AWARD REVIEW

Bacterial communication through membrane vesiclesMasanori Toyofukua,b

aFaculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan; bMicrobiology Research Center for Sustainability(MiCS), University of Tsukuba, Tsukuba, Japan

ABSTRACTBacteria can communicate through diffusible signaling molecules that are perceived bycognate receptors. It is now well established that bacterial communication regulates hun-dreds of genes. Hydrophobic molecules which do not diffuse in aqueous environments alonehave been identified in bacterial communication, that raised the question on how thesemolecules are transported between cells and trigger gene expressions. Recent studies showthat these hydrophobic signaling molecules, including a long-chain N-acyl homoserine lac-tone signal produced in Paracoccus denitrificans, are carried by membrane vesicles (MVs). MVswere thought to be formed only through the blebbing of the cell membrane, but newfindings in Pseudomonas aeruginosa and Bacillus subtilis revealed that different types ofMVs can be formed through explosive cell lysis or bubbling cell death, which findings havecertain implications on our view of bacterial interactions.

ARTICLE HISTORYReceived 11 March 2019Accepted 7 April 2019

KEYWORDSQuorum sensing; membranevesicle; explosive cell lysis;bubbling cell death; biofilm

In 1970 it was reported that Aliivibrio fischeri,a marine bacterium present in bobtail squid(Euprymna scolopes) emits light in a population den-sity-dependent manner [1]. Such population density-dependent bacterial communication is called quorumsensing [2]. In general, quorum sensing is consideredto regulate extracellular public goods which providesocial benefits to the surrounding cells. N-acyl homo-serine lactone (AHL) signals are mainly used in pro-teobacteria of Gram-negative bacteria in quorumsensing. AHLs consist of a lactone ring and an acylside chain with length ranging from 4 to 20 carbonsand can be modified [3]. Genome data suggest thatover 150 species of Gram-negative bacteria havehomologs of the AHL synthase gene (luxI). Thereare also some cells that do not have luxI-homologor have mutated luxI-homolog, but possess the LuxR-homolog receptor proteins suggesting that they usethe signaling molecules of other bacteria [4].

Some bacteria produce several signals which sig-naling systems intergrade by regulating each other.For example, Pseudomonas aeruginosa, an opportu-nistic pathogen, produces mainly two types of AHLsC4-HSL and 3-oxo-C12-HSL and also a quinolonesignal, Pseudomonas quinolone signal (PQS). Thesesignals are global transcription factors that are eachrecognized by different receptors and affect theexpression of over 200 genes, involving genes relatedto the pathogenicity of the bacterium [5].

The canonical quorum sensing model assumes thatthe signaling molecules freely diffuse in and out of thecells. However, hydrophobic signals such as long-chain

AHLs are used in many bacteria, which do not diffusefreely in aquatic environments and accumulate in thecells. New findings indicate that these signals are trans-ported to cells by membrane vesicles (MVs) [6–9]. MVsrange from 40 to 400 nm in size and they are producedbymost bacteria [10–12]. MVs have various roles such ashorizontal gene transfer, cell-to-cell communication andserve as a decoy against threats such as phage and mem-brane targeting antibiotics, thereby increasing the cellsurvival. They can transport virulence factors to hostcells and also stimulate the immune response whichtrait is applied in vaccine development. MVs have alsobeen identified in natural environments, includingoceans and activated sludge, and are reported to contri-bute to the element cycle of the ecosystem [8,13].

MVs were originally thought to be produced by theblebbing of the membrane [10]. Recent findings showthat MVs can also be formed through the lysis of thecells [14–16]. These different routes of MV formationlead to different types of MVs that will serve differentbiological roles [17]. In this review, I will introducesome recent progress in research that may be of useon understanding the diversity of bacterial MVs.

MV-mediated signal delivery

Recent research has clarified that MVs are involved inquorum sensing. This was first shown in PQS deliveryby MVs in P. aeruginosa. PQS trigger MV formation byintercalating into the outer leaflet of the outer mem-brane [18–20]. The generated MVs carry PQS that canbe further utilized by the cells. This suggests that MVs

CONTACT Masanori Toyofuku toyofuku.masanori.gf@u.tsukuba.ac.jpThis review was written in response to the author’s receipt of the JSBBA Award for Young Scientists in 2018.

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY2019, VOL. 83, NO. 9, 1599–1605https://doi.org/10.1080/09168451.2019.1608809

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might also contain signals other than PQS that arehydrophobic. AHLs which are highly hydrophobicand accumulate in the cell envelope have been reportedin a number of bacteria [21,22]. In general, the hydro-phobic property of AHLs depends on acyl side-chainlength [23]. While short-chain AHLs were shown tofreely diffuse out of the cells, long-chain AHLs requiretransporters to pass the membrane [24–27].

Paracoccus denitrificans, a denitrifying bacterium thatis frequently identified in activated sludge, producesC16-HSL, which is highly hydrophobic (Figure 1(a)). Ithad been shown that most of the C16-HSL producedremains in the cell membrane [21], and has becomea question of how C16-HSL is released from the cellsand disperse into aquatic environments.

Analysis of P. denitrificans has revealed that itproduces MVs to which C16-HSL is strongly asso-ciated [8] (Figure 1(b,c)). Importantly, C16-HSL con-tained in the MVs is passed to the cell, where itregulates quorum sensing-controlled gene expression.As the induction of MV production results in anincreased extracellular C16-HSL concentration, itappears that C16-HSL release from the cells is largelydependent on MVs [8].

