Paracoccidioides lutzii sp. nov.: biological and clinical implications.

Paracoccidioides lutzii, formerly known as 'Pb01-like' strains in the P. brasiliensis complex, is proposed as a new species based on phylogenetic and comparative genomics data, recombination analysis, and morphological characteristics. Conidia of P. lutzii are elongated, different from those of P. brasiliensis. P. lutzii occurs in the central and northern regions of Brazil. Studies comparing P. brasiliensis and P. lutzii may have significant clinical consequences for the diagnosis and treatment of paracoccidioidomycosis.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Caspofungin affects growth of Paracoccidioides brasiliensis in both morphological phases.

Five Paracoccidioides brasiliensis isolates were grown in the presence of caspofungin (0 to 1 µg/ml). Inhibition of the yeast phase ranged from 20 to 65%, while in the mycelial form it ranged from 75% to 82%. Such variability was loosely related to the amount of cell wall ß-1,3-glucan. No association with point mutations in the ß-1,3-glucan synthase was detected. Caspofungin induced physical changes and cytoplasmic deterioration in both fungal phases.

Expression of Paracoccidioides brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant demonstrates its functionality as a chitin synthase gene.

We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall.

Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis.

In this report we identified orthologues of fungal AGS1, RHO1, RHO2, RAC1 and CDC42 genes in the dimorphic fungus Paracoccidioides brasiliensis. Based on its homology to known fungal sequences, P. brasiliensis Ags1 was identified as an alpha-1,3-glucan synthase, while Rho1, Rho2, Rac1 and Cdc42 proteins were classified into the Rho1, Rho2, Rac1 and Cdc42 subgroups of fungal Rho GTPases, respectively. Of them, Rho1 is one of two subunits of a putative beta-1,3-glucan synthase complex, the other being the synthase itself (Fks1), while Rho2 has been associated to the alpha-1,3-glucan synthase (Ags1). Expression studies showed that mRNAs levels of RHO2 and AGS1 kept a direct relationship but the levels of RHO1 and FKS1 did not. P. brasiliensis RHO1 successfully restored growth of Saccharomyces cerevisiae rho1 mutant under restrictive temperature conditions. Chemical analyses of P. brasiliensis alpha-1,3-glucan, synthesized by Ags1p, indicated that it is essentially a linear polysaccharide, with <3% of alpha-1,4-linked glucose branches, occasionally attached as single units to the alpha-1,3-backbone.

Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus.

Paracoccidioidomycosis (PCM) is a systemic disease endemic to most of Latin America, with greatest impact in rural areas. The taxonomic status of one of the best studied Paracoccidioides isolates (Pb01) as P. brasiliensis remains unresolved due to its genomic differences from the other three previously described phylogenetic species (S1, PS2 and PS3; Carrero et al., 2008. Fungal Genet. Biol. 45, 605). Using the genealogic concordance method of phylogenetic species recognition (GCPSR) via maximum parsimony and Bayesian analysis, we identified a clade of 17 genotypically similar isolates, including Pb01, which are distinct from the S1/PS2/P3 clade. Consistent with GCPSR, this "Pb01-like" group can be considered a new phylogenetic species, since it is strongly supported by all independent and concatenated genealogies. "Pb01-like" species exhibit great sequence and morphological divergence from the S1/PS2/PS3 species clade, and we estimate that these groups last shared a common ancestor approximately 32 million years ago. In addition, recombination analysis revealed independent events inside both main groups suggesting reproductive isolation. Consequently, we recommend the formal description of the "Pb01-like" cluster as the new species Paracoccidioides lutzii, a tribute to Adolpho Lutz, discoverer of P. brasiliensis in 1908.

Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration.

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583nt open reading frame, interrupted by three introns of 82, 87 and 97bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4kb from the 5' UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen.

By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.

Paracoccidioides brasiliensis AND Paracoccidioides lutzii, A SECRET LOVE AFFAIR.

To commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field.

[Mechanisms of degradation of the fungal ornithine decarboxylase]. / El peculiar mecanismo de degradación de la ornitina decarboxilasa fúngica.

