Intersection of phosphate transport, oxidative stress and TOR signalling in <i>Candida albicans</i> virulence

<div><p>Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of <i>Saccharomyces cerevisiae</i> and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of <i>Candida albicans</i> Pho84 attenuated virulence in <i>Drosophila</i> and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): <i>pho84-/-</i> cells were no more susceptible than wild type <i>C</i>. <i>albicans</i> to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. <i>pho84-/-</i> mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in <i>pho84-/-</i> cells, while <i>SOD3</i> overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of <i>SOD3</i> expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating <i>SOD3</i> transcription was observed in <i>PHO84</i> wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on <i>pho84-/-</i> cells, though, because overexpressing <i>SOD3</i> did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of <i>S</i>. <i>cerevisiae</i> Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in <i>pho84-/-</i> null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing <i>C</i>. <i>albicans</i> to oxidative stress.</p>