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 of WT and a mutant series in PHO84 pinned onto YPD with +/+ −/+ −/− other than haploinsufficiency affect growth during Pi depletion, like vehicle or 12 ng/mL rapamycin. PHO84 , JKC915; pho84 , JKC1583; pho84 , −/− + the feedback loops between expression of high- and low-affinity Pi JKC1450; pho84 / , JKC1588. (B) Cells as in A pinned onto YNB with 0 or 11 mM transporters characterized in detail in the S. cerevisiae PHO regulon Pi. (C) Separate Western blots of the same samples for P-S6, total Rps6, and tu- bulin of WT (+/+; JKC915), pho84 null (−/−; JKC1450), and PHO84 reintegrant (28). In liquid media with 1 mM Pi, growth of pho84 cells was close (−/−/+; JKC1588) cells grown in YNB with 0, 0.22, or 11 mM KH2PO4 for 90 min. to the WT (SI Appendix,Fig.S1D). Expression of the C. albicans Dens, densitometric ratio of P-S6 vs. tubulin signal. (D) Strains as in A spotted at PHO84 homologrestoredgrowthonmediumlackingPito equidistant points around agar plates with spot edges imaged. Compare with SI S. cerevisiae pho84 mutants (SI Appendix,Fig.S1E), indicating Appendix,Fig.S2A. (Scale bar: 1 mm.) (E) S. cerevisiae and C. albicans WT and functional orthology. WT cells secrete acid phosphatase in response pho84 null cells grown in SC medium with 0.22 mM (low Pi) or 11 mM Pi (high Pi) overnight assayed for free and total Pi; pho85 null cells as controls, which to low ambient Pi to mobilize covalently bound Pi from their envi- + + hyperaccumulate Pi. Ca / (C. albicans PHO84/PHO84 PHO85/PHO85), JKC915; ronment, and this response was used for decades in studies of the −− −− −/− Capho84 / , JKC1450; Capho85 / , CaLC1919 grown in 20 μg/mL doxycycline S. cerevisiae PHO regulon (29). C. albicans pho84 mutants, like + − − overnight; Sc (S. cerevisiae PHO84 PHO85), BY4741; Scpho84 , EY2960; Scpho85 , those in S. cerevisiae (24), inappropriately derepressed acid phospha- from ref. 37. Error bars: SDs of three technical replicates. *P < 0.01; **P < 0.05. tasesecretioninhighambientPi(SI Appendix,Fig.S1F), consistent with a conserved role of C. albicans Pho84 in the PHO regulon. Hyphal morphogenesis was defective in pho84 mutants on yeast C. albicans Pho84 Is Required for the Normal TORC1 Response to Pi extract peptone dextrose (YPD) agar medium with 10% serum, Availability. We then examined the relationship between Pho84 and Spider medium, and RPMI 1640 (Fig. 1D and SI Appendix, Fig. TORC1. To test whether rapamycin hypersensitivity is caused by de- −− S2A), whereas the mutant and the WT grew equally in these media creased TORC1 kinase activity in the pho84 / mutant, we monitored when the hyphal temperature signal was absent (SI Appendix, Fig. the phosphorylation state of ribosomal protein S6 (P-S6), which we previously showed is controlled by TORC1 signaling (9). Null mutants S2B). Although many signaling pathways converge on morpho- in PHO84 had a weaker P-S6 signal than the WT during Pi refeeding genesis, these findings are formally consistent with defective reg- at every Pi concentration of the media, although they responded to ulation by TORC1 (2). increasing Pi concentrations with an increasing P-S6 signal (Fig. 1C). Pho84, therefore, is required for normal anabolic TORC1 signaling, Pi Content of Cells Lacking Pho84 Is Diminished. We asked if and TORC1 activity responds to ambient Pi availability. C. albicans TORC1 activity may be down-regulated in response to decreased intracellular Pi in pho84 mutants, analogous to the re- Heterozygous and Homozygous Deletion Mutants in PHO84 Are sponse of S. cerevisiae TORC1 to decreased intracellular amino Defective in Hyphal Morphogenesis. TORC1 regulates hyphal mor- acids. Using pho85 mutants as controls known to hyperaccumulate phogenesis in C. albicans (2–7, 30), an important virulence determinant. intracellular Pi (31), we found that intracellular Pi concentrations

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1617799114 Liu et al. Downloaded by guest on September 29, 2021 from the ACT1 promoter. This GTR1-GTP allele suppressed the − − TORC1 signaling defect of pho84 / cells apparently equally to the overexpressed WT GTR1 (Fig. 2 D and E). Overexpression of GTR1 or GTR1-GTP did not increase phosphorylation of S6 in WT cells (Fig. 2E). These findings are consistent with the model that, in C. albicans, Gtr1 indirectly or directly conveys a Pi signal to TORC1 and links Pho84 to TORC1 signaling. We examined the relationship of Pho84 to TORC1 activity in the model yeast S. cerevisiae.Apho84 null mutant in the S288C genetic background (37) was hypersensitive to rapamycin (SI Appendix,Fig. S3A) at an intermediate ambient Pi concentration (1 mM). Of note, the rapamycin phenotype was highly responsive to the Pi concen- tration of pregrowth media. Rapamycin hypersensitivity was not suppressed, but the S. cerevisiae Sch9 phosphorylation state (36) (SI Appendix,Fig.S3B) and P-S6 signal intensity, which in S. cerevisiae, also responds to TORC1 activation (Fig. 3 and SI Appendix,Fig. S3C), were recovered by constitutively active Gtr1 in pho84 null cells. These findings suggest that Pi homeostasis and TOR sig- Fig. 2. Gtr1 links Pho84 to TOR in C. albicans.