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The role of bacterial outer membrane vesicles for intra-and interspecies delivery

James Berleman1,2** and Manfred Auer1*1Life Sciences Division, Lawrence Berkeley NationalLaboratory, 1 Cyclotron Rd., Berkeley, CA 94720, USA.2Department of Biology, St Mary’s College,212 Brousseau Hall, Moraga, CA 94556, USA.

Summary

An increasing number of Gram-negative bacteria havebeen observed to secrete outer membrane vesicles(OMVs). Many mysteries remain with respect to OMVformation, the regulation of OMV content and mode oftargeting and fusion. Bacterial OMVs appear to serve avariety of purposes in intra- and interspecies microbialextracellular activities. OMVs have been shown tomediate cell-to-cell exchange of DNA, protein andsmall signalling molecules. The impact of such mate-rial exchanges on microbial communities and patho-genic processes, including the delivery of toxins athigh concentration through OMVs, is discussed. Thisrather recent aspect of microbial ecology is likely toremain an important area of research as an in-depthunderstanding of OMVs may allow new approaches forcombating bacterial infections and provide new routesfor selective drug delivery.

Introduction

In the course of evolution bacteria, just as their eukaryo-tic competition, have evolved elaborate mechanisms tocolonize and survive in their respective niches, be it inan aqueous, terrestrial or in a host organism environ-ment (van Elsas et al., 2011). Bacteria must defendthemselves in their niche from the takeover of otherspecies or from immune responses of their host.Encounters between different species likely provoke ter-ritorial disputes, with either mutual tolerance, hostilestand-offs or annihilation of one species by its competi-

tor (Vetsigian et al., 2011). Hence one expects theco-evolution of a wealth of measures and countermeas-ures in interspecies disputes (Ferrer and Zimmer, 2012).Pathogenesis is one such example of a complex host–bacteria interplay, where a bacterial species not onlyattacks the host tissue, but also evades immuneresponse mechanisms.

Intraspecies communication is an important part ofmicrobial community life (Ng and Bassler, 2009), ena-bling the coordination of behaviour through cell-to-cellsignalling, allowing bacterial biofilms to act as a multicel-lular organism, either through predation or by turning onvirulence factors once a sufficient quorum has beensensed (Berleman and Kirby, 2009; Li and Tian, 2012).Homoserine lactone secretion into the extracellularspace has been shown to regulate gene expressionacross bacterial populations (Schuster and Greenberg,2006), but the mechanism of extracellular secretionof soluble small molecules has a number of disadvan-tages, including the fast dilution of the signal, and itspossible degradation by secreted enzymes (Boyer andWisniewski-Dyé, 2009). Therefore the danger of signifi-cant material waste is real and open broadcasting ofone’s presence could alert competing organisms. Outermembrane vesicles (OMVs) are an alternative vehicle forcell–cell signalling in bacteria that have recently gainedrecognition (Mashburn-Warren et al., 2008; Remis et al.,2010; Whitworth, 2011).

Outer membrane vesicles formed by bacteria canmediate intercellular exchange events including cell–cellsignalling, protein and DNA exchange and have evenbeen shown to kill cells (Kadurugamuwa and Beveridge,1996; Shetty et al., 2011; Evans et al., 2012; J.P. Remis,D. Wei, A. Gorur, J. Haraga, S. Allen, H.E. Witkowska,J.W. Costerton, J.E. Berleman and M. Auer, submitted).While these structures were identified several decadesago (Mayrand and Grenier, 1989), their importancehas gained recognition in the past 15 years (Beveridge,1999; Mashburn-Warren et al., 2008). Membrane vesiclesprovide a useful vector for vaccines and may elucidatenew forms of drug delivery (MacDonald and Beveridge,2002; Collins, 2011; Sanders and Feavers, 2011).

Received 2 November, 2012; accepted 3 November, 2012. Forcorrespondence. *E-mail mauer@lbl.gov; Tel. (+1) 510 486 7702;Fax (+1) 510 486 6488; **E-mail jeberleman@lbl.gov; Tel. (+1) 510486 7702; Fax (+1) 510 486 6488.

