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Phosphate Is the Third Nutrient Monitored by TOR in Candida Albicans and Provides a Target for Fungal-Specific Indirect TOR Inhibition

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Phosphate Is the Third Nutrient Monitored by TOR in Candida Albicans and Provides a Target for Fungal-Specific Indirect TOR Inhibition Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition Ning-Ning Liua, Peter R. Flanaganb, Jumei Zenga, Niketa M. Jania,1, Maria E. Cardenasc, Gary P. Moranb, and Julia R. Köhlera,2 aBoston Children’s Hospital and Harvard Medical School, Boston, MA 02115; bDivision of Oral Biosciences, School of Dental Science, Trinity College Dublin, Dublin 2, Ireland; and cDepartment of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710 Edited by Alexander D. Johnson, University of California, San Francisco, CA, and approved April 27, 2017 (received for review October 28, 2016) The Target of Rapamycin (TOR) pathway regulates morphogenesis TORC1 pathways are conserved (4), but there are important dif- and responses to host cells in the fungal pathogen Candida albicans. ferences between these species as well. Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth A small Ras-like GTPase upstream of TORC1, Rheb in mammals and proliferation in response to nitrogen and carbon source availabil- (14) and Rhb1 in S. cerevisiae (15), also responds to nutritional ity. Our unbiased genetic approach seeking unknown components of signals. Rheb is a central activator of mammalian TORC1 and TORC1 signaling in C. albicans revealed that the phosphate trans- modulated by the TSC1/TSC2 complex (16, 17), whereas in porter Pho84 is required for normal TORC1 activity. We found that S. cerevisiae, Rhb1 seems to play a minor role in nutritional signaling. mutants in PHO84 are hypersensitive to rapamycin and in response to In C. albicans, Rhb1 is required for normal tolerance to rapamycin phosphate feeding, generate less phosphorylated ribosomal protein and phosphorylation of ribosomal protein S6, a readout of TORC1 S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the activation (9, 18). C. albicans Rhb1 coregulates expression of genes TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in important in nitrogen source uptake (18) and virulence (19). Unlike Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective S. cerevisiae, C. albicans also has a TSC2 homolog with mutant in the P-S6 response to phosphate. Overexpression of Gtr1 and a Q67L phenotypes that are consistent with a conserved GTPase-activating MICROBIOLOGY constitutively active Gtr1 mutant suppresses TORC1-related activity of C. albicans Tsc2 for Rhb1 (18). Saccharomyces cerevisiae pho84 defects. In mutants, constitutively Given the differences between the S. cerevisiae and C. albicans active Gtr1 suppresses a TORC1 signaling defect but does not rescue TORC1 pathways, we used a forward genetic approach to find rapamycin hypersensitivity. Hence, connections from phosphate components of C. albicans TORC1 signaling. Using our mariner homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae transposon mutant collection (20), we isolated a rapamycin hy- . The converse direction of signaling from TORC1 to the persensitive mutant in a C. albicans homolog of PHO84, the gene PHO regulon previously observed in S. cerevisiae was genetically encoding the major S. cerevisiae high-affinity phosphate (Pi) shown in C. albicans using conditional TOR1 alleles. A small molecule transporter. inhibitor of Pho84, a Food and Drug Administration-approved drug, Having identified a connection between C. albicans Pho84 and inhibits TORC1 signaling and potentiates the activity of the antifun- the C. albicans TOR pathway in a forward genetic screen, we gals amphotericin B and micafungin. Anabolic TORC1-dependent pro- characterized this link between phosphate homeostasis (PHO) and cesses require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84. Significance ribosomal protein S6 phosphorylation | antifungal | amphotericin B | The human fungal pathogen Candida albicans uses the Target of echinocandin | drug potentiation Rapamycin (TOR) signaling pathway to contend with varying host environments and thereby, regulate cell growth. Seeking C. albicans rganisms that fail to maximize growth in response to abundant unknown components of the TOR pathway, we Onutrients can be outcompeted by those that do. Conversely, identified a phosphate importer, Pho84, and its molecular link to organisms that fail to cease growth and induce survival programs Target of Rapamycin complex 1 (TORC1). Because phosphorus is a during stress and starvation lose viability. The Target of Rapamycin critical element for anabolic processes, like DNA replication, ribo- (TOR) signaling pathway is conserved