(A) Western blot of WT (SC5314), naling are linked in S. cerevisiae as in C. albicans, although specific − − gtr1 / (YGM367), and pENO1-GTR/GTR1 (YGM365) cells grown in YNB with molecular connections seem to have divergently evolved in these −/− 0 and 0.22 mM KH2PO4 for 90 min. (B) Western blot of WT (SC5314), rhb1 two fungi. − − (CCT-D1), and rhb1 / /pADH1-RHB1 (CCT-OE1) cells grown in YNB with 0 or

0.22 mM KH2PO4 for 90 min. (C)GrowthinYPDwith4ng/mLrapamycinor TORC1 Modulates the PHO Regulon. Because TORC1 not only re- vehicle. OD600 monitored every 15 min. Blue, WT overexpressing GTR1 sponds to nutrient availability but also, directs nutrient uptake (e.g., −/− −/− (JKC1596); cyan, pho84 with vector (JKC1598); green, pho84 over- by regulating expression of amino acid and ammonium transporters), expressing GTR1 (JKC1600); yellow, WT with vector (JKC1594). (D) Cell dilutions we questioned whether it may play a similar role in phosphate ac- pinned onto YPD with vehicle or 12 ng/mL rapamycin. Strains WT with vector

(JKC1594; 1), WT overexpressing GTR1 (JKC1596; 2), WT overexpressing GTR1- quisition. Given known discrepancies between rapamycin exposure MICROBIOLOGY − − − − GTP (JKC1619; 3), pho84 / with vector (JKC1598; 4), pho84 / overexpressing and physiological TOR modulation (38–41), we examined this po- GTR1 (JKC1600; 5), and pho84−/− overexpressing GTR1-GTP (JKC1616; 6). tential connection genetically. Repressible tetO was used to control Δ1–381 (E) Western blot of cells grown in YNB with 0.22 mM KH2PO4 for 90 min. Strains expression of C. albicans TOR1 or a hypomorphic TOR1 1–6asinD. Dens, densitrometric ratio of P-S6 vs. tubulin signal intensity. encoding a protein lacking the first 381 amino acids which form protein–protein interaction repeat domains. The effect of TOR1 − − were lower in pho84 / null than WT cells in low- and high-Pi– depletion on expression of PHO84 was then examined. containing media, although the difference was substantially less When WT cells were transferred from overnight cultures into than in the homologous S. cerevisiae mutant–WT pair (Fig. 1E). freshrichmedium,PHO84 mRNA levels dropped in accordance − − ’ Diminished intracellular Pi concentrations of C. albicans pho84 / with the PHO regulon s response to availability of fresh Pi sources. cells may be responsible for the decreased TORC1 activation state, In cells depleted of either the WT or the N-terminally truncated possibly in addition to the lack of a putative TORC1-activating TOR1 allele, PHO84 expression also decreased but to a significantly lesser extent (SI Appendix,Fig.S4A). Full-length TOR1 permitted function performed specifically by Pho84. Δ – greater PHO84 expression than the TOR1 1 381 allele, suggesting Gtr1 Links Pho84 to TORC1 in C. albicans. Seeking a molecular link that structural perturbation of TORC1 by truncation of Tor1 af- between Pho84 and TORC1 activity, we considered the possibility fects its inhibitory as well as activating functions. Active TORC1, that Gtr1 may connect Pho84 to TORC1. GTR1 was first described signaling nutritional repletion, hence contributes input to the PHO for its functional and physical proximity to S. cerevisiae PHO84 (32, regulon to down-regulate Pi starvation responses, whereas loss of 33), and its product later was characterized as a component of the TORC1 activity conveys a starvation signal to dampen these responses TORC1-activating EGO complex (12, 13, 34, 35). We found that (SI Appendix,Fig.S4A). Similarly, overexpression of Gtr1 and − − the P-S6 response of gtr1 / cells to phosphate refeeding was Gtr1-GTP blunted up-regulation of secreted acid phosphatase in − − blunted (Fig. 2A). To determine whether this is an unspecific effect pho84 / cells (SI Appendix,Fig.S4B), supporting the model that, of decreased upstream TORC1 signaling, mutants in another small in response to Pi, TORC1 signaling down-regulates the PHO reg- TORC1-activating GTPase, RHB1, were tested; rhb1 mutants ulon to integrate its activity with availability of other nutrients. responded to Pi refeeding like the WT (Fig. 2B), suggesting that a Pi signal to TORC1 is transmitted specifically through Gtr1. If Gtr1 acts downstream of Pho84 