Ornithine decarboxylase (ODC) is the first enzyme in polyamine biosynthesis in numerous living organisms, from bacteria to mammalian cells. Its control is under negative feedback regulation by the end products of the pathway. In dimorphic fungi, ODC activity and therefore polyamine concentrations are related to the morphogenetic process. From the fission yeast Schizosaccharomyces pombe to human, polyamines induce antizyme synthesis which in turn inactivates ODC. This is hydrolyzed by the 26S proteasome without ubiquitination. The regulatory mechanism of antizyme on polyamines is conserved, although to date no antizyme homology has been identified in some fungal species. The components that are responsible for regulating polyamine levels in cells and the current knowledge of ODC regulation in dimorphic fungi are presented in this review. ODC degradation is of particular interest because inhibitors of this pathway may lead to the discovery of novel antifungal drugs.

Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.

α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified.

Expression of Paracoccidioides brasiliensis AMY1 in a Histoplasma capsulatum amy1 mutant, relates an α-(1,4)-amylase to cell wall α-(1,3)-glucan synthesis.

In the cell walls of the pathogenic yeast phases of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum, the outer α-(1,3)-glucan layer behaves as a virulence factor. In H. capsulatum, an α-(1,4)-amylase gene (AMY1) is essential for the synthesis of this polysaccharide, hence related to virulence. An orthologous gene to H. capsulatum AMY1 was identified in P. brasiliensis and also labeled AMY1. P. brasiliensis AMY1 transcriptional levels were increased during the yeast phase, which correlates with the presence of α-(1,3)-glucan as the major yeast cell wall polysaccharide. Complementation of a H. capsulatum amy1 mutant strain with P. brasiliensis AMY1, suggests that P. brasiliensis Amy1p may play a role in the synthesis of cell wall α-(1,3)-glucan. To study some biochemical properties of P. brasiliensis Amy1p, the enzyme was overexpressed, purified and studied its activity profile with starch and amylopeptin. It showed a relatively higher hydrolyzing activity on amylopeptin than starch, producing oligosaccharides from 4 to 5 glucose residues. Our findings show that P. brasiliensis Amy1p produces maltooligosaccharides which may act as a primer molecule for the fungal cell wall α-(1,3)-glucan biosynthesis by Ags1p.

Genus paracoccidioides: Species recognition and biogeographic aspects.

BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. METHODOLOGY/PRINCIPAL FINDINGS: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11-32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. CONCLUSIONS/SIGNIFICANCE: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides.

Experimental medical mycological research in Latin America - a 2000-2009 overview.

An overview of current trends in Latin American Experimental Medical Mycological research since the beginning of the 21(st) century is done (search from January 2000 to December 2009). Using the PubMed and LILACS databases, the authors have chosen publications on medically important fungi which, according to our opinion, are the most relevant because of their novelty, interest, and international impact, based on research made entirely in the Latin American region or as part of collaborative efforts with laboratories elsewhere. In this way, the following areas are discussed: 1) molecular identification of fungal pathogens; 2) molecular and clinical epidemiology on fungal pathogens of prevalence in the region; 3) cell biology; 4) transcriptome, genome, molecular taxonomy and phylogeny; 5) immunology; 6) vaccines; 7) new and experimental antifungals.

Synthesis, in vitro antifungal activity and mechanism of action of four sterol hydrazone analogues against the dimorphic fungus Paracoccidioides brasiliensis.