in eukaryotes and integrates some biogenesis, translation, and membrane biosynthesis, TORC1 multiple channels of information regarding the cells’ nutritional monitors its availability in regulating these processes. By de- and physical environment to induce either growth and proliferation pleting the central kinase in the TORC1 pathway, we showed or stress and survival responses (1). In the human fungal pathogen that TORC1 signaling modulates regulation of phosphate acqui- Candida albicans, TOR participates in regulating morphogenesis sition. A Food and Drug Administration-approved small molecule (2–7) and responses to host cells (8). To control growth, C. albicans inhibitor of Pho84 inhibits TORC1 signaling and potentiates the TOR also integrates signals of carbon source availability from the activity of the antifungals amphotericin B and micafungin. PKA pathway with nitrogen source status, its primary nutritional – Author contributions: N.-N.L., P.R.F., J.Z., G.P.M., and J.R.K. designed research; N.-N.L., P.R.F., input (9). Similar TOR PKA intersections have been reported in J.Z., N.M.J., and J.R.K. performed research; M.E.C. contributed new reagents/analytic the model yeast Saccharomyces cerevisiae (10, 11). tools; N.-N.L., P.R.F., J.Z., M.E.C., G.P.M., and J.R.K. analyzed data; and N.-N.L. and J.R.K. In S. cerevisiae, Target of Rapamycin complex 1 (TORC1), which wrote the paper. is susceptible to inhibition by rapamycin, is activated by preferred The authors declare no conflict of interest. nitrogen sources, such as glutamine and leucine. Leucine activates This article is a PNAS Direct Submission. TORC1 through the Exit from G0 (EGO) complex by inducing 1Present address: Division of Molecular Biology, Maryland Department of Health and GTP loading of one of its subunits, the small GTPase Gtr1 (12). Mental Hygiene Laboratories Administration, Baltimore, MD 21205. Leucine also promotes TORC1 activity through leucine–tRNA 2To whom correspondence should be addressed. Email: [email protected]. synthase Cdc60, which physically interacts with Gtr1 (13). Many This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transcriptional regulators responsive to S. cerevisiae and C. albicans 1073/pnas.1617799114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1617799114 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 the cell’s central growth control module. We found that we can indirectly target C. albicans TORC1 using small molecule Pho84 inhibitors, one of which is a Food and Drug Administration (FDA)- approved antiviral drug, and that the antifungals amphotericin B and micafungin are potentiated by Pho84 inhibitors. Results A Screen of Haploinsufficient Transposon Mutants for Altered Rapamycin Susceptibility Identified a PHO84 Ortholog. We screened our het- erozygous mutant collection of mariner-transposon insertions marked with our dominant selectable marker NAT1 (20, 21) for altered rapamycin susceptibility. We isolated a transposon mutant hypersensitive to rapamycin, in which the transposon disrupts the promoter of orf19.655 67 bp upstream of the predicted trans- lational start site (SI Appendix,Fig.S1A). This ORF encodes a protein with 66% amino acid identity to S. cerevisiae Pho84 and 55% amino acid homology to the Piriformospora indica PiPT phosphate transporter with a crystal structure that was recently described (22) (SI Appendix,Fig.S1B). According to the Candida Genome Database (CGD) nomenclature, we called this ORF C. albicans PHO84 and used the CGD sequence for additional analysis (23). To confirm that rapamycin hypersensitivity of the transposon mutant was linked to the disrupted PHO84 locus, two independent heterozygous deletion mutants and their homozygous null derivatives were constructed. Mutant phenotypes in these two lineages were the same, and one was chosen for additional char- acterization (SI Appendix, Fig. S1C). These mutants were also rapamycin hypersensitive (Fig. 1A), confirming that PHO84 is required for normal tolerance of rapamycin. The cytoplasmic membrane protein Pho84 is the major Pi transporter in S. cerevisiae (24–26). PHO84 expression is con- trolled by the PHO regulon, a homeostatic system that maintains Pi availability for metabolism and growth in fluctuating external Pi conditions (27). C. albicans pho84 mutants, like those in the S. cerevisiae homolog (28), failed to grow on medium without in- organic phosphate (Fig. 1B). Heterozygous and reintegrant cells, Fig. 1. C. albicans PHO84 is required for rapamycin tolerance, growth during Pi apparently haploinsufficient for rapamycin tolerance (Fig. 1A), starvation, normal TORC1 activity and hyphal morphogenesis, and Pi homeostasis. grew robustly on 0 Pi medium (Fig. 1B), indicating that mechanisms (A) Cell dilutions
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