Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Abstract

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.

Identification of factors involved in dimorphism and pathogenicity of Zymoseptoria tritici

“…For instance, in Sporothrix shenkii a link between G-protein mediated signalling and PLA2 has been evidenced. The heterotrimeric G-proteins seem to interact with the cytosolic phospholipase A2 (cPLA2), participating in the control of dimorphic switch [ 55 ]. Hence, using cPLA2 inhibitors, this enzyme was shown to affect stimulation of yeast-to-hyphal transition in S .…”

Sporothrix schenckii and Sporotrichosis

“…2) are smooth, tan, or cream colored (130). Some molecular aspects implicated in proliferation and maintenance of this morphological form of S. schenckii involve calcium/calmodulin-dependent protein kinases (260) and a signaling pathway involving the interaction between a cytosolic phospholipase and protein G. Studies have proven that this pathway is necessary for the reentry of S. schenckii yeast cells into the budding cycle, suggesting its function in the control of dimorphism in this fungus and for the maintenance of the yeast form (259).…”

Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

“…For the construction of the bait plasmid, ssg-2 cDNA was obtained from poly A + RNA, transcribed and amplified by RT-PCR using the Ready-to-Go™ Beads (Amersham Biosciences) as described [55], cloned and used to transform competent S. cerevisiae yeast cells (Y187). Competent S. cerevisiae yeast cells were transformed using the YEASTMAKER™ Yeast Transformation System 2 from Clontech (BD Biosciences, Clontech Laboratories Inc.).…”