only eight P. brasiliensis transcriptome. A gene homolog to PIG-L, HSPs had been previously reported in this pathogen. which codes an enzyme catalyzing the second step of GPI synthesis, was present in the transcriptome. In addition, Transporters and insights on drug resistance. In P I G - O , which codes an enzyme involved in the EtNP the analysis of microbial genome, specially those pathoge- transfer to the first and third mannose residues of the GIP nic ones, the identification of transmembrane protein anchor, was identified. Therefore, most of the genes invol- transporters are of special interest. This is an important ved in GPI biosynthesis were identified in P. brasiliensis, class of proteins once they can play a pivotal role in six remaining to be identified or to be described as being microbial survival and pathogenesis, including, drug resis- inexistent in this parasite. tance. In the P. brasiliensis trancriptome we found 26 Attachment of the GPI to a protein involves clea- groups (including singlets and contigs) that show a high vage of the pre-protein at a hydrophobic aminoacid similarity degree with transporters proteins, 22 that pro- sequence, followed by the attachment of the cleaved bably coding for ABC (ATP Binding Cassette) transpor- sequence to the fully assembled GPI via a transamidase ters and four that probably code for MFS - Major reaction. Human GPI transamidase is a protein complex, Facilitator Superfamily [32]. Among ABC transporters we containing five homologous subunits (GPI8, PIG-S, PIG- found, for example, Candida albicans CDR 1 [85] and T, and PIG-U) [57]. All of those encoding transamidases CDR 2 [97], S. cerevisae P D R 5 [45] and A. nidulans ESTs were detected in the analysis of the P. brasiliensis ATRF [3] orthologs. Regarding MSF we were also able to transcriptome with the exception of ESTs encoding PIG-U. identify a C. albicans M D R 1 [23] ortholog, also pointed Cleavage of GIP anchor by phospholipases (PL) is out as an important virulence gene. All of those genes are a mean of selective protein release. In P. brasiliensis, a related in these fungi to azole resistance. These findings potent PLC capable of selectively hydrolyze GPI anchor along with the recent reported ketoconazole resistant was detected [56]. Two open reading frames with high P. brasiliensis isolation from PCM patients [53] advice us sequence homology to phosphatidylinositol specific to this pathogen resistance potential emergence. phospholipase C (PI-PLC) and phospholipase D (PLD) of Aspergillus nidulans and Aspergillus oryzae respectively, Oxidative stress response. Oxidative stress is an were revealed in the P. brasiliensis transcriptome. imbalance between oxidants and antioxidants in favor of the former [100]. It can be caused by reactive oxygen spe- •- Molecular chaperones. Molecular chaperones or cies (ROS) such as the superoxide radical (O 2 ), hydrogen • stress proteins were first described as heat shock proteins peroxide (H2O2), hydroxyl radical (HO ), and peroxynitri- (HSPs), since they are over-expressed in response to heat te (ONOO-), a product of the reaction between superoxide shock. Other environmental parameters, such as oxidative with nitric oxide (NO). Enzymatic and non-enzymatic [72], osmolarity [99] and cold shock [59] stresses, also defense systems function to avoid the biological damage induce the expression of molecular chaperones. Another caused by ROS. Intracellular parasites should have antio- type of chaperone is the carbohydrate trehalose, which is xidants to protect against endogenously formed ROS and produced at high levels in response to several stresses in against ROS produced by the host cells. The enzymatic S. cerevisiae and other organisms. This molecule stabili- defense system present in P. brasiliensis include catalase, zes proteins and biological membranes, being thus consi- superoxide dismutase, cytochrome c peroxidase and dered a chemical chaperone. Its synthesis and degradation peroxiredoxin [26]. is tightly controlled in response to heat stress (for review Glutathione (GSH, γ-L- g l u t a m y l -L- c y s t e i n y l g l y c i- see [110]) and it is also produced by P. brasiliensis, ne) is an abundant thiol tripeptide that takes part in the according to our transcriptome data analysis [77]. metabolism of ROS. It protects against oxidative stress by Molecular chaperones are involved in protein fol- maintaining the cytosol of the cells more reduced. GSH ding and renaturing and are also implicated in other biolo- may react with the hydroxyl radical directly, producing gical processes, including the dimorphic transition of water [101]. Its synthesis occurs by the consecutive action fungi and immunopathogenicity of infectious diseases. of γ-glutamyl-cysteine synthetase and glutathione synthe- The P. brasiliensis HSP70 is preferentially expressed in tase. Genes coding both enzymes were identified in the the yeast phase [37]. Recombinant P. brasiliensis HSP60 P . b r a s i l i e n s i s transcriptome, as well as that coding for was recognized by 75 serum samples of infected patients, glutathione reductase (GR), responsible for reducing oxi- which suggests the usefulness of HSP60 singly or in asso- dized glutathione (GSSG) back to GSH using NADPH as ciation with other recombinants antigens in the PCM diag- an electron donor. However, a glutathione peroxidase nosis [35]. (GPx) gene is probably absent in the parasite’s transcrip- In the P. brasiliensis transcriptome we have identi- tome. A substitute for this enzyme may be a 1-Cys peroxi- fied 438 ESTs (184 in mycelium and 253 in yeast) which redoxin with GPx activity [26]. were clustered in 48 distinct chaperones or co-chaperones Glutathione S-transferases (GST) conjugate GSH transcripts [43]. These genes were classified in families, to DNA and lipid hydroperoxides, as well as mutagens, corresponding to three small chaperones, nine HSP40s, carcinogens, and other toxic chemical substances [61]. ten HSP60s, seven HSP70s, five HSP90s, four HSP100s These enzymes interact with kinases and have GSH- and ten other chaperones. Calnexin, the cytoplasmic dependent peroxidase and isomerase activities [74]. hsp60 (cct7) and the sba1 co-chaperone genes were over P. brasiliensis has a GST homolog belonging to the class expressed in mycelium cells, while the co-chaperone cpr1, Omega [13] and a microsomal counterpart of this enzyme. Transcriptome of P. brasiliensis Andrade RV, et al. 209

