The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram‐negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.


INTRODUCTION
All living organisms release extracellular vesicles as part of their normal growth [1]. In Gram-negative bacteria, extracellular vesicles were initially identified as being produced when a portion of the outer membrane was liberated from the bacterial cell resulting in the production of outer membrane vesicles (OMVs) [2]. It is now well established that all Gram-negative bacteria produce OMVs, and their production appears to occur via a conserved mechanism known as budding [3,4]. OMV release by budding occurs when the outer membrane of the bacterium bulges out due to changes in the composition or structure of the outer membrane, resulting in a part of the membrane budding off to form an OMV that is then released from the bacterium [3,5,6]. There are various known factors that can increase bacterial production of OMVs, which include protein modifications, lipid remodeling, and external stress factors [7]. More recently, a novel mechanism of OMV biogenesis was discovered in Pseudomonas aeruginosa and subsequently in Escherichia coli and Shewanella vesiculosa, known as explosive cell lysis [8][9][10]. In P. aeruginosa, explosive cell lysis can occur during conditions of bacterial stress and is mediated by cryptic prophage tailocins located in the R-and F-type tailocin gene cluster in the P. aeruginosa genome [8]. Induction of explosive cell lysis causes the rounding of P. aeruginosa which eventually explode, releasing cellular components that are then packaged within membrane fragments as they fuse to create new membrane vesicles (MVs) [8]. Furthermore, it is now understood that Gramnegative bacteria can release MVs consisting of either a single membrane as a result of blebbing from the bacterial outer membrane or containing both an outer and inner membrane bilayer which are referred to as outer inner membrane vesicles (OIMVs) and are produced as a result of bacterial cell lysis [4,[11][12][13]. Therefore, Gram-negative bacteria produce MVs predominately via two main mechanisms of biogenesis, involving either their release from the outer membrane during normal growth via budding, or their formation as a result of explosive cell lysis, and we collectively refer to all of these types of vesicles as OMVs.
Bacterial OMVs, irrespective of their mechanism of biogenesis, can package a range of biological cargo originating from their parent bacterium. Specifically, OMVs can package DNA [14], RNA [15], and proteins [16] and can therefore act as a delivery vehicle for transporting bacterial material to the surrounding environment [17], neighboring cells [18], and to host cells [19][20][21][22][23]. To better understand the biological functions of OMVs, the proteome of OMVs from a range of pathogenic bacteria has been investigated revealing that OMVs contain a range of virulence proteins [24][25][26][27], and can therefore contribute to mediating pathogenesis in the host [28,29]. More recently, it has been identified that bacteria can alter the protein composition of their OMVs to ultimately modify their functions. For example, bacteria can selectively enrich the packaging of virulence proteins into OMVs to increase their targeted delivery into host cells [30]. In addition, the proteome of OMVs can be altered by a range of factors such as nutrient availability [31], antibiotic stress [32], and planktonic versus biofilm

Significance of the Study
In this study, we examined if the proteome of P. aeruginosa OMVs was determined by their mechanism of biogenesis.
We identified significant differences in the production of OMVs when produced by one defined mechanism of biogenesis, as well as significant changes in the proteome of OMVs compared to their parent bacteria. Importantly, we found that OMVs produced via distinct mechanisms of biogenesis have significantly different proteomes when compared to one another. Furthermore, these proteomic differences state of growth [33] which highlights the ability of bacteria to tailor the cargo composition of OMVs in response to changes in their environment. Furthermore, the bacterial growth stage from which OMVs are isolated, and the size of OMVs can also determine their protein content and functions [34,35]

OMV production
For OMV production, all P. aeruginosa strains were grown aerobically

Nanoparticle tracking analysis
OMV size and concentration was determined using the nanoparticle tracking analyser ZetaView® basic PMX-120 NTA (Particle Metrix, Germany) as previously described [20]. OMVs were diluted in DPBS, and the concentration of OMVs contained within 1 mL of each diluted sample was determined by NTA, which typically contained approximately

Qubit™ fluorometric quantification
The DNA, RNA, and protein content of OMVs were quantified using the Qubit™ high sensitivity DNA assay, high sensitivity RNA assay, or protein assay, respectively (ThermoFisher, USA) and were measured using a Qubit™ 3.0 fluorometer. The DNA, RNA, and protein concentrations of OMVs were then normalized to 1 × 10 8 OMVs.
Data was acquired using Xcalibur software v4.0 (Thermo Fisher Scientific, USA). The MS-based proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository and are available via ProteomeXchange with identifier PXD032996.

