Analysis of Mold and Yeast Phosphoproteomes in the Dimorphic Fungus Penicillium marneffei

Abstract

Penicillium marneffei is a dimorphic fungus capable of multinucleate mold growth at 25°C and unicellular yeast growth at 37°C. Mostly prevalent in Southeast Asia, P. marneffei is pathogenic in immunocompromised individuals as the cause of penicilliosis. The switch from mold to yeast is a prerequisite for pathogenicity and the molecular mechanism responsible for such change is under investigation. As the organism changes in cell morphology, molecular modifications are made in the processes that affect both cell growth and cell differentiation. Both of these programs rely greatly on the post translational modification of proteins known as protein phosphorylation. A combination of the activity of both protein kinases and phosphatases produces entities known as signaling pathways which enable the cell to coordinate cellular events and respond to external signals. The current phosphorylated state of all proteins in a cell at a given time point is known as the phosphoproteome. This study is the first to produce a phosphoproteomic analysis of mold and yeast cells in P. marneffei. To study phosphoproteins in P. marneffei, whole cell proteins were extracted from mold and yeast cells. Proteins were then quantified using the Bradford assay and were separated by size using one dimensional gel electrophoresis. Gels were stained using Pro-Q Diamond Phosphoprotein Gel Stain for detection of phosphoproteins and comparative analysis was performed using Quantity One. Cells were also treated with rapamycin to determine morphological and phosphoproteomic changes brought about by the inhibition of the protein kinase TOR.