Pseudomonas aeruginosa leucine aminopeptidase influences early biofilm composition and structure via vesicle-associated anti-biofilm activity

Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both non-self P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture. Importance Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug- resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially-secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight as to how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.


Importance 23
Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug-24 resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In 25 order to treat these infections more successfully, the mechanisms of bacterial biofilm 26 2 development must be elucidated. While both bacterially-secreted aminopeptidase and outer 27 membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the 28 contributions of each of these factors to the steps in biofilm generation have not been well 29 studied. This work provides new insight as to how these bacterial components mediate the 30 formation of a robust, drug-resistant extracellular matrix and implicates outer membrane 31 vesicles as active components of biofilm architecture, expanding our overall understanding of P. 32 aeruginosa biofilm biology. 33

Introduction 37
The Gram-negative bacterium Pseudomonas aeruginosa is a prominent opportunistic 38 pathogen capable of causing both acute and chronic disease in a variety of compromised hosts. 39 P. aeruginosa is most well known as a leading cause of morbidity and mortality in cystic fibrosis 40 (CF) patients 1 . In these individuals, the pathogen establishes chronic, biofilm-based infections 41 that may persist for decades in the unique, mucous-rich environment of the CF lung. In addition 42 to lung tissue, P. aeruginosa forms biofilms on a wide range of substrates relevant to human 43 infection, including corneal and skin tissue, as well as soil and water reservoirs and hospital 44 surfaces, which can contribute to infection initiation and spread 2 . 45 P. aeruginosa is considered a model organism for the study of biofilm formation, and 46 many of the cellular and matrix components contributing to this mode of growth have been 47 studied previously 3-5 . The process of forming biofilm communities has also been characterized, 48 revealing distinct growth phases. First the bacteria settle and attach onto a suitable host tissue 49 or abiotic surface, then they begin to form stationary microcolonies and secrete extrapolymeric 50 substance (EPS) to form a dense matrix. This matrix contains a variety of polysaccharides, with 51 Psl and Pel polysaccharides present in all strains, and alginate found in mucoid P. aeruginosa 52 isolates 6 . It also contains proteins, lipids, extracellular outer membrane vesicles (OMVs), and 53 extracellular DNA (eDNA) 4,5,7 ; however, the mechanisms by which many of these secreted 54 bacterial components specifically affect microcolony growth and development remain unclear. 55 As with other extracellular pathogens, P. aeruginosa secretes many of its virulence 56 determinants into the extracellular milieu to affect interactions with the host, including those in 57 the biofilm matrix. Interestingly, many of the virulence factors secreted specifically by P. 58 aeruginosa demonstrate proteolytic activities 8- 10 . These enzymes, including elastase, protease 59 IV, and alkaline protease, act on both bacterial and host proteins to directly impact host-60 pathogen interactions. For example, purified elastase has been found to cleave fibrin, laminin, 61 various immunoglobulins, and components of the complement system 11 . Their activity not only 62 8 164

qRT-PCR 165
Pellicle and coculture biofilms were grown for the indicated times as described. For coculture 166 biofilms, supernatants were removed and 1 mL of Trizol was added to each sample to lyse both 167 bacterial and A549 cells. For pellicle biofilms, 3 mL/well of cultures incubated in 6-well 168 polystyrene dishes (VWR) were used to obtain sufficient biofilm material for analysis over the 169 as outlined previously 28 ), LB+ 0.4% glucose, or tissue culture media (described above) with no 200 antibiotics. Cultures were grown to an OD 600 of 0.5 and diluted to OD 600 of 0.1. Aliquots of the 201 diluted culture (100 µL/well) were incubated in five 96-well flat bottom polystyrene dishes (VWR) 202 for 0, 2, 6, 10, or 24 h at 37°C with no shaking and evaluated using a static biofilm quantitation 203 assay 28 . Briefly, at the incubation endpoint, the plate was washed out using distilled water 204 (dH 2 0). After three washes, 150 µL of 1% aqueous Crystal Violet solution was added to each 205 well and the samples were incubated for 10 min at room temperature. The stained wells were 206 washed three times using dH 2 0, 200 µL of 30% aqueous acetic acid was added to solubilize the 207 dye, and the OD 495 was measured. 208 209

Pellicle biofilm imaging 210
Cultures were prepared as described above and diluted cultures were grown in 35 mm glass 211 bottom dishes for 5 h at 30°C. Media was removed and the pellicles were stained using 212 10 BacLight. The pellicles were imaged using a Zeiss 780 inverted confocal microscope and 213 biomass was calculated using Comstat 2.1. 214 215