2.9
Database searching and analysis RAW MS data was processed using MaxQuant [41] (v1.6.14.0) with its built-in search engine Andromeda. Tandem mass spectra were

Statistical analysis
Data analysis was performed using GraphPad Prism 9.4.1. All data are represented as the mean ± standard error of the mean (SEM) of three biological replicates. Statistical analyses were performed using data from three biological replicates, using the one-way analysis of variance (ANOVA) with Tukey's multiple-comparisons test or a Student's t-test as indicated.

The mechanism of P. aeruginosa OMV biogenesis determines the number of OMVs produced
In this study we aimed to determine whether the mechanism of OMV biogenesis affects the production and protein composition of OMVs. To address this, we isolated OMVs from three P. aeruginosa strains that each have a distinct mechanism of OMV biogenesis, being P. aeruginosa PAO1 which produced OMVs by both budding and explosive cell lysis (WT-OMVs), P. aeruginosa PAO1Δlys which is unable to undergo prophage-mediated explosive cell lysis and therefore pro- Next, we examined if the mechanism of OMV biogenesis influenced the number of OMVs produced by P. aeruginosa. To do this, the overall quantity of OMVs produced by each P. aeruginosa strain with distinct mechanisms of OMV biogenesis was determined using ZetaView Nanoparticle Tracking Analysis (NTA; Figure 1D). Examination of the quantity of OMVs produced by each P. aeruginosa strain revealed that significantly fewer OMVs were produced by P. aeruginosa PAO1Δlys that produces B-OMVs by budding only, compared to the P. aeruginosa strain that produced WT-OMVs (p < 0.05, Figure 1D), consistent with previous findings [8]. Quantification of the number of bacteria within the cultures from which OMVs were isolated revealed that there were no differences in the number of bacteria present in the cultures of each of the three P. aeruginosa strains examined, suggesting that the significant reduction in the amount of B-OMVs produced was not attributed to reduced bacterial growth ( Figure 1E). However, as B-OMVs are produced by the endolysin mutant P. aeruginosa PAO1Δlys, a reduction in the amount of B-OMVs produced compared to WT-OMVs suggests that the endolysin, and therefore explosive cell lysis, significantly contributes to the generation of WT-OMVs by P. aeruginosa (p < 0.05, Figure 1D). In addition, we did not see an increase in the quantity of E-OMVs produced compared to WT-OMVs, suggesting that explosive cell lysis does not increase OMV production by P. aeruginosa ( Figure 1D). Furthermore, examination of the size distribution of OMVs produced by all three P. aeruginosa strains using NTA revealed that OMVs produced by all mechanisms of biogenesis ranged between approximately 50 to 400 nm in size ( Figure 1F) with no statistical differences in the size of OMVs produced by all three P. aeruginosa strains observed ( Figure S1).
Bacterial OMVs contain a range of cargo, including DNA, RNA, and protein. Therefore, we next examined the contribution of the mechanism of OMV biogenesis in regulating the quantity of DNA, RNA, and protein associated with OMVs. Quantification of bacterial cargo asso-ciated with P. aeruginosa OMVs revealed that WT-OMVs, B-OMVs, and E-OMVs all contained DNA, RNA, and protein ( Figure 1G-I). However, E-OMVs were associated with significantly more DNA when compared to both WT-OMVs and B-OMVs (p < 0.001; Figure 1G) which could be due to the release of cytoplasmic DNA cargo by bacteria during explosive cell lysis that can be packaged in E-OMVs [8]. In addition, there were no significant differences in the quantity of RNA or proteins associated with OMVs produced by different mechanisms of biogenesis ( Figure 1H,I). Collectively, our data shows that the mechanism of P. aeruginosa OMV biogenesis determines the quantity of OMVs produced by P. aeruginosa and their DNA cargo, but does not affect the overall size distribution of OMVs or their overall quantity of RNA and protein cargo.

