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

BACKGROUND

Sporothrix 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.

RESULTS

In this work we describe a new G protein alpha 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.

CONCLUSIONS

A new G protein alpha 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 alpha 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.