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Environmental Microbiology (2013) 15(2), 347–354 doi:10.1111/1462-2920.12048

Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

OMV function

Outer membrane vesicles appear to be a scaffold for avariety of extracellular activities, rather than mediate asingle specific process (Mashburn and Whiteley, 2005). Inthis respect, they are quite distinct from other extracellularorganelles, such as Type III needles of Pseudomonasaeruginosa or Yersinia pestis, which transfer specifictoxins into a host cell (Büttner, 2012), or the Type IVsystem in Agrobacterium tumefaciens that functions soefficiently in transferring the T-DNA between bacteria andplant cells (Tomlinson and Fuqua, 2009). So while there isample evidence for OMVs as a cell–cell transport vehicle,the details of their cargo and the mechanism of biogen-esis from the budding of the vesicle from the membrane,to targeting and fusion of the vesicle to the recipient cell ismuch less understood.

We endeavour here to highlight a few of the systemsthat have been studied and examine them in terms of thetype of molecule exchanged, bound or transferred. Whiledoing so it is important to keep in mind the functions thatcan be provided by exchanging proteins, DNA, carbohy-drate or lipid molecules through OMV. Only DNA is likelyto provide a heritable change in the receiving cell and onlyprotein is likely to provide enzymatic function, whereas allof these molecules can potentially act as a cell–cell signal.

Regulation of vesicle biogenesis

There are several reports indicating that vesicles form asa response to membrane stress (Kulp and Kuehn, 2010;Collins, 2011). Examination of a number of stressors inPseudomonas putida indicates that the vesicle formationresponse is similar in all cases and leads to increased OMhydrophobicity and cell–cell packing (Baumgarten et al.,2012). In P. aeruginosa, 2-heptyl-3-hydroxy-4-quinolone(PQS) biosynthesis has been shown to stimulate OMVproduction, through promoting increased membrane cur-vature (Fig. 2A) (Schertzer and Whiteley, 2012). This indi-cates that in some instances vesicle production isregulated by factors other than stress. PQS also regulatesthe production of P. aeruginosa virulence factors as acell–cell signal and it has the same vesicle-inducingimpact on outer membranes when it is supplied exog-enously as it does when synthesized by cells (Dubern andDiggle, 2008). Schertzer and Whiteley (2012) propose amodel where interactions of PQS with the LPS leaflet ofthe outer membrane, but not the inner phospholipid leafletdrives the process vesicle biogenesis.

Mechanism(s) of vesicle biogenesis

To date no proteins have been identified to be clearlyinvolved in vesicle formation. Three mechanisms have

been proposed for vesicle formation: (i) the loss of outermembrane contact with the underlying peptidoglycanallowing excess lipid formation to induce vesicle forma-tion, (ii) local build-up of periplasmic proteins, literallypushing out the outer membrane, thus trapping largeamounts of protein inside the resulting vesicle, and (iii) theinvolvement of integral membrane proteins or possiblysmall molecules inside the outer membrane that inducecurvature of the membrane and hence vesiculation(reviewed in Kulp and Kuehn, 2010). The first mechanismwould imply a rather low protein occupancy of the interiorof the vesicles, and suggest that the vesicle protein com-position is somewhat similar to the outer membrane,whereas the second mechanism suggests high proteinoccupancy in the vesicles, and also a somewhat similarprotein composition of vesicles compared with outermembrane, whereas the third mechanism would predicta rather low protein occupancy in the interior of the vesi-cles and an enriched integral membrane protein andmembrane-associated composition. The latter mecha-nism would also suggest that most likely a comparisonbetween different species could yield candidate proteins,as conserved biogenesis proteins would be expected toshow up in proteomics analysis of OM vesicles from manydifferent species. Thus far no conserved components canbe identified through comparison of various proteomicsstudies (Kahnt et al., 2010; Elmi et al., 2012; Mendezet al., 2012). In addition to proteomics, better geneticstudies are required to help identify if there are any con-served loci for OMV formation genes, such as the mag-netosome island responsible for forming intracellularorganelles in magnetotactic bacteria (Komeili, 2012).

