Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of <i>C</i>. <i>albicans</i> Virulence Pathways

<div><p><i>Candida albicans</i> is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of <i>C</i>. <i>albicans</i> morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in <i>C</i>. <i>albicans</i> and similar effects were observed in other <i>Candida</i> species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.</p></div

GTP-Ras1 decrease by MB is independent of the GEF Cdc25, but depends on the GAP Ira2.

<p><b>(A)</b> and <b>(B)</b> Colony morphology and western blot analysis of the <i>cdc25</i>/<i>cdc25</i> and <i>ira2</i>/<i>ira2</i> strains compared to WT (BWP17). <b>(C)</b> Cellular morphology of the <i>ira2</i>/<i>ira2</i> strain. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s001" target="_blank">S1A Fig</a> panels 23 and 24 for cellular morphology of <i>cdc25</i>/<i>cdc25</i>. Scale bar = 10 μm. <b>(D)</b> Intracellular ATP measurement of the <i>ira2</i>/<i>ira2</i> strain compared to the WT (BWP17). Mean ± SD are shown. *p<0.05. (A) and (B) Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is reported. (A) to (D) Cells were grown for 24 h on YNBAGNP with and without MB at 37°C.</p

Effects of respiratory inhibitors on cellular metabolism indicate increased AMP kinase activity, but decreased Ras1 signaling is independent of AMP kinase.

<p><b>(A)</b> Venn diagrams of significantly altered metabolites of cells treated with MB, PYO, or AA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s008" target="_blank">S1 Table</a>) showed that the majority of metabolites are changed similarly in cells treated with different respiratory inhibitors. Cells were grown for 24 h on YNBAGNP at 37°C. <b>(B)</b> Significant changes in pyruvate catabolism, sterol, fatty acid, and lyso-phospholipid levels in cells treated with MB, PYO, AA. <b>(C)</b> WT (SN250) and <i>snf4</i>/<i>snf4</i>, lacking a functional AMP kinase, grown for 24 h on YNBAGNP at 37°C (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s001" target="_blank">S1A Fig</a> panels 19 to 22) and analyzed for total and GTP-bound Ras1 levels and <b>(D)</b> intracellular ATP. (B) Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is shown. (D) Mean ± SD are shown. *p<0.05.</p

MB inhibits Ras1 activity and Ras1-dependent filamentation.

<p><b>(A)</b> Colonies of the wild type (<u><i>RAS1</i></u>/<i>RAS1</i>, SC5314), <i>ras1</i>/<i>ras1</i>, and <i>ras1</i>/<i>ras1</i>+<i>RAS1</i> were grown with vehicle or with 1.5 μM MB on YNBAGNP at 37°C for 24 h (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s001" target="_blank">S1A Fig</a> for microscopy images of cells from all colonies). Scale bar = 10 μm. <b>(B)</b> Expression levels of hypha-specific genes (HSG) and yeast-specific genes (YSG) with and without MB or Ras1 grown under conditions listed in (A). Nanostring analysis was used to determine transcript levels. <b>(C)</b> Model of the canonical Ras1 signaling pathway in <i>C</i>. <i>albicans</i>. Ras1 cycles between an active GTP- and an inactive GDP-bound state in response to the activities of the GEF (guanine nucleotide exchange factor) and GAP (GTPase-activating protein), Cdc25 and Ira2, respectively. GTP-Ras1 binds to the adenylate cyclase, Cyr1, and triggers cAMP production. cAMP activates protein kinase A (PKA) which results in the activation of hyphae specific genes resulting in the morphological switch from yeast to hyphae. <b>(D)</b> Analysis of the total Ras1 protein and GTP-Ras1 fraction of <i>C</i>. <i>albicans</i> strain CAF2 grown on YNBAGNP at 37°C (inducing) or YNBGP at 30°C (non-inducing) for 24 h with and without MB. Pma1 levels were shown as a loading control. Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is shown.</p

Ras1 signaling depends on total intracellular ATP levels.