Psl and matrix protein staining 216
To stain the biofilm extrapolymeric substance, 100 µg/mL HHA-FITC lectin (USA Biologicals) 217 was added to cocultures 2 h before the imaging timepoint, and the samples were incubated at 218 37°C with 5% CO 2 . Pellicle samples were treated with 100 µg/mL HHA-TRITC (USA Biologicals) 219 lectin for 2 h before the final imaging timepoint. For protein staining, pellicles were stained with 220 Ruby FilmTracer SYPRO Ruby Biofilm Matrix Stain (ThermoFisher Scientific) for 5 min before 221 imaging, as described in the reagent protocol. All samples were imaged using a Zeiss 780 222 inverted confocal microscope and staining was quantified using Comstat 2.1. 223

OMV isolation and supernatant fractionation 225
Bacterial cultures were grown overnight in LB at 37°C, diluted 1:100 into 1.5 L LB, and grown 226 (37°C, shaking) to OD 600 0.9-1.1. Cells were pelleted (10,000xg for 15 min 4°C), and 227 supernatants were collected and concentrated to 100 mL using tangential flow with a 10 kDa 228 samples were further concentrated using Millipore 10,000 MWCO centrifugal filters, and the 235 soluble proteins were separated from the OMVs using an iodixanol (Optiprep, Sigma) density 236 gradient. Concentrated proteins and OMVs were adjusted to 50% Optiprep/HEPES in 2 mL, and 237 applied to the bottom of the gradient tube, and subsequently layered with 2 mL of 40%, 2 mL of 238 35%, 4 mL of 30%, and 2 mL of 25% Optiprep/HEPES. The gradient was centrifuged overnight 239 at 40,000xg, 4°C in an ultracentrifuge (SW 40 Ti swing bucket rotor, Beckman Coulter). 240 Gradient fractions were removed from the top in 1 mL aliquots, and the lipid and protein content 241 for each fraction were identified using FM4-64 and Bradford assays as described previously 29 . 242 The OMV-containing fractions (typically 1-5) and soluble protein fractions (typically 7-12) were 243 pooled and diluted with HEPES, then centrifuged at 40,000xg for 2 h to pellet OMVs and 244 proteins and to remove Optiprep. Protein and OMV pellets were resuspended in HEPES, and 245 sterile-filtered (0.4 µm PVDF, VWR). 246 247 rPaAP purification 248 rPaAP expression, purification, and refolding were carried out as described previously 15 , and the 249 refolded protein was concentrated using 10,000 MWCO Amicon filter units (Millipore-Sigma). 250 The concentrated material was then dialyzed using the same method described above, applied 251 to a S200 Sephadex ion exchange column, eluted with a gradient of 0-2 M NaCl, pH 7.6, at a 252 flow rate of 0.02 mL/min, and 18-2 mL fractions were collected. Fractions were assayed for 253 leucine aminopeptidase activity as described 30 , and analyzed by SDS-PAGE and Ruby staining 254 as described above. Fractions containing pure rPaAP were pooled and concentrated using 255 Amicon 10,000 MWCO filter units to ~0.5 µg/mL. 256 257

