A soluble α-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS–polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man₉GlcNAc₂ to produce Man₈GlcNAc₂ isomer B and mannose as a function of time of incubation up to 12 h at 37°C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man₇GlcNAc₂ and probably Man₆GlcNAc₂. These two products were also observed when Man₈GlcNAc₂ isomer B instead of Man₉GlcNAc₂ was used as substrate. Other oligosaccharides, such as Man₆GlcNAc₂-Asn, Man₅GlcNAc₂-Asn, and the α1,3- and α1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an α1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.

In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.

Sporothrix schenckii is one of the causative agents of sporotrichosis, a worldwide-distributed mycosis that affects humans and other mammals. The interest in basic and clinical features of this organism has significantly increased in the last years, yet little progress in molecular aspects have been reported. Gene expression analysis is a set of powerful tools that helps to assess the cell response to changes in the extracellular environment, the genetic networks controlling metabolic pathways, and the adaptation to different growth conditions. Most of the quantitative methodologies used nowadays require data normalization, and this is achieved measuring the expression of endogenous control genes. Housekeeping genes, whose expression is assumed to suffer minimal changes regardless the cell morphology, the stage of the cell cycle or the presence of harsh extracellular conditions are commonly used as controls in Northern blotting assays, microarrays, and semi-quantitative or quantitative RT-PCR. Since the biology of the organisms is usually species specific, it is difficult to find a reliable group of universal genes that can be used as controls for data normalization in experiments addressing the gene expression, regardless the taxonomic classification of the organism under study. Here, we compared the transcriptional stability of the genes encoding for elongation factor 1A, Tfc1, a protein involved in transcription initiation on Pol III promoters, ribosomal protein L6, histone H2A, β-actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase, UAF30, the upstream activating factor 30, and the transcription initiation factor TFIID subunit 10, during the fungal growth in different culture media and cell morphologies. Our results indicated that only the gene encoding for the ribosomal protein L6 showed a stable and constant expression. Furthermore, it displayed not transcriptional changes when S. schenckii infected larvae of Galleria mellonella or interacted with immune cells. Therefore, this gene could be used as control for data normalization in expression assays. As a proof of concept, this gene was used to assess the expression of genes encoding for glycosidases involved in the protein N-linked glycosylation pathway, a histidine kinase whose expression is regulated during the fungal dimorphism, and a glycosidase that participates in sucrose assimilation.

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Sporothrix schenckii and Sporothrix brasiliensis are causative agents of sporotrichosis, a mycosis that affects humans and other mammals. Adhesins are considered among the primary virulence factors of these species; however, their molecular identities are currently scarce. Here, we generated silenced mutant strains in PAP1, encoding a peptidorhamnomannan-associated protein, in both fungal species and compared their phenotypical characterization. PAP1-silenced mutants had normal growth, dimorphism, and morphology, suggesting the gene is not essential. The silenced strains showed defects in adhesion to HeLa cells, laminin, fibronectin, fibrinogen, type-I collagen, and type-II collagen. The S. schenckii silenced mutants showed a significant reduction in the adhesion to elastin, whilst the S. brasiliensis mutant showed impaired ability to bind thrombospondin 1 and to form biofilms. Both sets of PAP1-silenced mutants displayed defects in the cell wall composition, with reduced levels of cell wall rhamnose. The S. schenckii mutants showed a compensatory mechanism increasing cell wall mannose content, while the S. brasiliensis mutants increased cell wall protein levels in compensation to PAP1 silencing. Both mutant sets changed their ability to stimulate cytokine production in human peripheral blood mononuclear cells and monocyte-derived macrophages. In addition, phagocytosis of S. schenckii PAP1-silenced strains was significantly increased, while the S. brasiliensis PAP1-silenced strains were poorly phagocytosed. Finally, PAP1 silencing affected virulence in both species.

Sclerotium cepivorum Berk is the etiological agent of white rot disease that affects plants of the genus Allium. This fungus produces resistance structures called sclerotia that are formed by a rolled mycelium with a thick layer of melanin and it can remain dormant for many years in the soil. Current interest in S. cepivorum has arisen from economic losses in Allium crops in the agricultural sector. Melanin is a component that protects the sclerotia from adverse environmental conditions In many organisms, it plays an important role in the infectious process; in S. cepivorum, the pathway by which this component is synthetized is not fully described. By using infrared spectrophotometric assays applied direct to the sclerotia and a melanin extract followed by an NMR analysis and a tricyclazole melanin inhibition experiment, it allowed us to determine the dihydroxynaphthalene (DHN)-melanin pathway by which S. cepivorum performs its melanin synthesis. Moreover, we focused on studying scytalone dehydratase (SDH) as a key enzyme of the DHN-melanin synthesis. We obtained the recombinant SDH enzyme and tested its activity by a zymogram assay. Thereby, the S. cepivorum melanogenic route was established as a DHN pathway.