The glyceraldehyde-3-phosphate dehydrogenase homologue is differentially regulated in phases of Paracoccidioides brasiliensis: molecular and phylogenetic analysis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis. We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH. That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome. The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%). Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships. Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P. brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.     

Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.     

Comparison of transcription of multiple genes during mycelia transition to yeast cells of Paracoccidioides brasiliensis reveals insights to fungal differentiation and pathogenesis

The ascomycete Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The infection process of P. brasiliensis is initiated by aerially dispersed mycelia propagules, which differentiate into the yeast parasitic phase in human lungs. Therefore, the transition to yeast is an initial and fundamental step in the infective process. In order to identify and characterize genes involved in P. brasiliensis transition to yeast, which could be potentially associated to early fungal adaptation to the host, expressed sequence tags (ESTs) were examined from a cDNA library, prepared from mycelia ongoing differentiation to yeast cells. In this study, it is presented a screen for a set of genes related to protein synthesis and to protein folding/modification/destination expressed during morphogenesis from mycelium to yeast. Our analysis revealed 43 genes that are induced during the early transition process, when compared to mycelia. In addition, eight novel genes related to those processes were described in the P. brasiliensis transition cDNA library. The types of induced and novel genes in the transition cDNA library highlight some metabolic aspects, such as putative increase in protein synthesis, in protein glycosylation, and in the control of protein folding that seem to be relevant to the fungal transition to the parasitic phase.     

Ultrastructure of Dimorphic Transformation in Paracoccidioides brasiliensis

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.     

Characterization and functional analysis of the β-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 ( Pb Gel3p) is presented here. Pb Gel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis . The recombinant Pb Gel3p was overexpressed in Escherichia coli , and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of Pb Gel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1 Δ mutant. The results indicated that Pb Gel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.     

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.     

Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.