Complementation and detachment experiments 258
For biofilm complementation assays, CFS, OMV fractions, soluble fractions, and rPaAP 259 samples were prepared as described above, total protein was measured by Bradford assay, and 260 aminopeptidase activity was measured as described above. To examine the hypothesis that PaAP impacts bacterial microcolony formation, we adapted a 291 previously published biofilm coculture assay for use with A549 lung epithelial cells 26  To first establish the coculture conditions for our biofilm assays in which we would use a 297 clinical P. aeruginosa isolate (S470 WT), the confluent A549 cells were inoculated with 298 fluorescently labeled S470 and incubated for up to 6 h, and the A549 cells were assessed for 299 damage and breaks in the cell layer. We established that this biotic substrate could be used to 300 investigate clinical P. aeruginosa strain biofilm development through the early stages (up to 5 h 301 post infection, hpi) of microcolony formation when LDH activity remained low (Fig S1) as 302 substantial breakdown of these cellular layers occurred after 6h. We noted that once these 303 cytotoxic bacteria infiltrated the host cell layer, the microcolony structure also broke down, as 304 the bacteria were able to colonize the underlying tissue culture dish and migrate to the air-liquid 305 interface. These results were consistent with the previously described toxicity of P. aeruginosa 306 clinical strains to host cells 31 and time courses for static coculture biofilm models 26 . In addition, 307 we confirmed that the bacterial colonies exhibited biofilm properties, as they displayed gene 308 expression patterns similar to those documented for biofilm-associated cells (Fig S2) as well as 309 increased antibiotic resistance when compared to planktonically grown bacteria (Fig S3). 310 To examine the effect of PaAP on P. aeruginosa biofilm microcolony growth, we 311 quantitatively compared cocultures of fluorescent S470 WT to S470 ∆PaAP, a previously 312 described isogenic knockout strain (S470 ∆PaAP) that contains a disruption of PA2939, the 313 gene encoding PaAP 24 . We observed significant differences between the two biotic biofilms at 5 314 hpi. Interestingly, the PaAP mutant strain formed biofilms with substantially greater cellular 315 biomass compared to the WT strain (Fig 1A, B). We also examined S470 WT and ∆PaAP 316 microcolony morphologies (Fig 1D, E). In addition to their greater quantitative biomass, S470 317 ∆PaAP colonies displayed greater cellular organization. Even at this early point in biofilm 318 development, characteristic mushroom-shaped colony structures were observed for the PaAP 319 mutant. The S470 WT microcolonies, by comparison, showed very little cellular organization 320 and were packed less densely. Based on these data, we can conclude that the lack of PaAP not 321 only increases cellular biomass, but also cellular organization at the microcolony level during 322 early coculture biofilm development. 323 To confirm this PaAP-dependent phenotype in a different strain background, we 324 compared the biofilm characteristics of PAO1, a laboratory P. aeruginosa strain commonly 325 studied in vitro, and the isogenic PAO1 ∆PaAP strain that harbors a transposon insertion in 326 PA2939. We note that our lab previously showed that OMVs isolated from PAO1 cultures show 327 very low aminopeptidase levels compared to S470 and other clinical P. aeruginosa isolates 13 . 328 Nevertheless, as with the clinical strain phenotype, PAO1 ∆PaAP formed significantly more 329 biomass as compared to the WT counterpart (Fig 1A, C). Our observations indicate that PaAP 330 is important to biofilm development, even in strains with low endogenous PaAP expression, and 331 that its role may be relevant to a broad range of P. aeruginosa strains. 332

Increased biofilm cellular density induced by PaAP deletion coincides with PaAP 334 expression and increased EPS production 335
To detail the time course of the PaAP-dependent phenotype, we imaged the cocultures 336 at hourly intervals post infection. Directly following the wash step at 1 hpi and up to 3 hpi, we 337 observed a similar level of biomass from the S470 WT and ∆PaAP strains. A significant 338 phenotype did not develop until 4 hpi (Fig 2A), suggesting that the aminopeptidase is critical in 339 limiting microcolony growth and development rather than during the initial steps of colonization 340 and attachment. Based on this timeline, it is expected that the observed phenotype would 341 develop in parallel with the level of aminopeptidase expression. Indeed, previous reports found 342 PaAP to be regulated by the las quorum sensing system 14,32 , and thus PaAP expression would 343 be expected to increase as bacterial density increases in our experimental system. Using qRT-344 PCR, we examined the expression of PaAP in S470 WT biofilms over time under the coculture 345 assay conditions. Consistent with quorum sensing density-dependent expression, our results 346 show expression of PaAP mRNA at 1 hpi, increasing steadily over the next several hours (Fig  347   S4) and PaAP protein in cocultures at the 5 hpi time point (Fig 2B). Based on these data, it 348 appears that the biofilm limiting phenotype may depend on either a threshold level of 349 aminopeptidase or a specific microcolony developmental phase. 350 Bacterial fluorescence images from the coculture assay in Fig 1 revealed significant  351 density and structural differences between the biofilms formed by WT and ∆PaAP P. aeruginosa 352 strains, but we were also interested in whether PaAP affected the composition of the biofilm 353 matrix. The P. aeruginosa matrix is composed of polysaccharides, proteins, DNA, and vesicles 354 secreted by cells in the microcolony. We imaged and quantitated the polysaccharide content of 355 the matrix using a lectin specific for Psl, a major component of non-mucoid matrices. As seen in 356 Fig 2C and 2D, the S470 microcolonies contained significantly more Psl per cellular biomass. 357 This also correlated with a modest, but significant increase in colistin resistance (Fig 2E), on a late-stage phenotype, they also observed that PaAP deletion led to increased biomass in 373 early biofilms. By growing the bacteria planktonically in Jensen's media to mid-log phase and 374 diluting and inoculating the culture containers, we were able to replicate this phenotype with the 375 S470 clinical strain and corresponding PaAP deletion mutant (Fig 3A, B, C, Untreated). In 376 Jensen's media, the ∆PaAP strain formed pellicles with significantly greater cellular biomass. 377 Interestingly, this phenotype was not found with cultures grown in other media used frequently 378 in biofilm studies (M63 minimal media and LB + glycerol) or the cell culture media used in the 379 coculture experiments (both fresh and conditioned F-12K) (Fig S5). These data lead us to 380 conclude that PaAP-dependent effects in early biofilms are growth-condition and nutrient 381 specific. 382 To examine whether PaAP affected the matrix composition of abiotic biofilms, pellicles 383 grown in Jensen's media were stained for Psl. As with the coculture biofilms, the S470 WT 384 pellicles consisted of significantly greater exopolysaccharide per cellular biomass as compared 385 to ∆PaAP pellicles (Fig 3D, Untreated). We were also able to stain for matrix protein content 386 using this model since the background protein levels were much lower as compared to the 387 coculture biofilms. Compared to the ∆PaAP strain, the WT pellicles exhibited a significantly 388 lower protein to Psl ratio (Fig 3E, Untreated), strengthening our conclusion that PaAP 389 expression can cause substantial changes in matrix composition of early biofilms under the 390 stated growth conditions. 391 392

