Bet Hedging in Pdr5-mediated Drug Resistance and a Mechanism for its Regulation

Human health is increasingly threatened by the emergence of multiply drug resistant malignant organisms. Yet, our understanding of the numerous ways by which such resistance arises is modest. Here, we present evidence of a bet hedging strategy in the budding yeast, Saccharomyces cerevisiae, to counter the effects of cytotoxic drugs through the action of Pdr5, an ATP-binding cassette transporter. We have employed flow cytometry and fluorescent activated cell sorting to probe the expression levels of a GFP-tagged version of PDR5 in individual cells. The results obtained from these experiments demonstrate that each yeast population is variable in the levels of Pdr5 production, and a small subpopulation of cells produces this efflux pump at much higher quantities than the population average. Consequently, cells with high and low levels of Pdr5 grow differentially in presence and absence of cycloheximide, a cytotoxic drug. These properties are highly suggestive of a bet hedging strategy mediated by Pdr5 levels. We further link this bet hedging strategy to the transcriptional regulatory network of PDR5 consisting of two major transcription factors, Pdr1 and Pdr3. Our analysis suggests that a self-activating feedback loop acting on Pdr3 plays an important role in generation of the aforementioned subpopulation. Furthermore, our results point to a large difference in the activity of these two regulators wherein Pdr3 is notably stronger than Pdr1. The disparity in their activity could indicate a mechanism for generation of the observed proportions of subpopulations with regards to the level of Pdr5