in activating TORC1, its − − overexpression may suppress pho84 / phenotypes. GTR1 was − − overexpressed from the ACT1 promoter in WT and pho84 / C. albicans cells. Compared with rapamycin hypersensitive − − pho84 / cells transformed with the empty vector, the resulting − − pho84 / pACT1-GTR1 cells showed WT tolerance to rapamycin, suggesting recovery of their TORC1 signaling activity (Fig. 2 C and D). To investigate this possibility, TORC1 activity was tested − − directly by comparing the P-S6 signal of pho84 / cells trans- − − formed with the empty vector with that of pho84 / pACT1-GTR1 Fig. 3. A connection between Pho84 and TORC1 is conserved in S. cerevisiae. Western blot of PHO84 with vector (Y1597; 1), pho84 with vector (Y1599; 2), cells. Overexpression of GTR1 recovered Rps6 phosphorylation in −/− PHO84 with GTR1-GTP GTR2-GDP (Y1596; 3), and pho84 with GTR1-GTP GTR2- pho84 cells nearly to WT levels (Fig. 2E). A GTR1 mutant encoding GDP (Y1604; 4) cells grown in SC (-His-Ura) containing 0.22 and 11 mM KH PO Q67L 2 4 constitutively GTP-bound Gtr1 , homologous to S. cerevisiae for 90 min probed for P-S6, S6, and tubulin. Dens, densitrometric ratio of Q65L Gtr1 (12, 35, 36), was then constructed and overexpressed P-S6 vs. tubulin signal intensity.

Liu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 (Fig. 5) and antifungal potentiation as shown in our proof of prin- ciple experiments with PAA and Fos. Discussion Eukaryotic cell mass consists to 0.5–1% of phosphorus (51). However, phosphorus constitutes only 0.07% of the Earth’scrust (51), and only ∼15% of soil phosphorus is bioavailable in a soluble form (52). Fungi, like plants and bacteria, have sophisticated regulatory networks to manage Pi starvation. S. cerevisiae cells use the TORC1 and PKA pathways to ensure orderly cessation of growth when nitrogen or carbon sources become limiting (53, 54). C. albicans cells similarly use the TORC1 pathway for calibrated responses to distinct nitrogen sources, with modulating input from PKA according to carbon source type and concentration (9). Our study shows that C. albicans TORC1 monitors not only nitrogen and carbon source but also, Pi availability (Figs. 1C and 2 A and E). Whether Pho84 activates TORC1 indirectly by an in- creased intracellular Pi content, directly in a transceptor role, or through a combination of these inputs remains an open question. Our results establish the small GTPase Gtr1, first discovered in Fig. 4. Small molecule inhibition of Pho84 represses TORC1 and hyphal S. cerevisiae because of its mutant phenotypes’ resemblance to morphogenesis and potentiates amphotericin B and micafungin. (A) West- those of Pho84 (32), as an important element linking C. albicans ern blot of WT (SC5314) cells with vehicle (1), 100 μM Fos (2), 200 μM Fos (3), Pho84 and TORC1 (Fig. 2). How Gtr1, a subunit of the TORC1- + + − + and 400 μM Fos (4) and strains PHO84 / (JKC915; 5, 7, 9, and 11), pho84 / associated vacuolar membrane-residing EGO complex (55), re- − − (JKC1583; 6 and 8), and pho84 / (JKC1450; 10 and 12) grown in standard SC ceives information from Pho84 remains to be determined. Possible (7.3 mM Pi) for 60 min and probed for P-S6 and tubulin. Dens, densitrometric models include an activating signal to the TORC1-activating ratio of P-S6 vs. tubulin signal intensity. (B) WT (SC5314) spotted at equi- Seh1-associated complex activating TORC1 (SEACAT) complex distant points around RPMI agar (0.22 mM KH2PO4, pH 7) containing vehicle, 8 mM PAA, or 500 μM Fos grown at 37 °C, and spot edges were imaged. (55) through a sensor of cytosolic Pi or vacuolar polyphosphate or Compare with SI Appendix, Fig. S2A. (Scale bar: 1 mm.) (C) WT (SC5314) was a physical interaction between Pho84 and the EGO complex on exposed to vehicle, 500 μMFos,0.2μg/mL amphotericin B, and amphotericin endosomes while Pho84 is internalized during ambient Pi abundance

B plus Fos; OD600 in SC with 0.5 mM KH2PO4 at 30 °C was monitored every (56). We cannot exclude alternative explanations of our findings 15 min. (D) WT (SC5314) was exposed to vehicle, 500 μM Fos, 25 ng/mL (e.g., a TORC1 response only to intracellular Pi concentrations and micafungin, and micafungin plus Fos; OD600 in SC with 0.5 mM KH2PO4 at up-regulation of low-affinity Pi transporters during GTR1 over- − − 30 °C was monitored every 15 min. Dens, densitrometric ratio of P-S6 vs. expression in pho84 / null cells). If Gtr1 is activated by Pho84, this tubulin signal intensity. GTPase’s role in the input to TORC1 regarding the cell’sPistate seems to be specific (Fig. 2), because there was no perturbation of −/− Small Molecule Inhibitors of Pho84 Repress TORC1 and Potentiate TORC1 activation during