Tolerance to the antifungal drug fluconazole is mediated by tuning cytoplasmic fluidity

Treatment failure rates for fungal infections cannot be explained simply by increased rates of drug resistance. Antifungal drug tolerance, the ability of a susceptible isolate to grow in the presence of inhibitory drug concentrations, relies upon a broad set of stress response pathways and can contribute to antifungal drug treatment failures. As the physical properties of the cytoplasm are critical for diverse cellular processes, we investigated whether cytoplasmic mesoscale fluidity is linked to antifungal tolerance, taking advantage of a fluorescent microrheological probe. Here, we show either using fluconazole, a common drug that inhibits ergosterol biosynthesis, or mutants in the ergosterol biosynthesis pathway, that cytoplasmic fluidity decreased and could be reversed upon drug removal. Reducing ribosome concentration decreased drug tolerance and restored cytoplasmic fluidity, highlighting a link between tolerance and cytoplasmic crowding and/or viscosity. However, growth in fluconazole did not increase ribosome concentrations; rather growth in fluconazole increased the number of dormant, hibernating ribosomes, cytoplasmic protein concentration, viscosity and condensate formation. Specifically, growth in fluconazole resulted in a substantial increase in processing bodies (P-bodies), whose presence correlated with azole tolerance. Furthermore, we found a substantial increase in cell-cell heterogeneity in all biophysical, biochemical and molecular outputs analyzed. Together, our results reveal that changes in the physical properties of the cytoplasm occur in response to antifungal drug and suggest that these changes, as well as increased cell-cell heterogeneity, are crucial for survival in fungistatic drugs.