Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

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

ROS management and Sod3 expression are defective in cells lacking <i>PHO84</i>.

<p>(A) DCFDA-detectable ROS of cells unexposed to extrinsic oxidative stress, of strains <i>+/+</i>, JKC915; -/-, JKC1450 and -/-/+, JKC1588 diluted into SC medium with 0.22 mM, 1 mM and 11 mM Pi. Fluorescence intensity was measured after staining cells with 50 μM DCFH-DA. <i>-/-</i> versus +/+ at 0.22 mM Pi <i>p</i> = 0.0149. <i>-/-</i> versus +/+ at 1 mM Pi <i>p</i><0.0001. <i>-/-</i> versus <i>+/+</i> at 11 mM Pi <i>p</i><0.0001. <i>p</i> values per Student’s t-test. (B) DCFDA-detectable ROS production during exposure to 100 μM menadione (Mena). Strains as in A cultured overnight were diluted into SC medium (Loflo) and fluorescence intensity was measured as in A. <i>p</i> = 0.0002 for <i>-/-</i> versus <i>+/+</i>. (C) Superoxide dismutase (SOD) activity of strains as in A, grown in YPD medium with vehicle, 50 μM Menadione (Mena) and 0.8 mM bathocuproine disulfonic acid (BCS) for 8 hours; cell lysate in non-denaturing gel stained with nitroblue tetrazolium to detect SOD activity and with Coomassie blue to assess loading. (D) Western blot of strains as in A, grown in normal SC medium with vehicle, 3 mM MnCl₂ and 3 mM CuSO₄ for 13 hours, probed for Sod3 and loading control tubulin. <i>p</i> values were calculated using Student’s t-test. *<i>p</i><0.01; +<i>p</i><0.05; ns non-significant. A-D show representatives of at least 3 biological replicates; error bars SD of 3 technical replicates.</p

Overexpression of <i>SOD3</i> from a heterologous promoter significantly rescues ROS hypersensitivity of cells lacking Pho84.

<p>Cells were grown on YPD agar medium without or with 50 ng/ml doxycycline for 39 hrs, and were maintained in these doxycycline concentrations throughout the course of the experiment. Cells were inoculated at OD₆₀₀ 0.1 in YPD (glucose-containing medium that represses transcription from <i>pMAL2</i>), with vehicle DMSO (V) or 21 μM Plumbagin (P). OD₆₀₀ was monitored every 15 minutes for strains (A) wild type background: +/+, JKC915; +/+ <i>tetO-SOD3/SOD3</i>, JKC1738; +/+ <i>pMAL2-SOD3/SOD3</i>, JKC1776; (B) <i>pho84</i> null mutant background: -/-, JKC1450; -/- <i>tetO-SOD3/SOD3</i>, JKC1745; -/- <i>pMAL2-SOD3/SOD3</i>, JKC1780. A, B are representative of 3 biological replicates, error bars SD of 3 technical replicates.</p

Pho84 is required for resistance to killing by whole blood or neutrophils, in dependence on neutrophil ROS.

<p>(A) Percent survival of <i>C</i>. <i>albicans</i> cells after incubation with whole blood from healthy human volunteers; cells of genotypes <i>+/+</i>, JKC915; -/-, JKC1450 and -/-/+, JKC1588 were inoculated into blood and plated onto agar medium at the indicated time points for calculation of CFU/ml. <i>-/-</i> versus <i>+/+</i> at 5 h <i>p</i> = 0.008. (B) Percent survival of <i>C</i>. <i>albicans</i> cells after incubation with neutrophils, strains as in A, at M.O.I. 2 for 2 hrs and 5 hrs. <i>-/-</i> versus <i>+/+</i> at 5 h <i>p</i> = 0.04. (C) Human peripheral blood-derived neutrophils pretreated with different concentrations of N-acetyl-l-cysteine (NAC) were incubated for 90 min with strains as in A at M.O.I. 2. <i>-/-</i> versus <i>+/+</i> with vehicle <i>p</i><0.0001. (D) Human peripheral blood-derived neutrophils pretreated with 10 μM Diphenyleneiodonium (DPI) were incubated for 90 min with strains as in A at M.O.I. 2. Vehicle alone, DPI alone and neutrophil alone groups are controls. (E) Chronic Granulomatous Disease patient-derived neutrophils (CGD) were incubated for 2 hours with strains as in A at M.O.I. 2. <i>p</i><0.0001 for <i>-/-</i> in healthy control neutrophils (control) versus <i>+/+</i> in control, all others non-significant. <i>p</i> values per Student’s t-test. *<i>p</i><0.01; +<i>p</i><0.05; ns is non-significant. A-D representative of at least 3 biological replicates.</p

Virulence is attenuated in <i>C</i>. <i>albicans</i> cells lacking <i>PHO84</i>.

<p>(A) Percentage of surviving <i>Drosophila mlanogaster</i> after infection with cells of the <i>PHO84</i> genotypes +/+, JKC915; -/-, JKC1450 and -/-/+, JKC1588 respectively. The flies were injected with 50 nl fungal cell suspensions with a micro-injector. At least 3 (up to 7) biological replicates and 6–15 technical replicates per strain were performed. (B) Oral fungal burden of mice with oropharyngeal candidiasis was calculated by enumerating CFU per gram tongue tissue after 5 days of infection. Each dot represents the tissue fungal burden of one mouse. (C) Kaplan-Meier survival plot of mice with disseminated candidiasis. Eight mice per strain were injected with strains as in A respectively. (D) Kidneys from <i>+/+</i>, <i>-/-</i> and -/-/+ infected mice were isolated and sections stained with Gomori Methenamine Silver. Size bar 0.1 mm.</p

Pho84 activates TORC1 via Gtr1 to modulate Sod3 expression in combating neutrophil derived ROS.

<p>Signalling events with known molecular mechanisms in <i>C</i>. <i>albicans</i> are shown as green lines. Blue lines represent predicted activities based on our findings.</p

TORC1 activity contributes to ROS management and Sod3 expression.

<p>(A) DCFDA-detectable ROS measurement of strains, (1) <i>+/+</i> containing vector (JKC1594), (2) <i>-/-</i> containing vector (JKC1598), (3) <i>+/+</i> overexpressing <i>GTR1</i> (JKC1596), (4) <i>-/-</i> overexpressing <i>GTR1</i> (JKC1600). Cells cultured overnight in YPD were diluted into SC medium (Loflo) with vehicle or 8 ng/ml rapamycin for 1 hour, and fluorescence intensity was determined after staining cells with 50 μM DCFH-DA. <i>p</i> = 0.0252 for ratio of <i>-/-</i> with vector and <i>-/-</i> overexpressing <i>GTR1</i> versus ratio of wild type with vector and wild type overexpressing <i>GTR1</i>; all cells exposed to vehicle. <i>p</i> = 0.0014 for ratio of <i>-/-</i> with vector and <i>-/-</i> overexpressing <i>GTR1</i> versus ratio of wild type with vector and wild type overexpressing <i>GTR1</i>; all cells exposed to rapamycin. <i>p</i> values per Student’s t-test. (B) Western blot of strains as in A, grown in SC medium (Loflo) for 1 hour, probed for Sod3 and loading control tubulin. (C) Wild type (SC5314) cells were exposed to vehicle (v), 0.5 mM foscarnet (F) or 1 mM PAA (P) for 1 hour, and fluorescence intensity was determined as in A. p<0.001 for both 0.5 mM foscarnet versus vehicle and 1 mM PAA versus vehicle. A-C show representatives of at least 3 biological replicates; error bars SD of 3 technical replicates.</p