Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance

Abstract

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. IMPORTANCE Candida 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.

Phosphate in Virulence of Candida albicans and Candida glabrata

“…What other molecular alterations in pho84 null mutant cells are responsible for their hypersensitivity to host immune mechanisms? Another possible weakness of these cells is their defective cell wall [80].…”

“…Their outer phosphomannan cell wall layer is thinner, they contain less chitin and beta-1,6-glucan, and they are hypersensitive to cell wall stress [80]. The cell wall stressors to which cells lacking Pho84 are susceptible are varied and include beta-1,3-glucan synthase inhibition by the echinocandin micafungin, chitin synthase inhibition by nikkomycin, heat, and disruption of chitin-glucan bonds by Congo red [80]. Hypersensitivity of C. albicans wild type and pho84 null mutant cells to pharmacologic cell wall stressors is inversely related to their phosphate repletion state [80].…”

“…The cell wall stressors to which cells lacking Pho84 are susceptible are varied and include beta-1,3-glucan synthase inhibition by the echinocandin micafungin, chitin synthase inhibition by nikkomycin, heat, and disruption of chitin-glucan bonds by Congo red [80]. Hypersensitivity of C. albicans wild type and pho84 null mutant cells to pharmacologic cell wall stressors is inversely related to their phosphate repletion state [80]. This hypersensitivity corresponds to deficient cell wall stress signaling as assayed by the phosphorylation state of the cell wall integrity pathway MAP kinase Mkc1 [80].…”

“…Hypersensitivity of C. albicans wild type and pho84 null mutant cells to pharmacologic cell wall stressors is inversely related to their phosphate repletion state [80]. This hypersensitivity corresponds to deficient cell wall stress signaling as assayed by the phosphorylation state of the cell wall integrity pathway MAP kinase Mkc1 [80]. Mechanical weakness of the cell wall might contribute to pho84 null mutant cells' inability to withstand neutrophil hydrolytic enzymes [81].…”

“…Mechanical weakness of the cell wall might contribute to pho84 null mutant cells' inability to withstand neutrophil hydrolytic enzymes [81]. Cell wall integrity defects of pho84 null mutant cells cannot be rescued by artificial TORC1 activation [80], suggesting they may be independent of these cells' TORC1 signaling perturbation.…”