Pi refeeding in rhb1 cells (Fig. 2B). Antifungal Activity. S. cerevisiae Pho84 has been characterized as In S. cerevisiae, Pho84 signals to PKA as a transceptor (42, 57). A a Pi transceptor signaling to PKA through identification of point signal from Pho84 to TORC1 has not yet been described in mutations and small molecules that preferentially perturb transport, S. cerevisiae. We found that, in the genetic background S288C, loss signaling, or both (42, 43). Direct pharmacological inhibition of of Pho84 leads to rapamycin hypersensitivity and TORC1 in- C. albicans SI Appendix C. albicans TORC1 with rapamycin incurs too high a cost on host activation as it does in ( ,Fig.S3). Sch9 phosphorylation, like that of Rps6, is known to correspond with the immune function to be clinically useful (44). We tested whether TORC1 activation state (36, 58). Overexpression of constitutively blocking Pho84 with its known small molecule inhibitors phos- phonoformic acid [foscarnet (Fos)] and phosphonoacetic acid (PAA) (42), which we showed inhibit C. albicans growth in de- pendence on the presence of their target Pho84 (SI Appendix,Fig. S5 A and B), can indirectly inhibit C. albicans TORC1. Exposure of WT cells to the FDA-approved antiviral Fos inhibited Rps6 phos- phorylation (P-S6) in a dose-dependent manner (Fig. 4A)atFos concentrations attained in human plasma during antiviral therapy − + (45, 46). In heterozygous cells (pho84 / ) with haploinsufficiency phenotypes that likely reflect decreased copies of the drug target, Pho84, P-S6 was hypersensitive to Fos (Fig. 4A). In cells lacking the target Pho84, exposure to Fos did not further decrease the P-S6 signal (Fig. 4A). Pho84 inhibition with small molecules also recapitulated the hyphal growth defect seen in cells genetically depleted of PHO84 (Figs. 1D and 4B and SI Appendix,Fig.S5C). Potentiating existing antifungals is a promising strategy (47–49). Fos at concentrations reached in plasma during antiviral therapy (45, 46) and PAA potentiate activity of the antifungal amphotericin B at concentrations of the latter far below those in serum or tissue during standard dosing (50) (Fig. 4C and SI Appendix,Fig.S5D). Activity of the antifungal micafungin, belonging to the distinct drug Fig. 5. Pho84 activates TORC1 via Gtr1, and TORC1, in turn, modulates the class of echinocandins, was also potentiated (Fig. 4D). Because PHO regulon. Fos inhibits Pho84 and thereby, indirectly blocks TORC1 activity. Pho84 is not conserved in mammals, inhibition of Pho84 offers a Signaling events with known molecular mechanisms in S. cerevisiae are shown nonconventional approach to fungal-specific TORC1 inhibition as green lines. Blue lines represent predicted activities based on our findings.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1617799114 Liu et al. Downloaded by guest on September 29, 2021 active Gtr1 restores TORC1 activity in S. cerevisiae as assayed by experiment. Introduced mutations were confirmed by PCR spanning the up- Sch9 and Rps6 phosphorylation (Fig. 3 and SI Appendix,Fig.S3B). stream and downstream homologous recombination junctions of transforming However, in contrast to C. albicans, constitutively active Gtr1 did constructs and sequencing. Experiments with defined ambient Pi concentrations not suppress rapamycin hypersensitivity of S. cerevisiae pho84 null were performed in yeast nitrogen base (YNB) 0 Pi (ForMedium Ltd) with added KH PO to stated concentrations. Other media were used as in ref. 20. cells, indicating differences in the connections of PHO and TORC1 2 4 signaling between these fungi. In S. cerevisiae, control of entry into Screening Transposon Mutants for Altered Rapamycin Susceptibility. Our het- quiescence (G0) by the Rim15 kinase is coregulated by the PHO erozygous mutant collection containing a mariner transposon marked with pathway cyclin/cyclin-dependent kinase module Pho80/Pho85 as CaNAT1 (20) was used. Mutants were pregrown at room temperature in well as TORC1 (59), suggesting multiple levels of cross-talk be- 96-well plates containing 2× YPD with 8% glucose to minimize hyphal growth. tween phosphate-specific and global nutritional signaling pathways Cells were replicated to YPD agar with vehicle (90% ethanol) or 20 ng/mL in that organism, which remain to be explored in C. albicans. rapamycin. Clones showing less growth than the WT (SC5314) were isolated as In addition to the signal from Pho84 to TORC1, a signal in the rapamycin hypersensitive. The transposon insertion site was identified by opposite direction was seen from TORC1 to expression of PHO84 vectorette PCR (20). and the classic readout of the PHO regulon, the acid phosphatase (27) (SI Appendix,Fig.S4A), although clearly, TORC1 input con- Growth Assays. For cell dilutions spotted onto agar media as previously described (20), saturated overnight cultures were diluted in fivefold steps from an OD600 of tributes only to a fraction of the PHO regulon responses. TORC1 is 0.5. For growth curves in liquid media, saturated overnight cultures in YPD were well-known to regulate proteins required for acquisition of other washed once in 0.9% NaCl and diluted to an OD600 of 0.15 in 150 μLmediumin nutrients, like amino acids and ammonium, in S. cerevisiae (60, 61). flat-bottomed 96-well dishes. For growth assays, including those during drug

