BackgroundSporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii.ResultsThe presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90.ConclusionsUsing the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.

BackgroundSporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.ResultsIn this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid) and isotetrandrine (an inhibitor of G protein PLA2 interactions) were found to inhibit budding by yeasts induced to re-enter the yeast cell cycle and to stimulate the yeast to mycelium transition showing that this enzyme is necessary for the yeast cell cycle.ConclusionA new G protein α subunit gene was characterized in S. schenckii and protein-protein interactions studies revealed this G protein alpha subunit interacts with a cPLA2 homologue. The PLA2 homologue reported here is the first phospholipase identified in S. schenckii and the first time a PLA2 homologue is identified as interacting with a G protein α subunit in a pathogenic dimorphic fungus, establishing a relationship between these G proteins and the pathogenic potential of fungi. This cPLA2 homologue is known to play a role in signal transduction and fungal pathogenesis. Using cPLA2 inhibitors, this enzyme was found to affect dimorphism in S. schenckii and was found to be necessary for the development of the yeast or pathogenic form of the fungus.

Sporothrix schenckii is a pathogenic dimorphic fungus that grows as a yeast and as mycelia. This species is the causative agent of sporotrichosis, typically a skin infection. We report the genome sequence of S. schenckii, which will facilitate the study of this fungus and of the Sporothrix schenckii group.

BackgroundImportant biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins.ResultsUsing the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD), a glyceraldehyde-3-P dehydrogenase (GAPDH) and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS) and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein) family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment.ConclusionsThis study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association of G protein alpha subunits to transport molecules reinforces the role of G proteins in the response to environmental signals and also highlights the involvement of fungal G protein alpha subunits in nutrient sensing in S. schenckii. These interactions suggest that these permeases could function as transceptors for G proteins in fungi.

Following the publication of this article [1], it was brought to our attention that Fig. 7A lane 2 is identical to Fig. 7B lane 2 and Fig. 7B lane 4 is identical to Fig. 7C lane 4.