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

Abstract

70Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the 71 major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial 72 element in the phosphate homeostatic system of this model yeast. We found that loss of 73Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and 74 disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. 75Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species 76 (ROS): pho84-/-cells were no more susceptible than wild type C. albicans to neutrophils from 77 a patient with chronic granulomatous disease, or to those whose oxidative burst was 78 pharmacologically inhibited or neutralized. pho84-/-mutants hyperactivated oxidative stress 79 signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in 80 high as well as low ambient phosphate conditions. ROS accumulation correlated with 81 diminished levels of the unique superoxide dismutase Sod3 in pho84-/-cells, while SOD3 82 overexpression from a conditional promoter substantially restored these cells' oxidative stress 83 resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress 84 hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 85 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor 86 driving oxidative stress effects on pho84-/-cells, though, because overexpressing SOD3 did 87 not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has 88 further roles in oxidative stress resistance and virulence. Measurement of cellular metal 89 concentrations demonstrated that diminished Sod3 expression was not due to decreased 90 import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 91 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we 92 found its role in TORC1 activation to impact oxidative stress management: overexpression of 93 the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/-null 94 mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal 95 growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-96 approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence 97 support host defenses by sensitizing C. albicans to oxidative stress. 98 99 100 Author Summary 101 102Candida albicans is the species most often isolated from patients with invasive fungal disease, 103 and is also a common colonizer of healthy people. It is well equipped to compete for nutrients 104 with bacteria co-inhabiting human gastrointestinal mucous membranes, since it possesses 105 multiple transporters to internalize important nutrients like sugars, nitrogen sources, and 106 phosphate. During infection, the fungus needs...