TORC1 input to the PHO regulon may fine-tune the investment of exposure, OD600 readings were obtained every 15 mininaplatereader,andSDs energy and nutrients in Pi acquisition to match the overall state of of three technical replicates were calculated and graphed in Graphpad Prism. the cell. In S. cerevisiae, transcriptional regulation favoring Pi ac- Growth during drug exposure was assayed in synthetic complete (SC) medium. quisition is achieved through the transcription factor Pho4 (31). A Vehicle for Pho84 inhibitors PAA (284270; Sigma) and Fos (SC-253593A; Santa C. albicans homolog of Pho4, required for stress resistance (62, 63) Cruz Biotechnology) was water, and vehicle for amphotericin B (A9528; Sigma) and commensalism in a murine model (63), was recently shown to was DMSO. All panels shown represent at least three biological replicates. control C. albicans PHO84 expression (62). How C. albicans Pho4 Western Blots. Cell harvesting, lysis, and Western blotting were performed as may be coregulated by TORC1 remains to be determined. described in ref. 9. Antibodies are listed in SI Appendix,TableS1. At least three Although TORC1 monitors nutrient availability in mammals as biological replicates were obtained for each experiment shown. For densi-

in fungi, its relationship with the PHO regulon may have diverged MICROBIOLOGY + tometry, ImageJ (https://imagej.net/Downloads) software (open source) in these phyla. In fungi, H -Pi symport is the major form of Pi wasusedasinref.9. import, because the steep Pi concentration gradient at the plasma membrane imposes a high energetic demand on transport, which is Hyphal Morphogenesis Assay. Cells were revived from frozen stocks on solid YPD met by the electrochemical proton gradient generated by the overnight, washed, and resuspended in 0.9% NaCl to OD600 0.1. Variations + + P-type H ATPase(52).Inanimals,Na-Pi pumps predominate between single colonies and colony density effects were minimized by spotting (64). Humans largely consume Pi together with amino acids in food 3-μL cell suspension at four or six equidistant points using a template around consisting of other eukaryotes and their products, so that Pi star- the perimeter of an agar medium plate as in ref. 20. For small molecule Pho84 inhibitor effects on hyphal formation, Spider and RPMI were used vation tends to occur during starvation for protein (65). In contrast, (R8999-04; TOKU-E), the latter with 0.22 mM KH2PO4 buffered to pH 7 with Pi and nitrogen sources must be acquired independently by uni- 50 mM Mops. All panels shown represent at least three biological replicates. cellular organisms, and therefore, the connection that we discov- + ered between the fungal H -Pi symporter and the TORC1 pathway Acid Phosphatase Assays. As adapted from ref. 25, overnight cultures in SC

seems physiologically plausible. weredilutedtoanOD600 of 0.05 into YNB medium buffered to pH 4 with We found that TORC1 is indirectly repressed during Pho84 in- 50 mM sodium citrate containing 0 or 11 mM KH2PO4 and grown overnight. hibition with small molecules (Fig. 4 and SI Appendix,Fig.S5). P-Nitrophenyl Phosphate (N4645; Sigma) was added to washed cells to a con- Because TORC1 components are conserved between fungi and centration of 5.62 mg/mL. After 15 min at room temperature, the reaction was humans, whereas Pho84 has no human homologs, this result pro- stopped with Na2CO3 (pH 11) to a concentration of 0.3 g/mL, and OD420 and vides an option for indirect fungal-specific TORC1 inhibition not OD600 were measured. At least three biological replicates with three technical replicates each were obtained. previously explored in the search for new antifungals. Although TORC1 is only partially inhibited in this manner, other fungal- Intracellular Pi Assays. Free and total Pi was measured by colorimetric molybdate specific indirect TORC1 activators may provide synergistic targets. assay as described (68). Briefly, cultures were washed with distilled water twice, More immediately, Pho84 inhibitors potentiate the antifungal effect resuspended in 500 μL 0.1% Triton X-100, and lysed by glass bead homogeni- of micafungin and amphotericin B (Fig. 4 C and D), possibly per- zation. Lysate protein content was determined using a BioRad Protein Assay kit. mitting lower dosing of the latter “gold standard” broad-spectrum Free Pi was measured in unboiled lysate; then, total phosphate was measured – μ agent to obviate its often treatment-limiting toxicities (66). We used after boiling 3 30 g whole-cell lysate for 10 min in 0.5 M H2SO4. At least three two of multiple Pho84 inhibitors previously characterized (42) (Fig. biological replicates with three technical replicates each were obtained. 