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

Abstract

AbstractPseudomonas aeruginosaandCandida albicansare 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. 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. Analyses using KEGG-pathways and the previously published eADAGE method revealed severalP. aeruginosaresponses toC. albicans-produced ethanol including the induction of a non-canonical low phosphate response mediated by PhoB.C. albicanswild-type, but notC. albicans adh1Δ/Δ, inducesP. aeruginosaproduction 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 ofpstB,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 inC. albicans; theadh1Δ/Δ mutant had increased expression of genes regulated by Pho4, theC. albicanstranscription factor that responds to low phosphate which we confirmed by showing theadh1Δ/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 betweenP. aeruginosaandC. albicans.Author SummaryPseudomonas aeruginosaandCandida albicansare opportunistic pathogens that are frequently isolated from co-infections. Using a Dual-Seq approach in combination with genetics approaches, we found that ethanol produced byC. albicansstimulates the PhoB regulon inP. aeruginosaasynchronously with activation of the Pho4 regulon inC. albicans.In doing so, we demonstrate that eADAGE-based analysis can improve the understanding of theP. aeruginosaresponse to ethanol-producingC. albicansas 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 theP. aeruginosaresponse toC. 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.