A system has been developed for the quantitative analysis of gene expression within individual Candida albicans cells in infected tissue. The system is based on the plasmid pGFP, which contains the codon-optimized yeast enhanced green fluorescent protein (yEGFP; Cormack et al., 1997) cloned between a basal CaADH1 promoter and the ScCYC1 terminator on an integrating vector. Promoters were inserted into pGFP and GFP levels measured in individual cells by quantitative fluorescence microscopy. Analysis of pPCK1-GFP and pMET3-GFP fusions revealed that GFP folds rapidly following gene induction, and is turned over rapidly following gene repression. Hence, single cell fluorescence measurements are likely to reflect ongoing gene expression levels with reasonable accuracy. pACT1-GFP expression levels were relatively constant during growth of C. albicans in both yeast and hyphal forms, and during growth in vivo in the mouse model of systemic infection. Therefore, pACT1-GFP provides a useful control for this quantitative GFP-based system in future analyses of C. albicans molecular responses during fungal infections.
Copyright 2004 John Wiley & Sons, Ltd.