3.2
Global protein comparison analysis revealed that the proteome of OMVs produced via different mechanisms of biogenesis differ significantly to that of their parent bacteria Bacteria are known to selectively package cargo into OMVs for their delivery to surrounding bacteria or host cells [43,44]. To determine if there is preferential packaging of proteins into P. aeruginosa OMVs produced by different mechanisms of biogenesis, we performed a quantitative global protein comparison and enrichment analysis of all three P. aeruginosa bacterial strains and their OMVs. Proteins identified in P. aeruginosa bacteria and their OMVs were compared to a P. aeruginosa reference genome to reveal selective enrichment of proteins in OMVs [45]. We identified a total of 2983 proteins in samples obtained from all three P. aeruginosa strains and a total of 1820 proteins in the OMVs produced by all mechanisms of biogenesis, covering approximately 53% of all P. aeruginosa PAO1 predicted proteins ( Figure 2A; Table S1). There were 36 proteins only identified at a significant level in OMVs produced by all mechanisms of biogenesis compared to 1199 proteins only identified at a significant level in all three P. aeruginosa bacterial strains (Figure 2A; Tables   S2 and S3). Of the 36 proteins significantly enriched in OMVs produced by all mechanisms of biogenesis were proteins reported to be involved with the cell surface, or part of integral components of the cell membrane, suggesting that these proteins are localized to the outer membrane of P. aeruginosa (Figure 2A; Table S3). Specifically, OMVs produced by all mechanisms of biogenesis were found to be significantly enriched in proteins that comprise part of the ABC transport complex, which is known to facilitate OMV biogenesis [46], and numerous uncharacterized proteins which are thought to contribute to protein secretion and transport, suggesting a functional role for the release of OMVs by P. aeruginosa (Table S3). Furthermore, OMVs produced by all mechanisms of biogenesis were found to contain proteins involved in quinone binding, drug transmembrane activity, and metal ion transportation, indicating that P. aeruginosa OMVs may play a role in the secretion and transport of metal ions and other molecules to bacteria (Table S3)

