Project description:Across the tree of life, clonal populations-from cancer to chronic bacterial infections - frequently give rise to subpopulations with different metabolic phenotypes. Metabolic exchange or cross-feeding between subpopulations can have profound effects on both cell phenotypes and population-level behavior. In Pseudomonas aeruginosa, subpopulations with loss-of-function mutations in the lasR gene are common. Though LasR is often described for its role in density-dependent virulence factor expression, interactions between genotypes suggest potential metabolic differences. The specific metabolic pathways and regulatory genetics enabling such interactions were previously undescribed. Here, we performed an unbiased metabolomics analysis that revealed broad differences in intracellular metabolomes, including higher levels of intracellular citrate in LasR- strains. We found that while both strains secreted citrate, only LasR- strains, consumed citrate in rich media. Elevated activity of the CbrAB two component system which relieves carbon catabolite repression enabled citrate uptake. Within mixed genotype communities, we found that the citrate responsive two component system TctED and its gene targets OpdH (porin) and TctABC (transporter) required for citrate uptake were induced and required for enhanced RhlR signalling and virulence factor expression in LasR- strains. Enhanced citrate uptake by LasR- strains eliminates differences in RhlR activity between LasR+ and LasR- strains thereby circumventing the sensitivity of LasR- strains to quorum sensing controlled exoproducts. Citrate cross feeding also induces pyocyanin production in LasR- strains co-cultured with Staphylococcus aureus, another species known to secrete biologically-active concentrations of citrate. Metabolite cross feeding may play unrecognized roles in competitive fitness and virulence outcomes when different cell types are together.ImportanceCross-feeding can change community composition, structure and function. Though cross-feeding has predominantly focused on interactions between species, here we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes of Pseudomonas aeruginosa. Here we illustrate an example of how such clonally-derived metabolic diversity enables intraspecies cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa, was differentially consumed between genotypes, and this cross-feeding induced virulence factor expression and fitness in genotypes associated with worse disease.

Project description:UnlabelledIn Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections.ImportancePseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed in lasR mutants. Furthermore, we show that Anr regulates the production of several different secreted factors in lasR mutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections.

Dataset Information

Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Publications

<i>Pseudomonas aeruginosa lasR</i> mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

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