The design and synthesis of novel sterol hydrazone analogues (9, 10, 11 and 12) are described, followed by their evaluation as inhibitors of fungal growth, using Paracoccidioides brasiliensis as the biological tester. Compounds 9, 10, 11 and 12 generated a dose-dependent effect in fungal growth, particularly 9, 11 and 12, which were active at nanomolar concentrations (100 nM). When P. brasiliensis in its pathogenic yeast-like phase was treated individually with each of the aforementioned compounds at concentrations that reduced growth rate around 50%, the analysis of sterol composition in the resulting surviving cells demonstrated a 50% reduction of the final sterols brasicasterol and ergosterol, and concomitant increase in the levels of lanosterol. These results indicate that these compounds inhibit the enzyme Δ(24)-sterol methyl transferase (SMT), in a manner dependent on the stereochemical location of the hydrazone group. Compound 12, instead, induced a good antiproliferative activity not associated with blockage of any step in the pathway to sterol biosynthesis, suggesting a different mode of action. The X-ray crystal structure of H1 was determined to obtain information regarding the rings and side chain conformation of the sterol hydrazones. Comparison of the inhibitory effects of sterol hydrazones (9-12) and azasterols (AZA1-AZA3) on SMT with the molecular electrostatic potential, negative isopotential energy surfaces (-10 kcal/mol) and local ionization potential calculated via DFT methods, showed that changes in the electronic moiety introduced by the N and O atoms were not as important as the additional flexibility of the side chain introduced by an extra methylene group.

Paracoccidioides brasiliensis AND Paracoccidioides lutzii, A SECRET LOVE AFFAIR / Paracoccidioides brasiliensis e Paracoccidioides lutzii, um caso secreto de amor

SUMMARYTo commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field.

RESUMOPara comemorar o centenário de aniversário do Prof. Dr. Carlos da Silva Lacaz, os autores fazem um breve relato dos estudos sobre a biologia, ecologia e filogenia molecular que culminaram na revelação da espécie Paracoccidioides lutzii, que havia permanecido escondida por mais de um século ao lado de Paracoccidioides brasiliensis. O professor Lacaz exerceu papel central no desenvolvimento desta área do conhecimento, pois manteve interesse permanente nas pesquisas deste fungo e da paracoccidioidomicose, visando principalmente proporcionar benefícios aos pacientes acometidos por esta micose.

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy.

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of delta 24(28) sterol methyl reductase (SMR) and/or delta (24)-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.

The actin gene in Paracoccidioides brasiliensis: organization, expression and phylogenetic analyses.

PbrACT1, the gene responsible for the synthesis of actin in Paracoccidioides brasiliensis, was found as a single copy, organized into six exons and five introns. Its open reading frame (ORF) codes for a putative protein of 375 amino acids, with a molecular mass of 41.5 kDa and an isoelectric point of 5.6. Analysis of the nucleotide sequence revealed a high homology to other fungal actins, the presence of characteristic fungal actin sequences, and heat shock elements at the 5' untranslated region (UTR). Phylogenetic analyses with deduced amino acid sequences of fungal actins grouped P. brasiliensis within the phylum Ascomycota, order Onygenales, in concordance with a few previous reports. Patterns of expression through the temperature-induced morphological transitions from mycelial to yeast-like shapes and reverse, suggests that PbrACT1 is regulated in this process. The PbrACT1 gene sequence is available at the GenBank database under accession number AY383732.

Topology of charge density of flucytosine and related molecules and characteristics of their bond charge distributions.

The molecular charge distribution of flucytosine (4-amino-5-fluoro-2-pyrimidone), uracil, 5-fluorouracil, and thymine was studied by means of density functional theory calculations (DFT). The resulting distributions were analyzed by means of the atoms in molecules (AIM) theory. Bonds were characterized through vectors formed with the charge density value, its Laplacian, and the bond ellipticity calculated at the bond critical point (BCP). Within each set of C=O, C-H, and N-H bonds, these vectors showed little dispersion. C-C bonds formed three different subsets, one with a significant degree of double bonding, a second corresponding to single bonds with a finite ellipticity produced by hyperconjugation, and a third one formed by a pure single bond. In N-C bonds, a decrease in bond length (an increase in double bond character) was not reflected as an increase in their ellipticity, as in all C-C bonds studied. It was also found that substitution influenced the N-C, C-O, and C-C bond ellipticity much more than density and its Laplacian at the BCP. The Laplacian of charge density pointed to the existence of both bonding and nonbonding maxima in the valence shell charge concentration of N, O, and F, while only bonding ones were found for the C atoms. The nonbonding maxima related to the sites for electrophilic attack and H bonding in O and N, while sites of nucleophilic attack were suggested by the holes in the valence shell of the C atoms of the carbonyl groups.