OMVs produced by different mechanisms of biogenesis are enriched in unique subsets of proteins compared to their parent bacteria
We next determined if there were any differences in the enrichment of proteins packaged in OMVs produced by different mechanisms of biogenesis compared to their parent bacteria. Specifically, when compared to their parent bacteria, we found that WT-OMVs were significantly enriched in the tailocin endolysin PA0629, necessary for explosive cell lysis [8], and the tailocin protein PA0622 compared to their parent bacteria ( Figure 3A). Furthermore, WT-OMVs were also significantly enriched in the OMV biogenesis proteins TolQ, which is part of the Tol-Pal system that facilitates the production of OMVs by budding [49,50], and LptD which aids in the assembly of LPS at the outer membrane and can affect OMV biogenesis [51] (Figure 3A; Table S4). In addition, WT-OMVs were significantly enriched in various virulence proteins including the porins OprB, OprD, and OprF, the pyocyanin Pys2 [52], and the multidrug resistance proteins MexA and MexB [53] (Figure 3A; Table S4). Furthermore, AmpDh3 which degrades peptidoglycan has previously been identified in P. aeruginosa OMVs and gives rise to lytic activity on murein sacculi zymography [54]. AmpDh3 has also been reported to be enriched in OMVs produced as a result of explosive cell lysis, in addition to being induced by the SOS response [54]. We identified that AmpDh3 was also enriched in WT-OMVs compared to their parent bacteria, further suggesting that P. aeruginosa WT-OMVs may be predominately produced as a result of explosive cell lysis (Table S4).
Compared to their parent bacteria, the enrichment of these proteins in WT-OMVs suggests that P. aeruginosa WT-OMVs may contribute to enhancing bacterial virulence and pathogenesis.
We then compared the enrichment of proteins in B-OMVs to their parent bacteria to identify any proteins that would indicate the potential role of OMVs produced by budding. We found that B-OMVs were significantly enriched in the peptidoglycan degraders AmpDh2 and AmpDh3, the latter which has previously been identified in P. aeruginosa OMVs [54] (Figure 3B; Table S4). In addition, B-OMVs were found to be enriched in the proteolysis protein PA0328 which suggests that B-OMVs may be able to target and degrade bacterial material in the environment ( Figure 3B; Table S4). B-OMVs were also significantly enriched in numerous proteins with assigned functions in siderophore transport and metal binding including FpvA, FptA, and the aminopeptidase Iap ( Figure 3B; Table S4) suggesting that OMVs produced by budding may contribute to the acquisition of essential metal ions as previously identified for P. aeruginosa OMVs [55,56]. The enrichment of these proteins in B-OMVs indicates that OMVs released from the cell outer membrane via budding may contribute to bacterial survival via targeting competing bacteria and acquiring nutrients.
Furthermore, to understand if OMVs produced by explosive cell lysis were enriched in proteins that would indicate their function, we compared the enrichment of proteins in E-OMVs to their parent bacteria. Our analysis shows that E-OMVs were significantly enriched in the Sec protein translocase complex protein SecF, which aids in the incorporation of proteins into OMVs [57,58] and the multidrug resistance proteins MexA and MexB ( Figure 3C; Table S4). Furthermore, E-OMVs were also significantly enriched in the penicillin-binding protein MrcB and the porins OpdC and PA1271 suggesting that E-OMVs contain a range of cargo that may aid in the survival of bacteria within their environment ( Figure 3C; Table S3). Interestingly, as PA0629 is the known causative agent of explosive cell lysis in P. aeruginosa [8] and is overexpressed in our E-OMV producing bacteria, we postulated that E-OMVs would be enriched in this protein. However, proteomic analysis revealed that PA0629 levels were significantly reduced in E-OMVs compared to their parent bacteria ( Figure 3C