PaAP + OMVs, but not soluble aminopeptidase, exhibit anti-biofilm activity 393
Complementation of the PaAP deletion phenotype was pursued next. Based on its 394 tightly-controlled, quorum sensing-regulated expression and secretion pathways, PA2939 395 complementation using plasmid-based expression has proven to be unfeasible. We therefore 396 performed biochemical complementation experiments to test the ability of secreted PaAP to 397 reconstitute the original biofilm composition in ∆PaAP biofilms. Complementation was tested 398 using cell-free supernatants (CFS) from mid-log phase cultures of S470 WT and S470 ∆PaAP 399 after confirming the respective presence and absence of PaAP activity in the S470 WT and 400 S470 ∆PaAP CFS by Western blot (Fig 4A) and aminopeptidase activity assays (Fig S6). 401 Equivalent amounts (by total protein) of CFS were added to washed S470 ∆PaAP coculture 402 biofilms 1 hpi, and the cocultures imaged 5 hpi. Addition of the PaAP-containing S470 WT CFS 403 complemented the phenotype of ∆PaAP biofilms to WT biomass levels, while the S470 ∆PaAP 404 CFS resulted in an insignificant change (Fig 4B). We concluded that P. aeruginosa CFS 405 harbors a PaAP-dependent biofilm modulating activity. 406 We wondered if secreted PaAP itself was responsible for the activity and recalled that 407 after secretion by the type II secretory system, PaAP is found both associated with OMVs and 408 as a soluble protein 24 . Therefore, to locate the source of the activity in the coculture 409 supernatants, soluble proteins and OMV-bound factors were separated using iodixanol density 410 gradients, and consistent with our previous reports 13 , aminopeptidase activity was detected in 411 both the light-and heavy-density fractions, representing the OMV-bound and soluble forms of 412 PaAP (sPaAP), respectively. Notably, whereas treatment with the OMV-containing S470 WT 413 fractions was able to significantly inhibit cellular biomass development by S470 ∆PaAP and 414 partially restore relative polysaccharide levels in these biofilms, the heavier, sPaAP-containing 415 fractions had no noticeable effect on biomass (Fig 4C, Fig S7). As expected, neither treatment 416 at 1 hpi of OMV-containing or heavy fractions of the CFS from S470 ∆PaAP cultures altered the 417 ∆PaAP coculture biomass at 5 hpi (Fig 4C). PaAP + OMVs, but not ∆PaAP OMVs, were also 418 able to complement the biofilm phenotype observed in Jensen's media pellicles and could 419 restore matrix composition in these samples (Fig 3A, C-E, Treated). These data suggested 420 OMVs were responsible for the biofilm modulating activity in a PaAP-dependent manner, and 421 that solubly secreted PaAP was not. 422 To confirm that aminopeptidase alone was not capable of restoring these phenotypes, 423 we purified and refolded recombinant PaAP (rPaAP) expressed in E. coli 15 . Consistent with the 424 results using sPaAP, purified rPaAP was also incapable of disrupting the formation of coculture 425 biofilms by the ∆PaAP strain (Fig 4D). Finally, we tested whether addition of rPaAP or sPaAP 426 could reconstitute the biofilm inhibitory activity of the OMVs isolated from ∆PaAP cultures 427 (∆PaAP OMVs). These rPaAP/∆PaAP OMV and sPaAP/∆PaAP OMV preparations also failed to 428 inhibit biotic biofilm development by the S470 ∆PaAP strain (Fig 4D). These data lead us to 429 conclude that OMVs derived from a PaAP + strain, mediate cell aggregation and matrix 430 composition at early stages of biofilm microcolony development. The fact that ∆PaAP OMVs 431 and PaAP alone or in combination with ∆PaAP OMVs were unable to limit biofilm development 432 additionally suggests that the aminopeptidase is not directly causing changes in biofilm 433 architecture, but rather modifying OMV activity and thereby indirectly changing the biofilm 434 microcolonies. 24  In order to characterize the biofilm-remodeling mechanism of the OMVs, we studied the 439 kinetics of the vesicle-mediated anti-biofilm activity. OMVs were added to cocultures either 440 before inoculation (pre-treatment), at 1 hpi, or at 4.5 hpi, (0.5 h before the final imaging time 441 point). Table 1  To test whether PaAP + vesicles could disrupt preformed microcolony structures, OMVs 448 were not added to the A549/S470 ∆PaAP cocultures until 4.5 hpi, and the biofilms imaged 30 449 minutes later. As seen in Fig 5A and B, this brief PaAP + OMV treatment resulted in a significant 450 and very substantial decrease in biomass of a preformed biofilm. To examine whether this 451 biofilm reducing activity was specific to S470 strain-derived vesicles, we purified and tested 452 OMVs derived from the PAO1 and PA14 P. aeruginosa WT strains and their corresponding 453 ∆PaAP mutants. WT OMVs from both strains disrupted the preformed S470 ∆PaAP coculture 454 biofilms whereas OMVs from the isogenic mutants did not (Fig 5A, B). OMVs also mediated 455 biofilm reduction for S470 pellicles grown in Jensen's media but not for pellicles that did not 456 show the original phenotype (e.g. those grown in M63 minimal media or tissue culture cell 457 media) (Fig S8). 458 During respiratory infections biofilms are often found as polymicrobial communities, and 459 components secreted by one species can impact the microcolonies of other strains or species in 460 nearby environments. To determine whether the anti-biofilm activity of the OMVs is species-461 specific, A549 cell layers were inoculated with K. pneumoniae, a bacterium which is often found 462 20 with P. aeruginosa in cases of ventilator-associated pneumonia 34 . S470 WT or ∆PaAP OMVs 463 were added at 4.5 hpi, and the cocultures were stained at 5 h with Congo Red to examine both 464 cellular and matrix biomass. As seen in (Fig 5C) susceptibility; however, cells undergoing a signaled dispersal response will lose this resistance 474 as they gain motility and release from the biofilm colonies. To characterize which of these 475 occurs during vesicle-mediated cellular release from coculture biofilms, the released cells were 476 collected and assayed for colistin resistance (Fig 6A). These cells displayed significantly higher 477 antibiotic resistance compared to planktonically grown cultures, suggesting that the mechanism 478 behind OMV-mediated biofilm disruption and remodeling likely involves the detachment of cells 479 or cellular aggregates, rather than a programmed dispersal response. Known biofilm-detachment factors include not only polysaccharide hydrolases, but also 490 DNase, Proteinase K and various endogenous P. aeruginosa proteases 36 . Based on the 491 importance of endogenous proteases to biofilm development, we tested PaAP + and ∆PaAP 492 OMVs for overall protease activity. Interestingly, we found that PaAP + OMVs display higher 493 protease activity, which cannot be directly accounted for by the presence of aminopeptidase in 494 these samples, as demonstrated by the inclusion of an rPaAP control matched to 495 aminopeptidase activity levels in the PaAP + vesicles (Fig 6C). By pre-incubating the vesicle 496 samples with a protease inhibitor (PI) cocktail, we were able to eliminate both protease and anti-497 biofilm activity in the PaAP + OMVs (Fig 6C, D) without impacting aminopeptidase activity in the 498 samples (Fig S9). While future work will be required to fully elucidate the proteases involved in 499 this phenotype, these results suggest that PaAP expression in P. aeruginosa increases 500 endogenous protease activity that becomes localized to secreted OMVs, which can in turn lead 501 to OMV-induced cell detachment and contribute to remodeling of overall biofilm architecture. 502 503