<p><b>(A)</b> Intracellular ATP levels were measured in WT (CAF2) cells exposed to dinitrophenol (DNP), oligomycin (olig.) or vehicle (control). Mean ± SD are shown. *p<0.05. Cells were grown for 24 h on YNBAGNP at 37°C. <b>(B)</b> Colony images and total Ras1 and GTP-Ras1 western blot analysis of WT (CAF2) grown for 24 h on YNBAGNP at 37°C. <b>(C)</b> Western blot analysis and colony images of the <i>ssn3</i>/<i>ssn3</i> mutant compared to its reconstituted strain grown on YNBAGP at 30°C (yeast growth) for 24 h. (B) and (C) Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is shown.</p

MB effects on Ras1 signaling also occur in other <i>Candida</i> species.

<p><i>Candida parapsilosis</i> and <i>Candida tropicalis</i> were grown on YNBAGNP with and without 1.5 μM MB at 37°C for 24 h (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s001" target="_blank">S1A Fig</a> panels 9 to 12 for microscopy images of cells). Western blot analysis of total Ras1 and GTP-Ras1 levels are shown. Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is shown. Unlike <i>C</i>. <i>albicans</i>, these fungi grow as yeast in the absence and presence of MB.</p

GTP-Ras1 decrease by MB depends on the adenylate cyclase Cyr1.

<p><b>(A) (B) (C)</b> Colony morphology and western blot analysis of total Ras1 and GTP-Ras1 levels in a <i>cyr1</i>/<i>cyr1</i> mutant strain or a strain carrying a catalytically inactive <i>cyr1</i> allele (<i>cyr1</i>^<i>1334</i>) with MB or AA compared to their respective reconstituted strain are shown. Percent of the GTP-Ras1/total Ras1 ratio compared to control conditions is reported. Cells were grown for 24 h on YNBAGNP at 37°C. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005133#ppat.1005133.s001" target="_blank">S1A Fig</a> panels 25 to 32 for cellular morphology.</p

New model of the Ras1-Cyr1 signaling pathway.

<p>In the presence of MB or other respiratory inhibitors, cellular ATP levels are low. Under these conditions, GTP-bound Ras1 is rapidly turned over to GDP-bound Ras1 in a Cyr1-Ira2 dependent manner repressing the input of filamentation inducing signals and hyphal formation does not occur (upper panel). Under control conditions (no inhibition) ATP levels are high due to efficient oxidative metabolism and slow growth. In the presence of inducing signals GTP-bound Ras1 can accumulate probably because Cyr1 does not stabilize the interaction of Ras1 with Ira2. GTP-Ras1 can now effectively bind to and activate Cyr1 resulting in filamentation (lower panel). MDPs: muramyl dipeptides; HCO₃^¯: bicarbonate.</p

<i>Candida albicans</i> Ethanol Stimulates <i>Pseudomonas aeruginosa</i> WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

<div><p>In chronic infections, pathogens are often in the presence of other microbial species. For example, <i>Pseudomonas aeruginosa</i> is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with <i>Candida albicans</i> are common. Here, we show that <i>P. aeruginosa</i> biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus <i>C. albicans</i>. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted <i>P. aeruginosa</i> colonization of CF airway epithelial cells. <i>P. aeruginosa</i> production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by <i>C. albicans</i>. Using a <i>C. albicans adh1</i>/<i>adh1</i> mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of <i>P. aeruginosa</i> phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both <i>P. aeruginosa</i> and <i>C. albicans</i> has been associated with worse outcomes in cystic fibrosis.</p></div

<i>C. albicans</i> promotes <i>P. aeruginosa</i> strain PAO1 WT biofilm formation on airway epithelial cells in part through ethanol production.

<p><i>P. aeruginosa</i> PAO1 WT was cultured with a monolayer of ΔF508 CFTR-CFBE cells alone or with <i>C. albicans</i> CAF2 (reference strain), the <i>C. albicans adh1/adh1</i> mutant (<i>adh1</i>), and its complemented derivative, <i>adh1/adh1+ADH1</i> (<i>adh1-R</i>). Data are combined from three independent experiments with 3–5 technical replicates per experiment, (* represents a statistically significant difference (p<0.05) between indicated strains). Error bars represent one standard deviation.</p