Ultrastructural tomographic 3D studies of faithfully pre-served vesicles in Myxococcus xanthus biofilms revealedthat the vesicle lumen was relatively empty (Fig. 1), withproteins being found relatively sparse and close to themembrane (Palsdottir et al., 2009), suggesting that thesecond mechanism with its predicted dense array of pro-teins is unlikely to be correct. One prediction of the thirdbut not necessarily the first mechanism is that vesicleshave defined sizes, as any kind of integral membraneprotein or vesicular lumen protein is likely to pack best incertain vesicle sizes. Interestingly, geometric analysis ofvesicles both in 2D and 3D suggest that while there is nota single size for all vesicles, the size seems to be regu-lated (Palsdottir et al., 2009, J.P. Remis, D. Wei, A. Gorur,J. Haraga, S. Allen, H.E. Witkowska, J.W. Costerton, J.E.Berleman and M. Auer, submitted). It remains to be deter-mined whether there is a prokaryotic equivalent to theeukaryotic vesicle-inducing proteins such as clathrin,COPI/II and/or caveolins (Zanetti et al., 2012), and thuswhether there is a set of proteins that could allow fordifferent vesicle diameters via compositional and/or con-formational variability. While it is possible that some

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molecular machinery is responsible for pinching off vesi-cles only to stay behind in the outer membrane, one wouldexpect such molecular machineries to be part of thevesicle proteome. Comparative analysis of vesicle pro-teomics data has not yet yielded a clear candidate,but more mass spectrometry together with subsequentgenetic analysis may reveal such candidate proteins.Interestingly it was found that certain proteins areenriched or excluded from the OM, arguing for the thirdproposed mechanism (Bernardini et al., 2007).

Vesicles as a vector for DNA exchange

Vesicles produced by some bacteria can harbour DNAfragments derived from the chromosome and plasmids aswell as RNA (Yaron et al., 2000; Kesty et al., 2004; Mash-burn and Whiteley, 2005). A study of vesicle-packagedDNA from Ruminococcus species resulted in the obser-vation of linear dsDNA varying in size up to 90 kbp (Klieve

et al., 2005). Importantly for the ecological role of thisgenus in rumen microbial communities, transformation ofvesicle DNA resulted in the transfer of cellulolytic genes tonon-cellulolytic strains of Ruminococcus. Yaron et al.determined the transformation efficiency of enteropatho-genic Escherichia coli vesicle-packaged DNA to otherE. coli strains to be 103 transformants per mg of DNA. Astudy examining transformation of environmental vesicleDNA restored growth to an E. coli amino acid auxotroph ata frequency of ~ 10-5 (Velimirov and Hagemann, 2011).This study also indicated that the upper limit of vesicleDNA size could be as high as 370 kbp.

Thus, the incorporation of DNA into vesicles can occurfrom three routes. Cytoplasmic DNA can be packaged intovesicles and moved out of the cell through specific trans-port mechanisms. While the details of such a mechanismhave not to our knowledge not been deciphered in anysystem, there is sufficient evidence to warrant furtherinvestigation. However, in addition to questions of trans-port there is the question of which DNA gets selected forreplication and transport. It is worth reminding ourselvesthat not all organisms replicate through binary fission

Fig. 1. Ultrastructure of OMVs.A. Vesicle biogenesis observed at the cell pole in M. xanthus withTEM. Both the inner and outer membrane of the cell are observed,with discrete vesicles and membrane-blebbing captured.B. Cryo-tomography showing the arrangement of vesicles along thecell membrane in M. xanthus, with the electron dense vesicleexterior surrounding the lower density core. (Images courtesy ofRemis & Auer; Palsdottir et al., 2009.)

