Conditional antagonism in co-cultures ofPseudomonas aeruginosaandCandida albicans: An intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

Pseudomonas aeruginosaandCandida albicansare opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here, we describe the role of ethanol in mixed-species co-cultures by dual-seq analyses.P.aeruginosaandC.albicanstranscriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producingC.albicansor aC.albicansmutant lacking the primary ethanol dehydrogenase, Adh1. Analysis of the RNA-Seq data using KEGG pathway enrichment and eADAGE methods revealed severalP.aeruginosaresponses toC.albicans-produced ethanol including the induction of a non-canonical low-phosphate response regulated by PhoB.C.albicanswild type, but notC.albicans adh1 Delta/Delta, inducesP.aeruginosaproduction of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells and exhibits antifungal activity. Here, we show thatC.albicans adh1 Delta/Delta no longer activatesP.aeruginosaPhoB and PhoB-regulated phosphatase activity, that exogenous ethanol complements this defect, and that ethanol is sufficient to activate PhoB in single-speciesP.aeruginosacultures at permissive phosphate levels. The intersection of ethanol and phosphate in co-culture is inversely reflected inC.albicans;C.albicans adh1 Delta/Delta had increased expression of genes regulated by Pho4, theC.albicanstranscription factor that responds to low phosphate, and Pho4-dependent phosphatase activity. Together, these results show thatC.albicans-produced ethanol stimulatesP.aeruginosaPhoB activity and 5-MPCA-mediated antagonism, and that both responses are dependent on local phosphate concentrations. Further, our data suggest that phosphate scavenging by one species improves phosphate access for the other, thus highlighting the complex dynamics at play in microbial communities. Author summary Pseudomonas aeruginosaandCandida albicansare opportunistic pathogens that are frequently isolated from co-infections. Using a combination of dual-seq transcriptomics and genetics approaches, we found that ethanol produced byC.albicansstimulates the PhoB regulon inP.aeruginosaasynchronously with activation of the Pho4 regulon inC.albicans. We validated our result by showing that PhoB plays multiple roles in co-culture including orchestrating the competition for phosphate and the production of 5-methyl-phenazine-1-carboxylic acid; theP.aeruginosaphenazine response toC.albicans-produced ethanol depends on phosphate availability. The conditional stimulation of antifungal production in response to sub-inhibitory concentrations of ethanol only under phosphate limitation highlights the importance of considering nutrient concentrations in the analysis of co-culture interactions and suggests that the low-phosphate response in one species influences phosphate availability for the other.