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. The sequence of P. brasiliensis CHS3 is available at the GenBank database under Accession No. AF107623.2. Copyright © 2009 John Wiley & Sons, Ltd.     

Cloning and functional analysis of the orotidine-5'-phosphate decarboxylase gene (PbrURA3) of the pathogenic fungus Paracoccidioides brasiliensis

A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.     

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.     

Production of leukotriene B4 by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. The production of eicosanoids during fungal infection has been associated with the biology of these microorganisms and modulation of host immune response. The aim of our study was to evaluate whether P. brasiliensis strains with high or low virulence produce leukotriene B4 (LTB4), using endogenous and/or exogenous sources of arachidonic acid (AA). Moreover, we assessed whether this fungus might use the same metabolic pathway, described for mammalian cells, that involves the lipoxygenase (LOX) enzyme. The association between the production of this eicosanoid and fungus survival and growth was also evaluated. Our results showed that P. brasiliensis, irrespective of its virulence, produces high levels of LTB4 using endogenous AA. In addition, in cultures treated with exogenous AA, LTB4 levels were significantly higher, showing that this fungus also uses exogenous sources of fatty acids. Treatment with MK886, which blocks the activity of lipoxygenase, by inhibiting five-lipoxygenase-activating protein (FLAP) or with nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, resulted in a significant reduction in LTB4 levels, indicating that the fungus produces this eicosanoid by using the LOX pathway or an enzyme with biochemically similar function. The significant reduction in viability detected in cultures treated with these inhibitors was, however, restored by adding exogenous LTB4 , confirming the role of this eicosanoid in fungus survival. Moreover, the addition of LTB4 to cultures capable of producing LTs induces fungal growth. These results provide a foundation for additional studies on the contributions of LTB4 in P. brasiliensis virulence.     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N‐acetyl‐β‐D‐glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N‐acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFα and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the β‐1,4‐homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin‐affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS–PAGE and Western blot analysis revealed similarities between the N‐acetylglucosamine‐ and chitin‐bound fractions, confirmed by MALDI–TOF–MS of trypsinic peptides. Western blot of two‐dimensional gel electrophoresis of the yeast extract showed a major spot with M_r 70 000 and pI approximately 5.63. Morevover, an N‐acetyl‐β‐D ‐glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p (defective for filamentous growth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a beta-glucan linked protein that undergoes N-glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues.     

Yeast sequencing reports. Molecular cloning and sequencing of the hcs gene,which encodes 3-hydroxy-3-methylglutaryl coenzyme a synthase of Schizosaccharomyces pombe

We have cloned and sequenced the hcs gene, which is thought to encode a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase consisting of 447 amino acids, from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence of the hcs product of S. pombe has homology with the HMG-CoA synthase of rat (47·8%), chicken (49·2%), hamster (47·1%) and human cells (46·9%). One of the hcs genes was replaced with a marker gene in the diploid cell. No viable hcs-disrupted haploid was isolated after tetrad dissection, suggesting that the hcs gene is essential for growth. However the hcs-defective mutant could be grown on a medium containing 5 mg/ml mevalonate. These results strongly support that the hcs gene encodes HMG-CoA synthase and S. pombe contains a single copy of the hcs gene. The sequence of the hcs gene has been entered into the public data libraries under Accession Number U32187.     

New insights into a complex fungal pathogen: the case of Paracoccidioides spp.

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the 100 years since its discovery and in this article we discuss features of this fascinating fungal pathogen, including its biology, eco‐epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host–fungal interactions and how the host defence mechanisms against this pathogen work. Copyright © 2015 John Wiley & Sons, Ltd.     

α‐(1,4)‐Amylase,but not α‐ and β‐(1,3)‐glucanases,may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N‐glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N‐glycosylation of proteins such as N‐acetyl‐β‐d ‐glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)‐mediated inhibition of N‐linked glycosylation on α‐ and β‐(1,3)‐glucanases and on α‐(1,4)‐amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α‐ or β‐(1,3)‐glucanase production and secretion. Moreover, incubation with TM did not alter α‐ and β‐(1,3)‐glucanase activity in yeast and mycelium cell extracts. In contrast, α‐(1,4)‐amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N‐glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N‐glycans were essential for α‐(1,4)‐amylase activity involved in the production of malto‐oligosaccharides that act as primer molecules for the biosynthesis of α‐(1,3)‐glucan. Our results suggest that reduced fungal α‐(1,4)‐amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd.     

Cloning, characterization and expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis

We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Calnexin specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum, thus being an essential component of the complex that interacts with the folded state of nascent secreted glycoproteins. The PbCnx open reading frame was found in a 1701 base pair (bp) fragment that encodes a 567 amino acid protein with an estimated mass of 62 680 Da. Northern and Southern blot hybridizations showed that PbCnx is encoded by a single, or a low number of, gene copies. PbCnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA-encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx was recognized by 6 out of 10 sera from PCM patients, a result that rules out its possible consideration for further use in diagnosis. Using confocal microscopy with anti-PbCnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed.     

Construction of novel vectors for transformation of Lentinula edodes using a chitin synthase gene promoter

Novel vectors (pLCHS-hph and pChG-bar) containing expression unit driving hph as a selectable marker gene by chitin synthase gene promoter were constructed for Lentinula edodes transformation. Expression of the hph gene in random selected transformants was confirmed by RT-PCR method. Thus, both vectors are useful for L. edodes transformation.