Date Published: July 30, 2018
Publisher: Public Library of Science
Author(s): Ning-Ning Liu, Priya Uppuluri, Achille Broggi, Angelique Besold, Kicki Ryman, Hiroto Kambara, Norma Solis, Viola Lorenz, Wanjun Qi, Maikel Acosta-Zaldívar, S. Noushin Emami, Bin Bao, Dingding An, Francisco A. Bonilla, Martha Sola-Visner, Scott G. Filler, Hongbo R. Luo, Ylva Engström, Per Olof Ljungdahl, Valeria C. Culotta, Ivan Zanoni, Jose L. Lopez-Ribot, Julia R. Köhler, Xiaorong Lin.
Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila 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): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- 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 pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 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 S. cerevisiae 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 pho84-/- 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 C. albicans to oxidative stress.
Candida albicans is the most common invasive human fungal pathogen, whose infections carry a high mortality rate . It is also a widespread commensal, colonizing gastrointestinal mucous membranes of around half of healthy humans  and competing with myriad bacteria for nutrients shed by the host or extractable from the food stream [3, 4]. Sources of the macronutrients carbon, nitrogen and phosphate must be distributed between the host and its bacterial and fungal colonizers. During invasive disease, C. albicans uses the human as its source of nutrients and must withstand the host immune system .
Virulence attenuation in mutants of a single macronutrient transporter like Pho84 (Fig 1), one of 4 predicted cell-surface Pi transporters of C. albicans, is unusual and has not been reported to our knowledge. In addition to their hyphal growth defect (Fig 1D), we posit that pho84 null mutant cells’ diminished virulence is due to their hypersensitivity to neutrophil killing (Fig 2B–2D). When a neutrophil encounters an invasive pathogen, a broad array of noxious molecules and hydrolytic enzymes are exocytosed, released into the phagosome  or onto microbial cells during formation of extracellular traps . Among these, ROS are critical for killing C. albicans [16, 37, 38]. We found that pharmacologically scavenging neutrophil-generated ROS (Fig 2C), blocking neutrophil ROS production (Fig 2D), or exposing C. albicans to neutrophils from a patient with a genetic defect in ROS production (Fig 2E) rescued viability of pho84 null mutant cells in this interaction. These findings indicate that among the large neutrophil armamentarium , C. albicans cells without functional Pho84 are particularly sensitive to ROS. pho84 null mutant cells were hypersensitive in vitro to each of 3 distinct sources of exogenous ROS, plumbagin and menadione, which generate the superoxide anion, and to the peroxide source H2O2 (Fig 3A).