The mechanism of OMV biogenesis determines the protein composition of P. aeruginosa OMVs
To determine key differences in the proteome of OMVs produced by different mechanisms of biogenesis we examined the protein composition of P. aeruginosa OMVs produced naturally, by budding only, or predominately by explosive cell lysis. We revealed that WT-OMVs, B-OMVs, and E-OMVs contained a total of 1456, 865, and 1165 proteins, respectively, with 534 proteins common to OMVs produced by all mechanisms of biogenesis ( Figure 4A; Table S5) forming protein PfpI, and numerous 30S and 50S ribosomal subunit proteins (Table S5). Furthermore, WT-OMVs were highly abundant in cell wall biogenesis and peptidoglycan biosynthesis proteins including UvrA, LpxH, ArnB, MurB, and MurD, which suggests that WT-OMVs may be produced during cell membrane modification events (Table S5).
In comparison, the 154 proteins identified only in B-OMVs consisted of numerous nucleoid-localized proteins, including UvrD, MutL, and FtsK ( Figure 4A; Table S5). However, how these proteins can traverse to the outer membrane of the cell for packaging into OMVs via budding from the cell membrane is currently unknown and remains an important question to be elucidated. Additionally, only B-OMVs were found to contain the transcriptional regulatory protein GacA which controls the production of numerous P. aeruginosa virulence factors including the bactericidal compound pyocyanin [59,60], and the antimicrobial toxin protein Tse5 [61] (Table S5), as well as LasI, a component of the LasI-LasR quorum-sensing system which also regulates the expression of virulence genes in P. aeruginosa [62,63]. The inclusion of these proteins in B-OMVs suggests that OMVs produced by budding from the cell membrane are packaged with proteins that can contribute to the regulation of bacterial virulence and are potentially virulent themselves, which forms the basis of future research. Furthermore, we identified unique proteins in E-OMVs produced by explosive cell lysis that contribute to the toxicity of P. aeruginosa within host cells. Specifically, E-OMVs were highly abundant in the cytotoxic protein Exoenzyme T [64] and the virulence protein Hcp1 which is actively secreted by P. aeruginosa during chronic cystic fibrosis infections [65], suggesting that this type of OMV may contribute to the virulence and pathogenesis of P. aeruginosa in an infectious setting ( Figure 4A; Table S5). Additionally, only E-OMVs were significantly abundant in the proteins PA2269 and PA0454, both of which have predicted GOMF transmembrane transporter activity. Furthermore E-OMVs were enriched in the protein TatC which is responsible for both pyoverdine-mediated iron acquisition and bacterial growth inhibition [66], suggesting E-OMVs may have multiple roles in nutrient acquisition and antimicrobial activity. As we had identified a significant increase in the amount of DNA packaged in E-OMVs compared to both WT-OMVs and B-OMVs, we also examined whether E-OMVs contained proteins associated with DNA binding that were not identified in other OMVs types. Upon investigation, we identified numerous DNA binding proteins only in E-OMVs which included AmgR, PA4778, and PA4992, as well as the DNA polymerases PolA and DnaX (Table S5). This suggests that there is an increase not only in bacterial DNA packaged within E-OMVs but also DNA-associated proteins when OMVs are produced by explosive cell lysis.
Next, PCA was performed to examine the overall proteome of OMVs produced by different mechanisms of biogenesis ( Figure 4B). We identified that WT-OMVs, B-OMVs, and E-OMVs had distinct proteomes, as demonstrated by the three separate populations of OMVs by PCA ( Figure 4B). Notably, the spread of E-OMVs observed in the PCA indicates that E-OMVs have greater variation in their proteome than B-OMVs produced by budding from the cell membrane ( Figure 4B Figure 4C). Furthermore, both B-OMVs and E-OMVs were significantly depleted in a common subset of proteins compared to WT-OMVs, indicating that there are significant differences in the proteome of OMVs when produced predominately via one main mechanism of biogenesis ( Figure 4C). Collectively, our data highlights that OMV biogenesis mechanisms determine the proteins that are packaged within OMVs, highlighting that OMV biogenesis is an important regulator of OMV composition and potential downstream biological functions.