Fig. 2. OMV–membrane fusion.A. Interspecies transfer of the quinolone cell signal produced byP. aeruginosa, PQS, alters the membrane curvature of red bloodcells (right), relative to untreated cells (left).B. Intraspecies transfer of lipid dyes (red) between cells ofM. xanthus (recipient cells, green) is shown to require the TraABprotein system. (Images adapted from Pathak et al., 2012;Schertzer and Whiteley, 2012.)

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and if processes such as budding (Jogler et al., 2012)can be a successful mechanism for reproduction, thanintermediate mechanisms are likely of use as well. Alter-natively, DNA could conceivably end up in vesicles as aresult of cell death. Cell lysis is a common occurrence inany bacterial population, and the wide range of genomicfragments that end up packaged in vesicles seems toindicate general nuclease activity. In this scenario, bacte-rial cell death may play a beneficial role to the community,through some storage of heritable information. The thirdroute is that extracellular DNA may be passively taken upand stored in extracellular vesicles. In this case, there arestill many questions remaining on the process of uptake.Is incorporation with the OM a passive process? Howdoes DNA move across the much more tightly regulatedcytoplasmic membrane?

In addition to vesicles, membrane ‘nanotubes’ havebeen reported to mediate the exchange of DNA inbetween cells of Bacillus subtilis, which does not have anouter membrane (Dubey and Ben-Yehuda, 2011). Theauthors observed exchange of plasmid DNA and subse-quent transfer of antibiotic resistance genes. However, itis unclear how common this mechanism of horizontalgene transfer occurs outside of lab conditions. Wepresume the rate to be extremely low for two reasons: (i)the DNA exchanged was a very high-copy-numberplasmid, and (ii) B. subtilis is one of only a few organismsthat displays natural transformation, the nanotubesobserved may be a specific structure that mediates acell–cell variation of natural transformation, rather than ageneral bacterial structure. Further research is needed todetermine if this mechanism is more broadly utilized.

Vesicles as a scaffold for protein activities

While the exact determinants of extracellular vesicle pro-duction are unknown, some organisms produce vesiclesmore often than others. In M. xanthus for instance(Fig. 1A) prolific vesicle production has been observed(Palsdottir et al., 2009). Vesicles were observed withtethers to the OM and to each other in highly organizedarrangements (Fig. 1B). In addition, there is evidence forprolific transfer of lipids and lipoproteins between cells ofM. xanthus that requires the OmpA-like TraAB proteins(Fig. 2B) (Pathak et al., 2012). Two functions for vesiclesare proposed for M. xanthus: (i) as an intraspeciesmechanism for mediating signalling and social movement,and (ii) as an interspecies mechanism for exchange ofcell–cell killing factors (discussed in the next section).Myxococcus xanthus moves on surfaces by gliding motil-ity (Mauriello et al., 2010). While many mutations causedefects in motility, motility mutants at the tgl and cgl lociare conditional (Rodriguez and Spormann, 1999; Nudle-man et al., 2005; Pathak and Wall, 2012). These strains

can regain motility when incubated adjacent to strains thatproduce the missing protein. It was initially suggested thatOM from two colliding bacteria would merge and thustransfer proteins (Nudleman et al., 2005); however, itseems more likely that extracellular vesicles fulfil the roleof exchange of lipoproteins between neighbouring cells(Palsdottir et al., 2009; Remis et al., 2010). Further inves-tigations of the process of vesiculation and in the exist-ence of membrane bridges between cells are needed.

Extracellular vesicles have also been implicated inexo-enzyme activity as well as cell-to-cell exchange ofproteins. Uptake and cytoplasmic incorporation ofGFP-labelled extracellular proteins was observed in theplanctomycete, Gemmata obscuriglobus (Fuerst andSagulenko, 2010). While bacterial endocytosis was sug-gested as the mechanism, currently there is not enoughevidence to support such a claim. In Bartonella henselae,a mammalian pathogen, haem toxicity is mediated in partthrough the expression and packaging of haem-bindinghbpC protein into OMV (Roden et al., 2012). Overexpres-sion of the HbpC protein was shown to cause increasedOMV-bound haem in B. henselae. Interestingly, B. hense-lae requires haem as its sole iron source, yet it is not clearif the haem bound in OMVs is only a protective mecha-nism or if it is also a transport mechanism.

