Enterococcus faecalis Antagonizes Pseudomonas aeruginosa Growth in Mixed-Species Interactions.

Enterococcus faecalis is often coisolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their coprevalence, the polymicrobial interactions of these two species in biofilms and under iron-restricted conditions remain unexplored. Here, we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to l-lactate production during fermentative growth. We find that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced under iron-restricted conditions, resulting in increased lactic acid exported and, consequently, a reduction in local environmental pH. Together, our results suggest that E. faecalis synergistically inhibits P. aeruginosa growth by decreasing environmental pH and l-lactate-mediated iron chelation. Overall, this study emphasizes the importance of the microenvironment in polymicrobial interactions and how manipulating the microenvironment can impact the growth trajectory of bacterial communities. IMPORTANCE Many infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored under iron-restricted conditions. Here, we explore the polymicrobial interactions between commonly coisolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron and also lowers the environmental pH to antagonize P. aeruginosa growth under iron-restricted conditions. Our findings provide insights into polymicrobial interactions between bacteria and how manipulating the microenvironment can be taken advantage of to better control infections.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule.

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4-diones, but was unable to inactivate the PQS precursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation.