Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 degrees C and as yeasts (Y) at 37 degrees C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition.     

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.     

Evidence for positive selection in putative virulence factors within the Paracoccidioides brasiliensis species complex

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood-based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi.     

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.     

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.     

The Ajellomycetaceae, a new family of vertebrate-associated Onygenales

Phylogenies inferred from the analysis of DNA sequence data have shown that the Onygenales contains clades that do not correspond with previously described families. One lineage identified in recent molecular phylogenetic studies includes the dimorphic pathogens belonging to the genera Ajellomyces, Emmonsia and Paracoccidioides. To evaluate the degree of support for this lineage and determine whether it includes additional taxa, we examined relationships among the members of this clade and selected saprobic onygenalean taxa based on maximum-parsimony analyses of partial nuclear large RNA subunit (LSU) and internal transcribed spacer (ITS) sequences. A clade distinct from the Onygenaceae was found to encompass Ajellomyces (including the anamorph genera Blastomyces, Emmonsia and Histoplasma) and Paracoccidioides brasiliensis. The members of this lineage are saprobic and pathogenic vertebrate-associated taxa distinguished by their globose ascomata with coiled appendages, muricate globose or oblate ascospores, and lack of keratinolytic activity. Anamorphs are solitary aleurioconidia or irregular alternate arthroconidia. Based on molecular data and on morphological and physiological similarities among these taxa, we propose the new family, Ajellomycetaceae.     

Nutritional studies on Paracoccidioides brasiliensis: the role of organic sulfur in dimorphism

The nutritional requirements of the mycelial and yeast-like phases of the dimorphic fungus Paracoccidioides brasiliensis, a human pathogen, were investigated. For all nine isolates tested, mycelial cells were prototrophic, whereas yeast-like cells required a sulfur-containing amino acid for growth. Moreover, changing the source of nitrogen greatly affected the morphology of the yeast-like cells.     

<Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Infection Mimicking Recurrent Hodgkin Lymphoma: A Case Report and Review of the Literature

Paracoccidioides infection is a rare entity in the USA. This dimorphic fungus is found in Central and South America and is thought to be acquired by inhalation through the soil. We report a case of Paracoccidioides brasiliensis infection presenting as a clavicular bone lesion, peripancreatic mass, and various skin lesions. A 35-year-old man with a history significant for Hodgkin lymphoma presented with a left clavicular mass that was suspected clinically and radiologically as recurrent Hodgkin lymphoma. He was not experiencing any associated symptoms and was undergoing chemotherapy treatment for his known Hodgkin disease. On CT imaging, the mass was seen as a lytic bone lesion with an overlying soft tissue mass. This was biopsied and histologically diagnosed as a Paracoccidioides brasiliensis infection with associated necrotizing granulomatous inflammation. Also found on the CT scan was an enlarging peripancreatic mass which on endoscopic biopsy had similar histologic findings. In conclusion, this report presents a rare case of Paracoccidioides brasiliensis infection mimicking recurrent Hodgkin lymphoma.     

Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.     

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.