PQS-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa

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 anti-biofilm 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-to-cell communication, virulence factor delivery, and the 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. Our group has shown that the Pseudomonas Quinolone Signal (PQS) induces OMV formation in P. aeruginosa, and in other species, through a biophysical mechanism that is also active 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 could be 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. PQS has been implicated in early biofilm development, but we hypothesized that its function as an OMV inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and that effective biofilm dispersion is dependent on production of PQS-induced OMVs, which likely act as delivery vehicles for matrix degrading enzymes. These findings lay the groundwork for understanding the roles of OMVs in biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.


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PQS-induced OMV production has been shown to be driven by a biophysical mechanism that is 246 not signaling dependent (31-33). The exogenous addition of PQS to a ΔpqsR biofilm restored 247 dispersion to wild type levels (One-way ANOVA, Tukey's post-hoc test, p = 0.72) (Fig. 6).

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Microcolony void formation increased from 60.65 ± 3.12% to 77.09 ± 6.94% (One-way ANOVA, 249 Tukey's post-hoc test, p = 0.024) (Fig. 6). This indicates that PQS modulates dispersion using an 250 OMV-dependent mechanism that is separate from the PQS signaling network. milk, tributyrin, and DNA to assess protease, lipase, and DNase activity, respectively. In order to 263 acquire sufficient material for these analyses, planktonic OMVs were used. Addition of OMVs to 264 skim milk agar resulted in the formation of a 119.8 ± 36.1 mm 3 zone of clearing, while the addition 265 of vehicle control (MV buffer only) to skim milk agar resulted in the formation of a 0.1 ± 8.6 mm 3 266 zone of clearing (Student's two-tailed t-test, p = 0.0007) (Fig. 7A). This suggests that OMVs 267 contain enzymes that have protease activity. The addition of OMVs to tributyrin agar resulted in 268 the formation of a 211.1 ± 24.1 mm 3 zone of clearing versus the vehicle control that produced a 269 25.9 ± 11.2 mm 3 zone of clearing (Student's two-tailed t-test, p < 0.0001) (Fig. 7B). This suggests 270 that OMVs also contain enzymes that have lipase activity. Finally, the addition of OMVs and 271 vehicle control to DNase agar resulted in the formation of 182.1 ±85.5 mm 3 and 21.3 ±16.3 mm 3 272 zones of clearing, respectively (Student's two-tailed t-test, p = 0.010) (Fig. 7C). This indicates that 273 OMVs carry enzymes with DNase activity. Overall, these data support the idea that OMVs abilities.

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Biofilms have become a major health and economic concern due to their prevalence and  Fig. 1 and Fig. 2). High levels of OMV production during these initial stages measured by Lowry assay were not corroborated by nanoparticle tracking analysis, suggesting that the protein detected in these OMV preparations was not 299 representative of OMV concentration but likely the result of non-OMV-related protein 300 components. As a result, we predicted that PQS and OMVs were not significant effectors of 301 reversible and irreversible attachment. This notion was supported by our crystal violet attachment 302 assays, which demonstrated that ΔpqsA, ΔpqsH, ΔpqsR, and ΔpqsE mutants had wild type levels 303 of reversible attachment (Fig. 3) production inhibited bacterial attachment to plant surfaces, increased bacterial motility, and 309 enhanced plant mortality (86). In the face of these conflicting reports, it is interesting that we found 310 the pqsA mutant had increased irreversible attachment versus wild type at 24 hours (Fig. 3A).

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During early biofilm development attachment is required. Therefore, it might be beneficial for P. 312 aeruginosa to reduce PQS production at this time to avoid potential interference of PQS-induced 313 OMVs with cell attachment. Regardless, it is evident that the role of OMVs in early-stage biofilm 314 development remains unclear and will require further studies to elucidate.

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During maturation I and II, we saw that both PQS and OMV production were relatively low ( showed that enzymatic degradation of PQS resulted in increased iron availability and enhanced biofilm formation for early and mature biofilms (87). The latter report aligns with our observations 321 and offers an explanation as to why cells might reduce PQS production during biofilm maturation.

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It is important to note that although PQS production was reduced during maturation in our study, 323 it was not eliminated. The same was true for OMV production. It is likely that baseline levels of activity, and that these OMVs can associate with and lyse bacterial sacculi (47). These findings 352 support the proposition that OMVs carry degradative enzymes. Here, we report that purified 353 OMVs possess protease, lipase, and DNase activity (Fig. 7). A recent study by Esoda and Kuehn 354 also found that OMVs traffic the P. aeruginosa peptidase, PaAP, and can deliver the peptidase to and Sauer showed that eDNA degradation is required for dispersion of P. aeruginosa (72). In P. 360 acnes, secreted lipases have also been demonstrated to enhance the dispersion response (93).

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Delivery of these degradative enzymes using OMVs may increase the enzymes' efficacy, facilitate 362 specific targeting to sites of degradation, and reduce potential deactivation of the enzymes while

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Strains, growth conditions, and media 385 All experiments were carried out using P. aeruginosa strains described in Table 1. The ΔpqsE and 386 ΔpqsR clean-deletion mutant strains were constructed using the pEX18gm suicide vector (97), and vector (98). Primer sequences used for construction of the vectors can be found in Table S1. 389 Biofilm tube reactors were inoculated as described below. Planktonic cultures were inoculated to 390 an OD600 of 0.01 and grown at 37 ℃ with shaking at 250 rpm. Planktonic cultures were grown in 391 Lysogeny Broth (LB) or brain heart infusion medium (BHI). Planktonic cultures of strains carrying 392 the pJN105 vector were grown in the presence of gentamicin (50 µg/mL), while biofilm cultures 393 of the same strains were not.    453 Biofilms were grown as described above for up to 7 days, and native dispersion was assessed as 454 previously described (9, 67). Briefly, biofilm microcolonies were observed by transmitted light 455 using an Olympus BX60 microscope and a 20 × UPlanF Olympus objective. Images were captured 456 using a ProgRes CF camera (Jenoptik, Jena, Thuringia, Germany) and processed with ProgRes microcolonies that had developed an interior void. For each biological replicate, biofilms were 459 grown in 2 to 4 wells of a 24-well plate, and all microcolonies that had formed in these biofilms 460 were analyzed for dispersion. The total number of microcolonies analyzed for each strain and 461 condition are presented in Supplemental table 2. 463 In order to acquire enough material for enzymatic analysis, OMVs were harvested from planktonic 464 cultures as described above. OMV preparations were quantified using NTA and diluted to 2×10 11 465 particles/mL in MV buffer. 180 μL of OMVs were then added to wells punched in agar using a 466 method described previously (93). Agar plates impregnated with protein, lipid or DNA were 467 prepared, and wells were punched within the agar using the wide end of a 1000 μL pipette tip.