<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>P. aeruginosa</i> Δ<i>wspR</i> shows loss of swarm repression in the presence of ethanol.

<p><i>P. aeruginosa</i> strain PA14 WT, Δ<i>wspR</i>, and Δ<i>wspR</i> strains containing either plasmid-borne <i>wspR</i> or the empty vector were analyzed on swarm medium with and without 1% ethanol (EtOH) and with 0.02% arabinose (to induce <i>wspR</i> expression in the complemented strain). Images are representative of at least 5 experiments for each strain.</p

<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

Ethanol represses swarming and stimulates biofilm formation by <i>P. aeruginosa</i>.

<p><b>A</b>. <i>P. aeruginosa</i> strain PAO1 attachment to the bottom of a polystyrene plastic well after 6 hours in medium with and without 1% ethanol (EtOH). <b>B</b>. <i>P. aeruginosa</i> strain PA14 attachment to plastic as assessed by quantification of microcolonies per field in wells containing medium with or without ethanol for 7 h. Error bars represent the standard deviation (p<0.01 as determined by a student's t-test, N = 12). <b>C</b>. <i>P. aeruginosa</i> strain PA14 swarming in the absence and presence of 1% ethanol. Images are representative of results in more than ten independent experiments.</p

Ethanol significantly increases <i>P. aeruginosa</i> strain PAO1 WT biofilm formation on airway cells.

<p><b>A</b>. PAO1 WT, Δ<i>wspR</i> or Δ<i>wspA</i> were co-cultured with a monolayer of ΔF508 CFTR-CFBE cells at an MOI of 30∶1 in medium with or without 1% ethanol and imaged after 6 h. Pictures are representative of at least 3 separate experiments with similar results. <b>B</b>. The number of CFUs from cultures determined as described above. Significance determination was based on an ordinary one-way ANOVA followed by Sidak's multiple comparisons test for each intrastrain comparison; ***, <i>P</i><0.001. Error bars represent one standard deviation.</p

Our proposed model for the impacts of fungally-produced ethanol on <i>P. aeruginosa</i> behaviors.

<p>Our previous work has shown that <i>P. aeruginosa</i> phenazines increase fungal ethanol production. Here, we show that ethanol stimulates the Wsp system, leading to a WspR-dependent increase in c-di-GMP levels and a concomitant increase in Pel production and biofilm formation on plastic and on airway epithelial cells. In addition, ethanol altered phenazine production by promoting 5MPCA release and the accumulation of PCN.</p

Ethanol leads to higher levels of PCN crystal formation and 5MPCA derivatives.

<p><b>A</b>. <i>P. aeruginosa</i> strain PA14 wild type (WT) was grown on medium without and with 1% ethanol. With ethanol, PCN crystals form and the colony has a yellowish color likely attributed to reduced PCN. <b>B</b>. <i>P. aeruginosa</i> strain PA14 WT was cultured on lawns of <i>C. albicans</i> CAF2 (WT reference strain), the <i>C. albicans adh1/adh1</i> mutant, and its complemented derivative (<i>adh1/adh1+ADH1</i>); the PA14 Δ<i>phz</i> mutant defective in phenazine production was plated on the <i>C. albicans</i> CAF2 for comparison.</p

Ethanol acts through the Wsp system.

<p><b>A</b>. Attachment of <i>P. aeruginosa</i> strain PAO1 WT, Δ<i>wspR</i> and Δ<i>wspA</i> mutants to the bottom of a polystyrene dish during growth in M63 medium with glucose and casamino acids, and with vehicle (Control) or with 1% ethanol for 6 hours. <b>B</b>. The number of cells per field for each condition was enumerated. Images and data are representative of results from more than three separate experiments. Significance determination was based on an ordinary one-way ANOVA followed by Sidak's multiple comparisons test for each intrastrain comparison; ***, <i>P</i><0.001. <b>C</b>. Δ<i>wspR</i> and Δ<i>wspFR</i> strains expressing WspR-E253A-YFP were grown without and with 1% EtOH, and the number of cells with fluorescent clusters were counted out of a total of approximately 100 cells examined per condition across two experiments.</p

Ethanol increases c-di-GMP levels in <i>P. aeruginosa</i> strain PA14 WT but not in a Δ<i>wspR</i> mutant.

<p>c-di-GMP levels from cultures grown on swarm plates without (black) or with 1% ethanol (grey) were measured by LC-MS. Error bars represent one standard deviation (*,p<0.05, N = 5); ns, not significant).</p