OMVs produced by different mechanisms of biogenesis are differentially enriched in proteins compared to one another
Having identified that the mechanism of OMV biogenesis determines the overall proteome of OMVs, we next sought to determine if the mechanism of biogenesis affected the enrichment of proteins in OMVs. To do so, we compared WT-OMVs, B-OMVs, and E-OMVs to one another and identified a significant enrichment of proteins in each OMV type. Compared to naturally produced WT-OMVs, B-OMVs were significantly depleted in the pathogenic proteins MexA and HflK ( Figure 5A). In addition, the majority of proteins significantly enriched in B-OMVs compared to WT-OMVs were uncharacterized proteins with unknown functions, and those with known functions were involved in glutamate synthesis and flagella activity ( Figure 5A).
Next, the comparison of WT-OMVs to E-OMVs revealed that E-OMVs were significantly depleted in proteins that comprised part of the Tol-Pal complex ( Figure 5B). However, E-OMVs were significantly enriched in the tailocin protein PA0622, in addition to the ABC-type transporter protein PA3187 and the protein transporter protein PA2982. We then compared the enrichment of proteins in OMVs produced by a single mechanism of biogenesis. When compared to B-OMVs, we found E-OMVs were significantly enriched in the multidrug resistance proteins MexA, MexB, and OprM, and the porins PA3038 and PA4974, which indicates that explosive cell lysis is responsible for the capture of these proteins by E-OMVs and therefore E-OMVs may have a greater contribution to promoting bacterial survival and antimicrobial resistance, however this remains to be elucidated ( Figure 5C).
Finally, we performed functional enrichment analyses of the proteins significantly enriched in OMVs produced by different biogenesis mechanisms to better understand their potential functions. All three types of OMVs were significantly enriched in proteins with GOBP terms of protein localisation, cellular localization, siderophore transport, and iron ion transport ( Figure 5D). However, only WT-OMVs were significantly enriched in proteins attributed to the GOBP of proton, hydrogen, protein, and cation transport. In comparison, only E-OMVs were significantly enriched in proteins attributed to protein targeting and intracellular protein transport. Interestingly, B-OMVs were not significantly enriched in any unique proteins compared to WT-OMVs and E-OMVs ( Figure 5D). Examination of the GOCC of OMVs produced by all three strains of P. aeruginosa revealed that all OMV types were significantly enriched in cell outer membrane, outer membrane, and membrane proteins, as expected ( Figure 5E). Most importantly, B-OMVs were not enriched in proteins associated with cellular components of the membrane, plasma membrane, or protein complex, indicating that B-OMVs were depleted in proteins that do not localize to the outer membrane as expected, due to being derived from the outer membrane of their parent bacterium ( Figure 5E). Moreover, only E-OMVs were significantly enriched in intracellular proteins, supporting the concept that cytoplasmic components of bacteria can only be contained within OMVs produced via explosive cell lysis ( Figure 5E), which was also supported by the identification of a significant increase in DNA and the packaging of DNA associated proteins only in E-OMVs ( Figure 1G; Table S5). Comparing the GOMF between OMVs produced by different mechanisms of biogenesis revealed that the three types of OMVs may have distinct biological functions as some proteins were found to be only enriched in one type of OMV ( Figure 5F). Specifically, WT-OMVs were significantly enriched in a range of cation transmembrane transporter proteins that were not significantly enriched in B-OMVs or E-OMVs ( Figure 5F). Furthermore, B-OMVs were significantly enriched in serine hydrolase and peptidase proteins suggesting that B-OMVs may play a role in the degradation of amino acids ( Figure 5F), whereas only E-OMVs were significantly enriched in proteins that have a role in oxidoreductase activity on heme indicating they may contribute to bacterial nutrient acquisition during conditions of stress. Notably, all three OMV types were significantly enriched in proteins associated with roles in porin activity and receptor activity suggesting that despite differences in the packaging and enrichment of proteins in OMVs produced by different types of biogenesis, all OMVs may share some conserved biological roles ( Figure 5F). Overall, our results show that a shift from a naturally produced population of OMVs to OMVs produced by a single mechanism of biogenesis significantly alters the enrichment of proteins incorporated into OMVs, suggesting that bacteria may harness different mechanisms of OMV biogenesis to produce OMVs with bespoke functions.

CONCLUDING REMARKS
OMVs are known to package bacterial proteins that can be delivered to host cells or neighboring bacteria [35,67]. However, the multiple factors which can influence the packaging of select proteins into OMVs is yet to be fully elucidated. In this study, we aimed to understand the role of the mechanism of OMV biogenesis on regulating the production, composition, and proteome of P. aeruginosa OMVs. We identified that the mechanism of P. aeruginosa OMV biogenesis determined the number of OMVs produced, as significantly fewer OMVs were released via budding compared to the production of wild-type OMVs that are OMVs, including their function as delivery vehicles for bacterial contents [68,69], their role in inhibiting bacterial growth [54,67,70], and their contribution in mediating inflammation in disease settings [71,72]. Furthermore, identifying how bacteria may utilize the mechanisms of OMV biogenesis to influence the content of OMVs and potentially alter their functions, may allow for the development of novel techniques to tailor the cargo of OMVs for therapeutic applications. Finally, our findings highlight that the multiple mechanisms of OMV biogenesis can also explain how proteins from distinct bacterial compartments may enter OMVs, as E-OMVs were enriched in cytoplasmic proteins, whereas B-OMVs produced by budding from the outer membrane predominately contained proteins located in the outer membrane. These findings contribute to the growing understanding that not all OMVs package specific bacterial cargo, such as cytoplasmic cargo, due to the variations in the mechanism by which they are produced.