Conditional antagonism in co-cultures of Pseudomonas aeruginosa and Candida albicans: an intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here we describe the focal role of ethanol in mixed-species co-cultures by dual RNA-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analyses using KEGG-pathways and the previously published eADAGE method revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low phosphate response mediated by PhoB. C. albicans wild-type, but not C. albicans adh1Δ/Δ, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells. We first demonstrate that PhoB is required for this interaction and that PhoB hyperactivity, via deletion of pstB, leads to increased production of 5-MPCA even when phosphate concentrations are high, but only in the presence of ethanol. Second, we show that ethanol is only sufficient to promote 5-MPCA production at permissive phosphate concentrations. The intersection of ethanol and phosphate in co-culture is mirrored in C. albicans; the adh1Δ/Δ mutant had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate which we confirmed by showing the adh1Δ/Δ strain had elevated Pho4-dependent phosphatase activity. The dual-dependence on ethanol and phosphate concentrations for anti-fungal production highlights how environmental factors modulate microbial interactions and dictate antagonisms such as those between P. aeruginosa and C. albicans.Author SummaryPseudomonas aeruginosa and Candida albicans are opportunistic pathogens that are frequently isolated from co-infections. Using a Dual-Seq approach in combination with genetics approaches, we found that ethanol produced by C. albicans stimulates the PhoB regulon in P. aeruginosa asynchronously with activation of the Pho4 regulon in C. albicans. In doing so, we demonstrate that eADAGE-based analysis can improve the understanding of the P. aeruginosa response to ethanol-producing C. albicans as measured by transcriptomics: we identify a subset of PhoB-regulated genes as differentially expressed in response to ethanol. We validate our result by showing that PhoB is necessary for multiple roles in co-culture including the competition for phosphate and the production of 5-methyl-phenazine-1-carboxylic acid, and that the P. aeruginosa response to C. albicans-produced ethanol depends on phosphate availability. The conditional stimulation of virulence 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.