We take an interdisciplinary approach incorporating experimental evolution, genetics, genomics, proteomics, and models of fungal pathogenesis to dissect the molecular mechanisms governing fungal evolution, drug resistance, development, and disease. Below are highlights of some of our recent projects.
Global Gene Deletion Analysis Exploring Yeast Filamentous Growth. We provide the premier global and comparative analysis of filamentous growth of the model yeast Saccharomyces cerevisiae and the pathogenic yeast Candida albicans, identifying novel regulators of morphogenesis, biofilm formation and virulence, as reported in our manuscript published in Science (Ryan O. and Shapiro, R. S. et al. 2012. Science 337: 1353-6).
Lysine Deacetylases Hda1 and Rpd3 Regulate Hsp90 Function Thereby Governing Fungal Drug Resistance. We establish acetylation as a mechanism of post-translational control of Hsp90 function in fungi, functional redundancy between KDACs Hda1 and Rpd3, as well as a mechanism governing fungal drug resistance with broad therapeutic potential, as reported in our manuscript published in Cell Reports (Robbins, N., Leach, M. D., and Cowen, L. E. 2012. Cell Reports 2:878-88).
The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance. We provide the premier characterization of an Hsp90 co-chaperone in a fungal pathogen, establishing C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, and providing a new target for treatment of life-threatening fungal infections, as reported in our manuscript published in PLoS ONE (Shapiro, R. S. et al. 2012. PLoS ONE 7(9): e44734).
A Novel Calcineurin-Independent Activity of Cyclosporin A in Saccharomyces cerevisiae. We utilize systems level chemical genomic approaches to implicate key cellular pathways in a novel mechanism of antifungal drug synergy, as reported in our manuscript published in Molecular Biosystems (Singh-Babak, S. D. et al. 2012. Molecular Biosystems 8: 2575-84).
Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata. We provided the first global analysis of mutations that accompany the evolution of fungal drug resistance in the human host, identified the premier compensatory mutation that mitigates the cost of resistance to the echinocandins, the only new class of antifungal drug to reach the clinic in decades, and suggest a new mechanism of drug resistance with broad therapeutic potential, as reported in our manuscript published in PLoS Pathogens (Singh-Babak, S. D. et al. 2012. PLoS Pathogens 8(5): e1002718).
Pho85, Pcl1 and Hms1 Signaling Governs Candida albicans Morphogenesis Induced by High Temperature or Hsp90 Compromise. We recently established a new mechanism through which Hsp90 orchestrates morphogenesis of the leading fungal pathogen of humans, C. albicans, and defined novel regulatory circuitry governing this temperature-dependent developmental program, with broad implications for temperature sensing and virulence of microbial pathogens, as reported in our manuscript published in Current Biology (Shapiro, R. S. et al. 2012. Current Biology 22(6): 461-70).
Mapping the Hsp90 Genetic Interaction Network in Candida albicans Reveals Environmental Contingency and Rewired Circuitry. We recently performed the first chemical genomic screen to map interaction networks in the leading fungal pathogen of humans, C. albicans. We established environmental contingency in the first chaperone network of a fungal pathogen, novel effectors upstream and downstream of Hsp90, and network rewiring over evolutionary time, as reported in our manuscript published in PLoS Genetics (Diezmann S. et al. 2012. PLoS Genetics 8(3):e1002562.
Cdc28 Provides a Molecular Link Between Hsp90, Cell Cycle Regulation, and Morphogenesis in the Pathogenic Fungus Candida albicans. We recently discovered that filaments generated by compromise of Hsp90 function are neither pseudohyphae nor hyphae but closely resemble filaments formed in response to cell cycle arrest. Our results suggest that Hsp90 is instrumental in the regulation of cell division during yeast-form growth in C. albicans, and that it exerts its major effects during late cell cycle events, as reported in our manuscript published in Molecular Biology of the Cell (Senn H., Shapiro, R. S. and Cowen, L. E. 2012. Molecular Biology of the Cell 23(2):268-83.
A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis. We contributed C. albicans mutants to this collaborative study to confirm evolutionary conservation of a novel methytransferase. Lissina, E. et al. 2011. PLoS Genetics 7(10): e1002332.
Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms. We recently established a novel mechanism regulating biofilm drug resistance and dispersion, and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections. Impairment of Hsp90 function genetically or pharmacologically in the leading fungal pathogen of humans, C. albicans, transformed the most widely deployed class of antifungals, the azoles, from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Further, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. This work was published in PLoS Pathogens (Robbins N. et al. 2011. PLoS Pathogens 7(9): e1002257).
PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90. We recently established a new role for protein kinase C (PKC) signaling in drug resistance of the leading fungal pathogen of humans, Candida albicans. Initiated by a high-throughput drug screen, we subsequently use genetic analyses in C. albicans and the model yeast Saccharomyces cerevisiae to dissect the circuitry through which PKC regulates drug resistance, as reported in our manuscript published in PLoS Pathogens (LaFayette, S. L. et al. 2010. PLoS Pathogens 6(8): e1001069). Hsp90 regulates PKC signaling through the MAPK cascade by stabilizing the terminal MAPK, Mkc1, affecting drug resistance through both calcineurin and PKC signaling. Compromising C. albicans PKC signaling attenuates virulence in a murine model of disease, with broad therapeutic potential for life-threatening infectious disease.
Targeting Hsp90 as a Powerful, Broadly Effective Therapeutic Strategy for Diverse Fungal Infectious Diseases. We previously established that inhibition of Hsp90 in the leading fungal pathogen of humans, Candida albicans, blocks the evolution of resistance to drugs that target the cell membrane and renders resistant pathogens responsive to treatment in vitro. Our translational work in this area established broad therapeutic potential and was recently published in PNAS (Cowen LE et al. 2009. PNAS 106:2818-23). Inhibition of Hsp90 with molecules that are well tolerated in humans enhances the efficacy of antifungals in an invertebrate model of disease caused by C. albicans and by the most lethal mould, Aspergillus fumigatus. Genetic reduction of pathogen Hsp90 enhances the therapeutic efficacy of antifungals in a mouse model of disseminated fungal disease. This paves the way for the new therapeutics and was ranked as “Must Read” by Faculty 1000 (http://www.f1000biology.com/article/id/1148141/evaluation) and highlighted in a Commentary in PNAS (Semighini CP and Heitman J. 2009 PNAS 106:2971-2). Our current efforts in this area focus on drug screens to identify new molecules with the capacity to abrogate fungal drug resistance.
Hsp90 Orchestrates Temperature-Dependent C. albicans Morphogenesis via Ras1-PKA Signaling. The molecular basis of temperature dependence of the C. albicans morphogenetic switch from yeast to filamentous growth has remained a longstanding mystery. We discovered that Hsp90 is the enigmatic temperature sensor. Inhibition of Hsp90 induces the morphogenetic transition in the absence of external cues that normally regulate the transition. Hsp90 exerts a repressive effect on Ras1-protein kinase A (PKA) morphogenetic signaling such that elevated temperatures compromise Hsp90 function and relieve repression of the morphogenetic program. Consistent with the requirement for morphogenetic flexibility in virulence, depletion of C. albicans Hsp90 attenuates virulence in a murine model of disseminated disease. This work was recently published in Current Biology (Shapiro RS et al. 2009. Curr. Biol. 19:621-9), and was spotlighted in a Dispatch in Current Biology (Gow NAR. 2009. Curr. Biol. 19: R333-4) and a Highlight in Nature Reviews Microbiology (Molloy S. 2009. Nat. Rev. Microbiol. 4:405). Faculty 1000 ranked this paper as “Exceptional” (http://www.f1000medicine.com/article/mymrrb1v0927lgd/id/1160328). Current studies focus on elucidating the key molecular target(s) of Hsp90 and the alterations in cellular architecture accompanying this developmental transition.
Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast C. albicans via Calcineurin. We recently discovered an entirely new role for C. albicans Hsp90 in regulating resistance to the only new class of antifungal drug to reach the clinic in decades, the echinocandins, which target the cell wall. Hsp90 governs echinocandin resistance by regulating the function of the protein phosphatase calcineurin. Genetic reduction of Hsp90 enhances echinocandin efficacy in a murine model of disseminated disease, validating Hsp90 as a promising therapeutic target in combinatorial antifungal therapy. Beyond the clinical significance, this work is of broad interest in that we identify calcineurin as the first Hsp90 client protein in C. albicans. This work was recently published in PLoS Pathogens (Singh SD et al. 2009. PLoS Pathog. 5(7): e1000532). Faculty 1000 ranked this paper as “Must Read” (http://www.f1000biology.com/article/id/1164066). Current studies focus on dissecting the roles of Hsp90 and calcineurin in drug resistance of other fungal pathogens.
Metabolic Control of Antifungal Drug Resistance. We recently discovered a novel role for nutrient signaling in fungal drug resistance. We delineated genetic and environmental factors that mitigate the translation of genotype into resistance phenotype. This work revealed that compromising a global cellular regulator that couples growth and metabolism to environmental cues, Tor kinase, provides a powerful strategy to abrogate fungal drug resistance with broad therapeutic potential. This work was recently published in Fungal Genetics and Biology (Robbins N et al. 2009. Fungal Genet. Biol. doi:10.1016/j.fgb.2009.07.004). Our future studies in this area focus on delineating other environmental stress conditions that modulate drug resistance and phenotypic diversity.