4andSI Appendix,Fig.S5D), and more specific, noncompetitive inhibitors with more favorable therapeutic indices than Fos may be RT-PCR Expression Analysis. Cells were grown overnight in YPD medium with μ found through screening efforts. Because Pho84 homologs are 5 ng/mL doxycycline, diluted into YPD with 30 g/mL doxycycline, and harvested at time 0 and 2 and 4 h. RNA was extracted with the Direct-Zol RNA miniprep kit highly conserved among fungi, potentiating amphotericin B activity (R2051; Zymo Research). RT-PCR procedures were performed as indicated (69). through their inhibition may prove a viable therapeutic strategy for other fungal species less amenable to other antifungal agents. ACKNOWLEDGMENTS. We thank Gerald R. Fink, Erin K. O’Shea, Bin He, Charles Boone, Françoise Stutz, Kevin Struhl, and Fred Winston for plasmids Materials and Methods and S. cerevisiae strains; Leah Cowen, Chung-Yu Lan, and Rajini Rao for Strains and Culture Conditions. The C. albicans and S. cerevisiae strains, plasmids, C. albicans strains; Gerald R. Fink, Felix Lam, Valmik Vyas, Luke Whitesell, and primers used are described in SI Appendix,TablesS1andS2. C. albicans and Bin He for helpful conversations; Brian M. Hoffman, Valeria C. Culotta, and Amit R. Reddi for protocols; and Dennis Wykoff, Bin He, Robert Husson, strains were generated using HIS1 and ARG4 markers and strains as described in and Paula Watnick for critical reading of the manuscript. This work was funded ref. 67 as well as the CaNAT1 selectable marker as described in refs. 20 and 21. by National Institute of Allergy and Infectious Diseases Grants R21AI096054 Two independent heterozygotes were used to derive homozygous null and and R01AI095305. P.R.F. was supported by Science Foundation Ireland Grant reintegrant mutants of PHO84 as well as tetO-TOR1 mutants. Auxotrophies were 11/RFP.1/GEN/3044. M.E.C. was funded by National Cancer Institute Grant complemented, so that only prototrophic strains were compared in an R01 CA154499.

Liu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 1. Loewith R, Hall MN (2011) Target of rapamycin (TOR) in nutrient signaling and 36. Kingsbury JM, Sen ND, Maeda T, Heitman J, Cardenas ME (2014) Endolysosomal mem- growth control. Genetics 189:1177–1201. brane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell 2. Cutler NS, Pan X, Heitman J, Cardenas ME (2001) The TOR signal transduction cascade growth in Saccharomyces cerevisiae. Genetics 196:1077–1089. controls cellular differentiation in response to nutrients. Mol Biol Cell 12:4103–4113. 37. Giaever G, et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. 3. Bastidas RJ, Heitman J, Cardenas ME (2009) The protein kinase Tor1 regulates adhesin Nature 418:387–391. gene expression in Candida albicans. PLoS Pathog 5:e1000294. 38. Cox KH, Kulkarni A, Tate JJ, Cooper TG (2004) Gln3 phosphorylation and intracellular 4. Homann OR, Dea J, Noble SM, Johnson AD (2009) A phenotypic profile of the Candida localization in nutrient limitation and starvation differ from those generated by rapa- albicans regulatory network. PLoS Genet 5:e1000783. mycin inhibition of Tor1/2 in Saccharomyces cerevisiae. JBiolChem279:10270–10278. 5. Zacchi LF, Gomez-Raja J, Davis DA (2010) Mds3 regulates morphogenesis in Candida 39. Puria R, Zurita-Martinez SA, Cardenas ME (2008) Nuclear translocation of Gln3 in albicans through the TOR pathway. Mol Cell Biol 30:3695–3710. response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces 6. Lu Y, Su C, Wang A, Liu H (2011) Hyphal development in Candida albicans requires cerevisiae. Proc Natl Acad Sci USA 105:7194–7199. two temporally linked changes in promoter chromatin for initiation and mainte- 40. Puria R, Cardenas ME (2008) Rapamycin bypasses vesicle-mediated signaling events to nance. PLoS Biol 9:e1001105. activate Gln3 in Saccharomyces cerevisiae. Commun Integr Biol 1:23–25. 7. Lu Y, Su C, Liu H (2014) Candida albicans hyphal initiation and elongation. Trends 41. Kingsbury JM, Cardenas ME (2016) Vesicular trafficking systems impact TORC1-controlled Microbiol 22:707–714. transcriptional programs in Saccharomyces cerevisiae. G3 (Bethesda) 6:641–652. 