Vesicles as a vehicle for cell killingand pathogenesis

A number of pathogenic organisms have been shown toproduce vesicles and these vesicles may play crucialroles in pathogenesis. Pseudomonas aeruginosa formsbiofilms in chronic infections and was also observed inacute burn wounds, where vesicles are observed outsideof cells in the extracellular matrix (Fig. 3) (Schaber et al.,2007). Acinetobacter baumannii vesicle fractions wereshown to mediate host cell apoptosis, which depends onthe presence of the OmpA channel protein presumablydamaging the membrane gradient of the host cell (Jinet al., 2011). Cytolethal distending toxin from Aggregati-bacter actinomycetemcomitans was shown to depend onvesicle transport into both HeLa cells and human gingivalfibroblasts (Rompikuntal et al., 2012). Haemolytic activitywas detected from vesicle fractions of the pathogenKingella kingae, as well as detection of the RtxA toxin inboth vesicles and the extracellular fraction (Maldonadoet al., 2011). In each case the toxic factor differs, indicat-ing that OMVs can mediate the transmission of a varietyof factors.

In addition to bacteria–host pathogenicity, OMVs havebeen shown to play a role in cell–cell inhibition and killingamong competing bacterial species. There is evidencethat P. aeruginosa OMVs disrupt biofilm formation,swarming and iron uptake in P. putida (Fernández-Piñar

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et al., 2011). Our own results (J.P. Remis, D. Wei, A.Gorur, J. Haraga, S. Allen, H.E. Witkowska, J.W. Coster-ton, J.E. Berleman and M. Auer, submitted) suggest thatvesicles from M. xanthus have a cytotoxic effect on E. colicells. Similar results were obtained by Evans and col-leagues (2012) who observed increased cell killing byaddition of fusogenic GAPDH enzyme, indicating thatintegration of OMVs from M. xanthus into the E. coli OMpromotes delivery. This also indicates thatthe uptake of OMVs may be regulated by production offusogenic enzymes such as GAPDH. In the cases ofintraspecies transfer the exchange of useful proteinswould help promote competitiveness, whereas duringinterspecies exchange of toxic cargo, the production ofGAPDH by the host cell would be expected to reducesurvival.

Specificity of OMV content

With so much variety observed in the content and func-tion of OMVs, the issue that requires more research iswhether bacterial OMVs contain specifically addressedmessages dependent on neighbouring species. It isconceivable that M. xanthus cells when among non-competitors focuses on delivering ‘benign’ messages,such as social motility proteins. However, the OMVcargo may change when faced with stress, competitionor prey. It seems reasonable that friend–foe interactionsmay occur simultaneously, within a microbial communitysuch as soils and detritus. To discriminate between mes-sages sent by friend and foe OMVs would require theequivalent of a bar code-like distinction. Such a rolecould be played either by a unique protein composition(specific receptors) or by altering the precise lipopoly-saccharide (LPS) composition. It has been reported inother systems and our own data also suggest that thereis exquisite control of what makes it into OM vesicles

and it wouldn’t be surprising if both mechanisms occursimultaneously. It would be remarkable if one couldshow that vesicle content is tailored to the specific com-munity situation, e.g. that the vesicle content transitionsfrom friendly neighbourly help to hostile toxin release.How such a transition could be regulated, if it indeedexists, would be indeed a mystery.

