Several well-characterized fungal proteins act as prions, proteins capable of multiple conformations, each with different activities, at least one of which is self-propagating. Through such self-propagating changes in function, yeast prions act as protein-based elements of phenotypic inheritance. We report a prion that makes cells resistant to the glucose-associated repression of alternative carbon sources, [GAR+] (for “resistant to glucose-associated repression,” with capital letters indicating dominance and brackets indicating its non-Mendelian character). [GAR+] appears spontaneously at a high rate and is transmissible by non-Mendelian, cytoplasmic inheritance. Several lines of evidence suggest that the prion state involves a complex between a small fraction of the cellular complement of Pma1, the major plasma membrane proton pump, and Std1, a much lower-abundance protein that participates in glucose signaling. The Pma1 proteins from closely related Saccharomyces species are also associated with the appearance of [GAR+]. This allowed us to confirm the relationship between Pma1, Std1, and [GAR+] by establishing that these proteins can create a transmission barrier for prion propagation and induction in Saccharomyces cerevisiae. The fact that yeast cells employ a prion-based mechanism for heritably switching between distinct carbon source utilization strategies, and employ the plasma membrane proton pump to do so, expands the biological framework in which self-propagating protein-based elements of inheritance operate.

SUMMARY In experimental science, organisms are usually studied in isolation, but in the wild they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR+], a protein-based epigenetic element, allows yeast to circumvent this glucose repression and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR+]. [GAR+] is advantageous to bacteria because yeast cells make less ethanol, and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR+] presents a unique example of Lamarckian inheritance.

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.

SUMMARY [GAR+] is a protein-based element of inheritance that allows yeast (Saccharomyces cerevisiae) to circumvent a normal hallmark of their biology: extreme metabolic specialization for glucose fermentation. When glucose is present, even in trace quantities, yeast will not use other carbon sources. [GAR+] allows cells to circumvent this “glucose repression.” [GAR+] is induced in yeast by a factor secreted by bacteria inhabiting their environment. We report that the de novo rates of [GAR+] appearance correlate with the yeast’s ecological niche. Evolutionarily distant fungi possess similar epigenetic elements that are also induced by bacteria. As expected for a mechanism whose adaptive value originates from the selective pressures of life in biological communities, the ability of bacteria to induce [GAR+] and the ability of yeast to respond to bacterial signals have been extinguished repeatedly during the extended monoculture of domestication. Thus, [GAR+] is a broadly conserved adaptive strategy that links environmental and social cues to heritable changes in metabolism.

Summary The anti-phagocytic polysaccharide capsule of the human fungal pathogen Cryptococcus neoformans is a major virulence attribute. However, previous studies of the pleiotropic virulence determinant Gat201, a GATA-family transcription factor, suggested that capsule-independent antiphagocytic mechanisms exist. We have determined that Gat201 controls the mRNA levels of ~1100 genes (16% of the genome) and binds the upstream regions of ~130 genes. Seven Gat201-bound genes encode for putative and known transcription factors—including two previously implicated in virulence—suggesting an extensive regulatory network. Systematic analysis pinpointed two critical Gat201-bound genes, GAT204 (a transcription factor) and BLP1, which account for much of the capsule-independent antiphagocytic function of Gat201. A strong correlation was observed between the quantitative effects of single and double mutants on phagocytosis in vitro and on host colonization in vivo. This genetic dissection provides evidence that capsule-independent anti-phagocytic mechanisms are pivotal for successful mammalian infection by C. neoformans.