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

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

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

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

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