OMV–OM recognition and fusion

If OM vesicles are to be effective in material or signaltransfer they must interact specifically and finally fuse withtheir respective target cells. Fine 3D ultrastructural studiesof high-pressure frozen, freeze-substituted stained bio-films (Palsdottir et al., 2009) revealed tethers betweenOM vesicles and the M. xanthus OM that kept the vesiclesbound, yet 10–20 nm away from the OM surface. Sincecarbohydrates are not visible under such heavy metalstain sample preparation conditions, we have employedcryo-EM of M. xanthus whole mount bacteria and foundOM vesicles and OM to contain a ~ 5- to 10-nm-thick layerof what appears to be LPS (J.P. Remis, D. Wei, A. Gorur,J. Haraga, S. Allen, H.E. Witkowska, J.W. Costerton, J.E.Berleman and M. Auer, submitted) that could account forthe observed distance, suggesting that LPS could play arole in the OMV–cell OM recognition. However, while spe-cific cell–cell recognition seems plausible via LPS and cellsurface proteins, what triggers fusion is much less clear.None of the published proteomics studies has yetrevealed a clear candidate for OMV–OM fusion.

OMV as nano-sized drug delivery vehicles

Outer membrane vesicles have been successfully usedfor vaccination purposes as they are much more safe asan antigen compared with the entire bacterial cell andmay contain a sufficient number of cell-surface markers

Fig. 3. Vesicles in pathogenesis. Images of P. aeruginosa in infected mouse tissue viewed with TEM. Numerous bacteria (PaB, P. aeruginosabiofilm) are visible surrounding the blood vessel wall (VW); red blood cells (RBC) are apparent in the vessel lumen. Pseudomonas aeruginosavesicles are present mingled with extracellular matrix that appears as dark fibres between cells (arrow). Images are at the followingmagnifications: ¥5500 (A), ¥16 500 (B), ¥68 750 (C) and ¥110 000 (D) (Images adapted from Schaber et al., 2007).

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(LPS and proteins) to stimulate an immune response(Bernardini et al., 2007; Collins, 2011; Sanders andFeavers, 2011). Another application of OMVs hasreceived much less attention, namely its potential for cell-and species-specific drug delivery. Given the prevalenceof this delivery vehicle in bacterial biofilms, one couldutilize this system for the undiluted, high concentrationdelivery of cell toxins, which would only affect theintended target. If we had a detailed understanding of thetarget cell specificity of OMVs, we could mimic the designand synthetically produce OMV or even better, use appro-priately engineered bacteria themselves to produce largequantities of secreted OMV with a content and specificityof choice.

Concluding remarks

The field of OMVs, like many important subjects inmodern biology, has taken its journey from original disbe-lief of the validity of the presented data to acceptance thatbacterial outer membrane are not only real but likely havea central function in microbial physiology and communitylife. The documented delivery of vesicles for exchange ofDNA, proteins and small molecules warrants closer atten-tion. The fact that vesicles in some cases can fill most ofthe extracellular space in a biofilm (Palsdottir et al., 2009)suggests that they serve an important function, as theproduction of OMVs surely is energetically very costly. Ifvesicles are used for intraspecies cell–cell communica-tion, material sharing, and possibly even horizontal genetransfer, they immediately become a high-value targetfor pharmacological research. Knocking out a species-specific communication system and keeping antibiotics-sensitive strains from becoming resistant via horizontalgene transfer and exchange of genetic material (and/orproteins) would seem highly desirable. Since there isno obvious evolutionary link/protein similarity betweenprokaryotic and eukaryotic vesicle biogenesis systems,one could argue that effective strategies against bacterialvesicle biogenesis are less likely to have side-effects ineukaryotes. As we continue to make the shift in thinkingabout human health in terms of the microbial ecosystems,understanding vesicle production and exchange by bac-teria is one critical aspect to a better understanding ofmaintaining healthy microbial communities and prevent-ing pathogenesis.

Acknowledgements

This work is part of ENIGMA, a Scientific Focus AreaProgram supported by the US Department of Energy, Officeof Science, Office of Biological and Environmental Research,Genomics: GTL Foundational Science through contractDE-AC02-05CH11231 between Lawrence Berkeley National

Laboratory and the US Department of Energy, and LDRDsupport by the Director, Office of Science, of the US Depart-ment of Energy under contract DE-AC03-76SF00098 to M.A.and J.B.

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