Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition

Abstract

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans. Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1 Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae. The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes 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.ribosomal protein S6 phosphorylation | antifungal | amphotericin B | echinocandin | drug potentiation

Intersection of phosphate transport, oxidative stress and TOR signalling inCandida albicansvirulence

“…In contrast, pho84 null mutant cells were a mixture of pseudohyphal filaments and yeast with fewer hyphae (Fig. 1D), reflecting defective hyphal growth previously seen in vitro (8), while reintegrant cells grew as hyphae, pseudohyphae and yeast (Fig. 1D).…”

“…Null mutants in PHO84 are defective in TORC1 signaling and in hyphal growth (8). As C. albicans hyphal growth is a virulence determinant, we examined pho84 null mutant (-/-, pho84/pho84) cells’ virulence in a Drosophila melanogaster model (17).…”

“…A high-affinity Pi transporter of C. albicans , Pho84, is required for normal Target of Rapamycin (TOR) signalling and hyphal morphogenesis (8). Pharmacologic inhibition of Pho84 potentiates the activity of two major antifungal classes (8). Pho84 is one of four predicted C. albicans plasma membrane Pi transporters, so that redundancy of its activity would be expected, and a role of Pho84 in virulence could not be assumed a priori.…”

“…Pho84 is one of four predicted C. albicans plasma membrane Pi transporters, so that redundancy of its activity would be expected, and a role of Pho84 in virulence could not be assumed a priori. We previously observed failure of pho84 deletion mutants to appropriately induce hyphal growth in response to several in vitro conditions (8). Since hyphal growth is a known virulence determinant in C. albicans , we examined virulence of pho84 mutant cells first in a wild type Drosophila melanogaster model, then in two murine models.…”

A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis, to couple metabolism to cell cycle progression.

“…We first measured the intracellular Pi levels in WT cells, thiolation mutants ( uba4 Δ), cells lacking Tps2p ( tps2 Δ), and cells lacking tRNA thiolation as well as Tps2p ( uba4 Δ tps2 Δ). pho85 Δ cells were used as a control, since they exhibit intrinsically higher intracellular Pi levels (Liu et al , 2017). We observed that in cells lacking TPS2 ( tps2 Δ) intracellular Pi levels were substantially lower (∼75-80%) relative to WT cells (Figure 6B and S7A).…”