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.     

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection.     

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.     

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.     

A synthetic peptide selectively kills only virulent Paracoccidioides brasiliensis yeasts

This work was conducted to identify virulence biomarkers for Paracoccidioides brasiliensis (Pb), the fungus responsible for Paracoccidioidomycosis (PCM), a systemic disease endemic in Latin America. Measurement of mortality showed that all B10.A mice were killed after 250 days by the virulent Pb18 isolate while only one of the mice that received the attenuated counterpart died. Also, number of lung CFUs from virulent Pb18 inoculated mice were much higher when these isolates were compared. Phage display methodology allowed selection of three phages that specifically bound to virulent Pb18. Variability of p04 phage binding to different Pb isolates were examples of variability of expression by the fungus of its binding molecule, strongly suggesting p04 as a biomarker of virulence. In vitro, its derived peptide pep04 killed only virulent fungi, and confocal microscopy showed that it was internalized only by the virulent isolate. Pep04 blocked establishment of Pb infection in mice and virulent Pb18 pre-incubated with p04 showed significantly inhibited lung infection. Furthermore, infected mice treated with p04 showed highly significant reduction in lung CFUs. These findings firmly establish p04 as a biomarker of Pb virulence. Therefore, after proper peptide engineering, p04 may become a useful adjuvant for the distressing treatment of PCM.     

Chromosomal polymorphism,syntenic relationships,and ploidy in the pathogenic fungus Paracoccidioides brasiliensis

Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus.     

Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo Dasypus novemcinctus )

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo ( Dasypus novemcinctus ).Pulmonal inflammatory area was evaluated by intravenous inoculation of 10⁶ yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously. This revised version was published online in August 2006 with corrections to the Cover Date.     

Binding of laminin to Paracoccidioides brasiliensis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-virulence isolates

The pathogenic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM). This pulmonary mycosis, acquired by inhalation of airborne propagules, may disseminate to several internal organs and tissues, leading to severe disease. Adhesion to host cell components is the first step involved in dissemination of pathogens. Previous studies showed that laminin, the most abundant glycoprotein of the basement membrane, binds to P. brasiliensis yeast cells, enhancing their pathogenicity in the hamster testicle model. As PCM is primarily a pulmonary infection, we studied the influence of previous treatment of yeast cells with laminin on the course of the intratracheal infection of resistant and susceptible mice using high-virulence (Pb18) and low-virulence (Pb265) P. brasiliensis isolates. Laminin treatment did not alter fungal loads, delayed-type hypersensitivity reactions, levels of pulmonary cytokines and production of specific antibodies in any group of Pb18-infected mice. However, early in the infection, a less intense inflammatory reaction was detected in the lungs of the laminin-treated groups. In addition, laminin treatment of Pb265 resulted in a less severe infection as revealed by the lower fungal loads recovered from lungs. Antibody and cytokine levels, however, did not change after laminin treatment. Altogether, our results demonstrate that laminin binding to yeast cells diminishes P. brasiliensis pathogenicity. The lower inflammatory response observed with the virulent isolate and the decreased pulmonary fungal burden with the low-virulence isolate indicate an inhibitory effect of laminin treatment on P. brasiliensis infectivity and interaction with pulmonary host cells or extracellular matrix proteins.     

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P. brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Pb18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P. brasiliensis.     

Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos ( Dasypus novemcinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P. brasiliensis cells with different genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genome size and ploidy of Paracoccidioides brasiliensis reveals a haploid DNA content: flow cytometry and GP43 sequence analysis

The aim of this study was to evaluate genome size and ploidy of the dimorphic pathogenic fungus Paracoccidioides brasiliensis. The cell cycle analysis of 10 P. brasiliensis isolates by flow cytometry (FCM) revealed a genome size ranging from 26.3+/-0.1Mb (26.9+/-0.1fg) to 35.5+/-0.2Mb (36.3+/-0.2fg) per uninucleated yeast cell. The DNA content of conidia from P. brasiliensis ATCC 60855-30.2+/-0.8Mb (30.9+/-0.8fg) -showed no significant differences with the yeast form, possibly excluding the occurrence of ploidy shift during morphogenesis. The ploidy of several P. brasiliensis isolates was assessed by comparing genome sizing by FCM with the previously described average haploid size obtained from electrophoretic karyotyping. The analysis of intra-individual variability of a highly polymorphic P. brasiliensis gene, GP43, indicated that only one allele seems to be present. Overall, the results showed that all analysed isolates presented a haploid, or at least aneuploid, DNA content and no association was detected between genome size/ploidy and the clinical-epidemiological features of the studied isolates. This work provides new knowledge on P. brasiliensis genetics/genomics, important for future research in basic cellular/molecular mechanisms and for the development/design of molecular techniques in this fungus.     

Distribution of a corticosteroid-binding protein in Candida and other fungal genera

Using [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (Kd) in the various isolates ranged between 8 and 19 nM; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)-1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.     

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.     

Identification of transcription elements in the 5' intergenic region shared by LON and MDJ1 heat shock genes from the human pathogen Paracoccidioides brasiliensis. Evaluation of gene expression

The MDJ1/LON locus is conserved among pathogenic dimorphic fungi. We have mapped using DNase I footprinting and mobility shift assays three putative heat shock elements and one AP-1 binding domain (ARE) in the 5' intergenic region shared by PbMDJ1and PbLON (ML) from Paracoccidioides brasiliensis. The region bearing an ARE-like towards PbLON also has an opposite skn-1-like element. We studied genetically and pathogenically distinct isolates Pb18 and Pb3, where ML is polymorphic and the number of elements detected was higher. The functionality of the elements was suggested by the stimulatory response of both genes to heat shock and oxidative stress. Co-regulation occurred upon heat shock from 36 to 42 degrees C and, only in Pb3, also during mycelium to yeast transformation (26-36 degrees C). In Pb18, PbMDJ1 seemed to be preferentially expressed in yeast. Our study might help understand regulation of genes involved in fungal adaptation to the host.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.