Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance

ACKNOWLEDGMENTS We declare no conflicts of interest. We thank Jesús Pla for his kind gift of the anti-Mkc1 antibody and Kristin Moffitt and Richard Malley for generous advice in ELISA technology and use of the ELISA reader. We thank Tahmeena Chowdhury for scientific discussions leading up to this work. We thank the Candida Genome Database. N.-N.L., M.A.-Z., W.Q., and J.R.K. were supported by R21 AI137716 and by Boston Children’s Hospital Department of Pediatrics. M.A.-Z. was partially funded by the Alfonso Martin Escudero Foundation. J.D.-A. and O.L. were funded by the Boston Children’s Hospital Department of Pediatrics and U19 AI118608-01A1. N.A.R.G. was supported by the Wellcome Trust and the Medical Research Council Centre for Medical Mycology (MR/N006364/1).Peer reviewedPublisher PD

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

Cytosolic acidification is the first transduction signal of lactoferrin-induced regulated cell death pathway

In yeast, we reported the critical role of K+-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H+-ATPase. In the present study, the K+ channel Tok1p was identified as the K+ channel-mediating K+-efflux, as indicated by the protective effect of extracellular K+ (≥30 mM), K+-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K+-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH4Cl or nigericin which released a percentage of K+ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (~0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H+-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K+ channel, Tok1p, allowing an electrogenic K+-efflux. These ionic events, cytosolic accumulation of H+ followed by K+-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells

Candida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.Candida albicans surface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future

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

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

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

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