8. Park H, et al. (2009) Transcriptional responses of candida albicans to epithelial and 42. Popova Y, Thayumanavan P, Lonati E, Agrochão M, Thevelein JM (2010) Transport endothelial cells. Eukaryot Cell 8:1498–1510. and signaling through the phosphate-binding site of the yeast Pho84 phosphate 9. Chowdhury T, Köhler JR (2015) Ribosomal protein S6 phosphorylation is controlled by transceptor. Proc Natl Acad Sci USA 107:2890–2895. TOR and modulated by PKA in Candida albicans. Mol Microbiol 98:384–402. 43. Samyn DR, et al. (2012) Mutational analysis of putative phosphate- and proton- 10. Pedruzzi I, et al. (2003) TOR and PKA signaling pathways converge on the protein binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H(+) transceptor kinase Rim15 to control entry into G0. Mol Cell 12:1607–1613. and its effect on signalling to the PKA and PHO pathways. Biochem J 445:413–422. 11. Zurita-Martinez SA, Cardenas ME (2005) Tor and cyclic AMP-protein kinase A: Two 44. Cruz MC, et al. (2001) Rapamycin and less immunosuppressive analogs are toxic to parallel pathways regulating expression of genes required for cell growth. Eukaryot Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of – Cell 4:63–71. TOR. Antimicrob Agents Chemother 45:3162 3170. 12. Binda M, et al. (2009) The Vam6 GEF controls TORC1 by activating the EGO complex. 45. Chrisp P, Clissold SP (1991) Foscarnet. A review of its antiviral activity, pharmacoki- Mol Cell 35:563–573. netic properties and therapeutic use in immunocompromised patients with cyto- – 13. Bonfils G, et al. (2012) Leucyl-tRNA synthetase controls TORC1 via the EGO complex. megalovirus retinitis. Drugs 41:104 129. – Mol Cell 46:105–110. 46. Minor JR, Baltz JK (1991) Foscarnet sodium. DICP 25:41 47. 14. Yamagata K, et al. (1994) rheb, a growth factor- and synaptic activity-regulated gene, 47. Robbins N, et al. (2015) An antifungal combination matrix identifies a rich pool of encodes a novel Ras-related protein. J Biol Chem 269:16333–16339. adjuvant molecules that enhance drug activity against diverse fungal pathogens. Cell – 15. Urano J, Tabancay AP, Yang W, Tamanoi F (2000) The Saccharomyces cerevisiae Rheb Reports 13:1481 1492. G-protein is involved in regulating canavanine resistance and arginine uptake. J Biol 48. Butts A, et al. (2013) A repurposing approach identifies off-patent drugs with fun- Chem 275:11198–11206. gicidal cryptococcal activity, a common structural chemotype, and pharmacological – 16. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J (2005) Rheb binds and regulates properties relevant to the treatment of cryptococcosis. Eukaryot Cell 12:278 287. 49. Butts A, Palmer GE, Rogers PD (2017) Antifungal adjuvants: Preserving and extending the mTOR kinase. Curr Biol 15:702–713. – 17. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell the antifungal arsenal. Virulence 8:198 210. 50. Hamill RJ (2013) Amphotericin B formulations: A comparative review of efficacy and 149:274–293. toxicity. Drugs 73:919–934. 18. Tsao CC, Chen YT, Lan CY (2009) A small G protein Rhb1 and a GTPase-activating 51. Audesirk T, Audesirk G, Byers BE (1999) Biology: Life on Earth (Prentice-Hall, Upper protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall Saddle River, NJ), 5th Ed. integrity of Candida albicans. Fungal Genet Biol 46:126–136. 52. Johri AK, et al. (2015) Fungal association and utilization of phosphate by plants: 19. Chen YT, et al. (2012) Rhb1 regulates the expression of secreted aspartic protease Success, limitations, and future prospects. Front Microbiol 6:984. 2 through the TOR signaling pathway in Candida albicans. Eukaryot Cell 11:168–182. 53. Barbet NC, et al. (1996) TOR controls translation initiation and early G1 progression in 20. Shen J, Cowen LE, Griffin AM, Chan L, Köhler JR (2008) The Candida albicans pesca- yeast. Mol Biol Cell 7:25–42. dillo homolog is required for normal hypha-to-yeast morphogenesis and yeast pro- 54. Broach JR (2012) Nutritional control of growth and development in yeast. Genetics liferation. Proc Natl Acad Sci USA 105:20918–20923. 192:73–105. 21. Shen J, Guo W, Köhler JR (2005) CaNAT1, a heterologous dominant selectable marker 55. Panchaud N, Péli-Gulli MP, De Virgilio C (2013) SEACing the GAP that nEGOCiates for transformation of Candida albicans and other pathogenic Candida species. Infect TORC1 activation: Evolutionary conservation of Rag GTPase regulation. Cell Cycle Immun 73:1239–1242. 12:2948–2952. 22. Pedersen BP, et al. (2013) Crystal structure of a eukaryotic phosphate transporter. 