The Candida albicans high-affinity
phosphate transporter Pho84 is required for normal Target of
Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans' tolerance of two antifungal drug classes,
polyenes and
echinocandins.
Echinocandins inhibit biosynthesis of a major cell wall component,
beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond
echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including
nucleotides like
ATP and
nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from
phosphate starvation. Nonphosphoric precursors like nucleobases and
nucleosides were elevated. Outer cell wall
phosphomannan biosynthesis requires a
nucleotide sugar,
GDP-mannose. The
nucleotide sugar UDP-glucose is the substrate of
enzymes that synthesize two major structural cell wall
polysaccharides, beta-1,3- and
beta-1,6-glucan. Another
nucleotide sugar,
UDP-
N-acetylglucosamine, is the substrate of
chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and
phosphate starvation, potentiated pharmacological or genetic perturbation of these
enzymes. We posit that low substrate concentrations of beta-
d-glucan- and
chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products.
Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-
glucans or
chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.IMPORTANCECandida species cause hundreds of thousands of invasive
infections with high mortality each year. Developing novel
antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining
phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A
protein that imports
phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans
Nucleotide sugars, which are
phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing
enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing
enzymes may provide a strategy for antifungal
therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.