Experimental evolution of S. cerevisiae for caffeine tolerance alters multidrug resistance and TOR signaling pathways.

Caffeine is a natural compound that inhibits the major cellular signaling regulator TOR, leading to widespread effects including growth inhibition. S. cerevisiae yeast can adapt to tolerate high concentrations of caffeine in coffee and cacao fermentations and in experimental systems. While many factors affecting caffeine tolerance and TOR signaling have been identified, further characterization of their interactions and regulation remain to be studied. We used experimental evolution of S. cerevisiae to study the genetic contributions to caffeine tolerance in yeast, through a collaboration between high school students evolving yeast populations coupled with further research exploration in university labs. We identified multiple evolved yeast populations with mutations in PDR1 and PDR5, which contribute to multidrug resistance, and showed that gain-of-function mutations in multidrug resistance family transcription factors PDR1, PDR3, and YRR1 differentially contribute to caffeine tolerance. We also identified loss-of-function mutations in TOR effectors SIT4, SKY1, and TIP41, and show that these mutations contribute to caffeine tolerance. These findings support the importance of both the multidrug resistance family and TOR signaling in caffeine tolerance, and can inform future exploration of networks affected by caffeine and other TOR inhibitors in model systems and industrial applications.

Automated quantification of Candida albicans biofilm-related phenotypes reveals additive contributions to biofilm production.

Biofilms are organized communities of microbial cells that promote persistence among bacterial and fungal species. Biofilm formation by host-associated Candida species of fungi occurs on both tissue surfaces and implanted devices, contributing to host colonization and disease. In C. albicans, biofilms are built sequentially by adherence of yeast to a surface, invasion into the substrate, the formation of aerial hyphal projections, and the secretion of extracellular matrix. Measurement of these biofilm-related phenotypes remains highly qualitative and often subjective. Here, we designed an informatics pipeline for quantifying filamentation, adhesion, and invasion of Candida species on solid agar media and utilized this approach to determine the importance of these component phenotypes to C. albicans biofilm production. Characterization of 23 C. albicans clinical isolates across three media and two temperatures revealed a wide range of phenotypic responses among isolates in any single condition. Media profoundly altered all biofilm-related phenotypes among these isolates, whereas temperature minimally impacted these traits. Importantly, the extent of biofilm formation correlated significantly with the additive score for its component phenotypes under some conditions, experimentally linking the strength of each component to biofilm mass. In addition, the response of the genome reference strain, SC5314, across these conditions was an extreme outlier compared to all other strains, suggesting it may not be representative of the species. Taken together, development of a high-throughput, unbiased approach to quantifying Candida biofilm-related phenotypes linked variability in these phenotypes to biofilm production and can facilitate genetic dissection of these critical processes to pathogenesis in the host.