56. Lagerstedt JO, et al. (2002) Mutagenic and functional analysis of the C-terminus of Nature 496:533–536. Saccharomyces cerevisiae Pho84 phosphate transporter. FEBS Lett 526:31–37. 23. Inglis DO, et al. (2012) The Candida genome database incorporates multiple Candida 57. Giots F, Donaton MC, Thevelein JM (2003) Inorganic phosphate is sensed by specific species: Multispecies search and analysis tools with curated gene and protein in- phosphate carriers and acts in concert with glucose as a nutrient signal for activation – formation for Candida albicans and Candida glabrata. Nucleic Acids Res 40:D667 D674. of the protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 24. Bun-Ya M, Nishimura M, Harashima S, Oshima Y (1991) The PHO84 gene of Saccharo- 47:1163–1181. – myces cerevisiae encodes an inorganic phosphate transporter. MolCellBiol11:3229 3238. 58. Urban J, et al. (2007) Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol ’ 25. Wykoff DD, O Shea EK (2001) Phosphate transport and sensing in Saccharomyces Cell 26:663–674. – cerevisiae. Genetics 159:1491 1499. 59. Wanke V, Pedruzzi I, Cameroni E, Dubouloz F, De Virgilio C (2005) Regulation of 26. Auesukaree C, et al. (2004) Intracellular phosphate serves as a signal for the regula- G0 entry by the Pho80-Pho85 cyclin-CDK complex. EMBO J 24:4271–4278. – tion of the PHO pathway in Saccharomyces cerevisiae. J Biol Chem 279:17289 17294. 60. Cardenas ME, Cutler NS, Lorenz MC, Di Como CJ, Heitman J (1999) The TOR signaling ’ 27. Thomas MR, O Shea EK (2005) An intracellular phosphate buffer filters transient cascade regulates gene expression in response to nutrients. Genes Dev 13:3271–3279. – fluctuations in extracellular phosphate levels. Proc Natl Acad Sci USA 102:9565 9570. 61. Crespo JL, Powers T, Fowler B, Hall MN (2002) The TOR-controlled transcription ac- ’ 28. Wykoff DD, Rizvi AH, Raser JM, Margolin B, O Shea EK (2007) Positive feedback tivators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of – regulates switching of phosphate transporters in S. cerevisiae. Mol Cell 27:1005 1013. glutamine. Proc Natl Acad Sci USA 99:6784–6789. – 29. Vogel K, Hinnen A (1990) The yeast phosphatase system. Mol Microbiol 4:2013 2017. 62. Ikeh MA, et al. (2016) Pho4 mediates phosphate acquisition in Candida albicans and is 30. Lee CM, Nantel A, Jiang L, Whiteway M, Shen SH (2004) The serine/threonine protein vital for stress resistance and metal homeostasis. Mol Biol Cell 27:2784–2801. phosphatase SIT4 modulates yeast-to-hypha morphogenesis and virulence in Candida 63. Urrialde V, Prieto D, Pla J, Alonso-Monge R (2016) The Candida albicans Pho4 tran- – albicans. Mol Microbiol 51:691 709. scription factor mediates susceptibility to stress and influences fitness in a mouse com- ’ 31. Lee YS, Huang K, Quiocho FA, O Shea EK (2008) Molecular basis of cyclin-CDK-CKI mensalism model. Front Microbiol 7:1062. regulation by reversible binding of an inositol pyrophosphate. Nat Chem Biol 4:25–32. 64. Escoubet B, Djabali K, Amiel C (1989) Adaptation to Pi deprivation of cell Na-dependent 32. Bun-Ya M, Harashima S, Oshima Y (1992) Putative GTP-binding protein, Gtr1, asso- Pi uptake: A widespread process. Am J Physiol 256:C322–C328. ciated with the function of the Pho84 inorganic phosphate transporter in Saccharo- 65. Hearing SD (2004) Refeeding syndrome. BMJ 328:908–909. myces cerevisiae. Mol Cell Biol 12:2958–2966. 66. Mora-Duarte J, et al.; Caspofungin Invasive Candidiasis Study Group (2002) Comparison 33. Lagerstedt JO, Reeve I, Voss JC, Persson BL (2005) Structure and function of the GTP of caspofungin and amphotericin B for invasive candidiasis. N Engl J Med 347:2020–2029. binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae. 67. Noble SM, Johnson AD (2005) Strains and strategies for large-scale gene deletion studies Biochemistry 44:511–517. of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4:298–309. 34. Nakashima N, Noguchi E, Nishimoto T (1999) Saccharomyces cerevisiae putative G protein, 68. McNaughton RL, et al. (2010) Probing in vivo Mn2+ speciation and oxidative stress Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, resistance in yeast cells with electron-nuclear double resonance spectroscopy. Proc negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p. Genetics 152:853–867. Natl Acad Sci USA 107:15335–15339. 35. Kingsbury JM, Sen ND, Cardenas ME (2015) Branched-chain aminotransferases control 69. Singh AK, et al. (2016) Investigating essential gene function in Mycobacterium tuber- TORC1 signaling in Saccharomyces cerevisiae. PLoS Genet 11:e1005714. culosis using an efficient CRISPR interference system. Nucleic Acids Res 44:e143.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1617799114 Liu et al. Downloaded by guest on September 29, 2021