The Cu+ 2 and Fe+ 2 ions play an important role in Perspectives ROS production. Organisms need these them for trans- port, protection against oxidative stress, cell growth and Functional analysis of the genes described in the development. However, these ions can catalyze hydroxyl P. brasiliensis transcriptome will yield relevant informa- +2 + radical formation by the Fenton reaction: Fe + H2O2 + H tion about cellular differentiation, pathogenicity and/or +3 • → Fe + H2O + HO [51]. Therefore, the cellular levels of virulence as genetic tools become available for this patho- Fe +2 and Cu+2 should be carefully controlled. Noteworthy is gen. However, functional analysis by gene disruption in the presence of a frataxin gene homolog in P. brasiliensis. P . b r a s i l i e n s i s is hampered by the evidence that isolates In S. cerevisiae, frataxin functions both as a chaperone for have different number of chromosomes [42] and cells are Fe2+ when mitochondrial iron is limiting, and as a storage normally multinucleated. molecule for Fe3+ when iron is in excess [81]. In humans, Peptide analogues to human proteins have been defects in frataxin causes Friedreich ataxia (FRDA), a reported as potent anti-fungal agents [48]. The high severe neuro- and cardio-degenerative disease in humans, potency and low toxicity make these peptides strong can- in which mitochondria are unable to handle iron properly didates for the treatment of several mycoses. These dise- [62,91]. Studies on P. brasiliensis frataxin may help to ases are traditionally treated with amphotericin B and clarify the function of this protein in humans. azoles (fluconazole or itraconazole, among others) but The transcripton factors Yap1 and Skn7 function in these drugs are becoming less effective as drug resistance signaling pathways used by fungal cells to control the arise among several pathogenic fungi. However, therapeu- expression of genes involved in the oxidative-stress res- tic peptides are less likely to induce drug resistance; for ponse [76]. The presence of ESTs coding both factors in example, peptides derived from MSH (melanocite stimu- P. brasiliensis is important to preserve cellular viability lating hormone) have shown no toxicity in pre-clinical when ROS are produced endogenously or as a result of and in vitro studies [48]. host immune attack. Among the perspectives that we foresee as a consequence of the transcriptome project we would like to high- Virulence. The search for PbAESTs related to viru- light the following: lence genes was based on those orthologs that fitted mole- 1) In vivo expression analysis will be extended to the cular Koch´s postulate: Falkow’s postulate [29,41] and genes proposed to be involved in host-pathogen inte- made possible the annotation of 28 virulence PbAESts raction, including the differentially expressed genes in [43,105]. Among those groups are metabolism gene ortho- mycelium and yeast cells, heat shock, essential genes, logs (eight different groups), two PbAESTs that probably putative drug targets and virulence factors. cDNA code for ICL (isocitrate lyase) and MLS1 (malate syntha- microarray experiments are currently being carried out se) C. albicans glyoxylate cycle orthologs, that is upregu- in order to evaluate the in vivo expression profile of lated in P. brasiliensis yeast form [43]. This phenomenon these genes in macrophages and human pulmonary was also described for C. albicans during its intracellular epithelial cells infected by P. brasiliensis. phase [65], where it is thought to play a crucial function in 2) S. cerevisiae can be used as a tester model for gene nutritional furnish. A similar mechanism can be postula- functional analysis by trans-complementation as ted for P. brasiliensis into the macrophage, after phagocy- recently demonstrated [78]. tosis. Those genes related to cell wall biosynthesis and 3) RNA interference (RNAi), a natural process which stabilization is also reported as critical to pathogenesis. relies in the ability of the cell to target and destroy We were able to find six PbAESts that shares high simila- foreign genetic material, may represent an attractive rity degree with fungal cell wall biosynthesis that were alternative for gene analysis in P. brasiliensis. T h i s previously reported as important virulence genes approach has been successfully used in other fungi [21,22,69,106]. The disruption of these genes affects such as H. capsulatum, Cryptococcus neoformans and C. albicans survival and attenuates its virulence, so these Neurospora crassa [38]. The identification of five can be potential drug targets, also because cell wall is the N . c r a s s a orthologs (rrp-3, qde-2, sms-2, dcl-2 and key difference between fungal and human cell. During the recQ-2) in the P. brasiliensis transcriptome is an evi- infection, P. brasiliensis is submitted to oxidative stresses dence that RNAi may also occur in this fungus thus caused by reactive oxygen (ROI) and nitrogen (RNI) encouraging the development of an efficient strategy intermediates generated during immune response to infec- for functional gene analysis. tion. In our transcriptome we found at least four groups 4) In silico design of synthetic peptides based on ESTs that probably codes for enzymes that are able to ROI and sequences may generate new antifungal molecules for RNI detoxification, whose other fungi orthologs also fit the treatment of PCM. Falkow´s postulate, decreasing their virulence when disrupted, and reversing this phenotype when the null mutants were supplied with the disrupted gene. The other ten PbAests assigned as potential virulence genes have miscellaneous fungi orthologs [43], as for example: phospholipase, urease, multi drug resistance transporter, signal transduction genes and other diverse functions. 210 Rev Iberoam Micol 2005; 22: 203-212

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