The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.

Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion. Previous studies reported differences in size and proteomic cargo between planktonic and agar plate biofilm OMVs, suggesting possible differences in function between OMV types. In Pseudomonas aeruginosa interstitial biofilms, extracellular vesicles were reported to arise through cell lysis, in contrast to planktonic OMV biogenesis that involves the Pseudomonas Quinolone Signal (PQS) without appreciable autolysis. Differences in biogenesis mechanism could provide a rationale for observed differences in OMV characteristics between systems. Using nanoparticle tracking, we found that P . aeruginosa PAO1 planktonic and biofilm OMVs had similar characteristics. However, P . aeruginosa PA14 OMVs were smaller, with planktonic OMVs also being smaller than their biofilm counterparts. Large differences in Staphylococcus killing ability were measured between OMVs from different strains, and a smaller within-strain difference was recorded between PA14 planktonic and biofilm OMVs. Across all conditions, the predatory ability of OMVs negatively correlated with their size. To address biogenesis mechanism, we analyzed vesicles from wild type and pqsA mutant biofilms. This showed that PQS is required for physiological-scale production of biofilm OMVs, and time-course analysis confirmed that PQS production precedes OMV production as it does in planktonic cultures. However, a small sub-population of vesicles was detected in pqsA mutant biofilms whose size distribution more resembled sonicated cell debris than wild type OMVs. These results support the idea that, while a small and unique population of vesicles in P . aeruginosa biofilms may result from cell lysis, the PQS-induced mechanism is required to generate the majority of OMVs produced by wild type communities.

Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

25Bacterial biofilms are major contributors to chronic infections in humans. Because they are 26 recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. 27 Identifying factors involved in biofilm development can help uncover novel targets and guide the 28 development of anti-biofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, 29 and hospital acquired infections, and is also associated with aggressive biofilm formation in the 30 lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa 31 virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been 32 associated with cell-to-cell communication, virulence factor delivery, and the transfer of antibiotic 33 resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, 34 little is known about their biogenesis and function in biofilms. Our group has shown that the 35 Pseudomonas Quinolone Signal (PQS) induces OMV formation in P. aeruginosa, and in other 36 species, through a biophysical mechanism that is also active in biofilms. Here, we demonstrate 37 that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, 38 PQS and OMV synthesis are significantly elevated during dispersion, compared to attachment and 39 maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in 40 their ability to disperse, but this phenotype could be rescued by genetic complementation or 41 exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade 42 extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-43 induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled 44 degradation of biofilm matrix components. 45 46 Importance 47 48biofilm infections from protected sessile communities into released populations that are orders-of-49 magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit 50 increased acute virulence and dissemination phenotypes. A thorough understanding of the 51 dispersion process is therefore critical before this promising strategy can be effectively employed. 52 PQS has been implicated in early biofilm development, but we hypothesized that its function as 53 an OMV inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs 54 are differentially produced during Pseudomonas aeruginosa biofilm development and that 55 effective biofilm dispersion is dependent on production of PQS-induced OMVs, which likely act 56 as delivery vehicles for matrix degrading enzymes. These findings lay the groundwork for 57 understanding the roles of OMVs in biofilm development and suggest a model to explain the 58 controlled matrix degradation that accompanies biofilm dispersion in many species. 60It has long been appreciated that biofilms contribute to a majority of bacterial infection...