Discussion 504
We set out to investigate the role of the PaAP leucine aminopeptidase in biofilm 505 development, as PaAP is one of the most abundant proteins in the P. aeruginosa biofilm 506 extracellular matrix and one of the major protein components of OMVs from clinical P. 507 aeruginosa isolates 13,20 . When we compared coculture biofilms of wild-type P. aeruginosa and a 508 PaAP deletion mutant, we found the mutant developed early biofilms with a significantly higher 509 cellular biomass and a lower concentration of the matrix exopolysaccharide Psl, a phenotype 510 confirmed in specific pellicle biofilm growth conditions. The mutant phenotype included a less 511 robust biofilm architecture that rendered the cellular community more susceptible to the 512 antibiotic colistin. We further found that biofilm detachment activity resided in OMVs in 513 22 supernatant fractions from wildtype cultures, but not OMVs secreted by ∆PaAP cells. The effect 514 of PaAP expression was indirect, as neither recombinant nor purified soluble aminopeptidase 515 alone or in combination with ∆PaAP OMVs possessed detachment activity. Our data further 516 lead us to conclude that PaAP influences changes in biofilm architecture through proteolytic 517 activity, as WT OMVs exhibited higher proteolytic activity than ∆PaAP OMVs, and WT OMV-518 mediated detachment was sensitive to protease inhibition. OMVs have been implicated in the 519 process of biofilm formation 7,19 , but while their presence in these bacterial communities has 520 been documented, active roles for these particles and the bacterial products they carry have not on the involvement of PslG in biofilm disruption, which was also, intriguingly, PaAP-dependent 531 33 . However, in this case, biofilm disruption was found to take place in late log-stage biofilms 532 under nutrient-limiting conditions such that the lack of PaAP lead to cell lysis, releasing the 533 periplasmic PslG enzyme. By contrast, the biofilm phenotype we observed not only does not 534 depend on starvation, it is mediated by OMVs and is independent of PslG expression. 535 Additionally, AlgL and rhamnolipids, which have biosurfactant properties that can cause the 536 release of biofilm cells, were shown not to be involved in this effect. These results demonstrate 537 a distinct role for both PaAP and OMVs in the modulation of biofilm communities. 538

23
Other described mechanisms to influence bacterial detachment involve protease activity, 539 which is important in many aspects of P. aeruginosa biology 38 . Previous studies have implicated 540 endogenous proteases in biofilm detachment and the dissolution of cellular aggregates 39 , 541 specifically those with a protein rich matrix. Based on our data we can add new details to this 542 model: endogenous PaAP expression helps regulate protease activity in secreted vesicles 543 which, in turn, lead to the remodeling of biofilm architecture (Fig 7). The changes induced by 544 OMVs ultimately increase Psl/biomass ratios in early biofilm matrices and help to protect the 545 developing colony from disruption by antimicrobials. As for the specific protease(s) and 546 substrate(s) involved in his mechanism, protease expression and activation during P. 547 aeruginosa development is a highly complex, regulated process with many steps, including 548 expression, secretion, and processing. Additionally, many of these proteases are involved in the 549 activation and expression of others 15,38,40 . This complex system makes it challenging to specify 550 which protease(s) are affected by PaAP expression, whether this is the effect of one or several 551 endogenous proteases, and how PaAP increases vesicle-associated protease activity. The 552 protein substrate(s) for the OMV-activity are also unknown and also require further study, 553 however one interesting observation is that both early biofilms of P. aeruginosa and K. 554 pneumonia contain a substrate for the activity. 555 While some aminopeptidases have been implicated in pathogenesis phenotypes 41 , these 556 enzymes are generally thought to act during starvation responses, in which they can provide 557 necessary amino acids for cellular processes. This study, by contrast, presents a novel role for 558 a bacterial aminopeptidase, as PaAP impacts early biofilm formation processes early in 559 development and under nutrient-rich conditions. Additionally, because we have shown that this 560 phenotype is not directly caused by the vesicle-associated aminopeptidase, but rather other 561 vesicle-associated proteases, we have implicated PaAP in a more complex regulatory network 562 that influences the activity of bacterial molecules and structures important to the formation of 563 24 robust biofilm structures. Future work on this project will seek to elucidate the mechanism by 564 which PaAP influences OMV-mediated biofilm maturation.