Binary signaling through MVs in bacteria

Importantly, the amount of C16-HSL associated in oneMV particle was above the threshold concentration that

triggers the quorum-sensing response in a cell [8]. Thisimplies that MV-driven quorum sensing system is fun-damentally different as opposed to the canonicalquorum-sensing system. The canonical quorum sensingsystem assumes a free diffusion of the signals that coor-dinate gene expressions of the local population. In con-trast, signals packed in MVs would become binary andtherefore be transported more unevenly between cells,leading to heterogeneous gene expressions in the popu-lation. In addition, the binary signaling system wouldallow long-distance signaling, as signal concentrationwould be maintained over time and through space,while free-diffusion of the signals results in infinitedilution of the signal.

Another advantage of using MVs in bacterial com-munication is that MVs can uptake signals from theenvironment and transport them to the target bac-teria [7] (Figure 1(c)). P. denitrificans can respond toother long-chain AHL signals produced by otherbacteria in addition to its own signal, C16-HSL.MVs produced by this bacterium were shown tosequester C12-HSL to C18-HSL signals from theenvironment, which could be further used by thisbacterium as its own signal. Since MVs produced byP. denitrificans have a propensity of self-recognitionand quickly fuse to cells of the same type than toother bacteria, the signals can be kept away fromother bacteria. Thus, P. denitrificans can use the sig-nals produced by other bacteria and such signal

100nm

a

b cLong-chain AHLs

C16-HSL released through MVs

Uptake by MVs

Figure 1. AHL signal delivery through MVs in P. denitrificans. (a) P. denitrificans produce C16-HSL. (b) MVs isolated fromP. denitrificans. Reproduced from Ref [8]. (c) Involvement of MVs in quorum-sensing of P. denitrificans. C16-HSL produced byP. denitrificans can be released to the environment via MVs and delivered to the targeting cell. MVs can also uptake free long-chain AHLs from the environment that can be used by P. denitrificans.

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piracy may be advantageous for this bacterium totrigger the quorum-sensing response in polymicrobialcommunities [28].

MV formation according to the environment

It has been shown in various bacteria that MVs areheterogenous despite being released under the sameconditions, suggesting that they may be produced bymultiple routes. P. aeruginosa has undergone muchresearch as a model bacterium for studying MV forma-tion [29]. This bacterium forms biofilms in which MVscan be isolated from. One feature of biofilms is that thebacterial cells are covered in a slimy substance called theextracellular matrix, which is composed of substancessuch as polysaccharides, extracellular DNA and pro-teins [30,31]. This extracellular matrix plays a crucialrole in cell-to-cell and cell-to-surface adhesion.A comprehensive analysis of extracellular matrix pro-teins using a proteomics approach revealedmanymem-brane proteins originating from MVs in P. aeruginosabiofilms matrix [32]. Further analysis showed that theseMVs derived from biofilms have different protein pro-files compared to planktonic cells, suggesting that thecontent of MVs reflects the physiology of the bacteria[32,33]. While MVs derived from planktonic cells carryvirulence factors such as LasA, LasB and alkaline pro-tease B, MVs derived from biofilms carries many pro-teins related to iron acquisition such as FptA [32]. Therole of these proteins in MVs is yet to be investigated.

MV production in P. aeruginosa under anoxicconditions

P. aeruginosa has a versatile metabolism that allows thisbacterium to conserve energy using nitrogen oxides aselectron acceptors of the respiratory chain in theabsence of oxygen [34,35]. This process is called deni-trification where NO3

− or NO2− is reduced to nitrogen

gases (NO, N2O and N2). P. aeruginosa is a completedenitrifying bacterium that possesses all sets of enzymesthat reduce NO3

− to N2 (Figure 2(a)). Denitrification isalso important for nitrogen cycling of the ecosystem.Four sequential steps are involved in denitrificationwhere each step is catalyzed by NO3

− reductase (NAR),NO2

− reductase (NIR), NO reductase (NOR), and N2

O reductase (NOS), respectively. The expressions ofthese enzymes are regulated in response to low-oxygen and the presence of the nitrogen oxides.Interesting, cbb3-type cytochrome c oxidases that sup-port aerobic respiration is also highly expressed underanoxic conditions, and were found to induce NO accu-mulation [36]. The NO accumulation leads to cell elon-gation in P. aeruginosa under anoxic conditions that areessential for biofilm formation under this condition[37], and have a large impact on the ecology of thisbacterium [38,39]. In addition to physicochemical

conditions, quorum-sensing control identificationactivity in P. aeruginosa [40,41] (Figure 2(a)). 3-oxo-C12-HSL regulates denitrification through C4-HSL sig-naling by repressing the gene expression of denitrifica-tion genes [40]. In contrast, PQS affects enzyme activitydirectly even in the absence of its cognate receptor PqsR[41]. The direct influence of PQS on the respiratorychains adds information on the multifunctionality ofthis signaling molecule [42,43]. It was later shown thatPQS affects the enzyme activity and growth of bacteriaother than P. aeruginosa [44]. The QS regulation ofdenitrification is also involved in biofilm formation ofthis bacterium [45,46] (Figure 2(b)).

The canonical route for MV formation in Gram-negative bacteria is the blebbing of the outer membrane(Figure 3). As already explained above, in P. aeruginosa,PQS is one of the major factors that induce membraneblebbing by intercalation into the outer leaflet of theouter membrane. MVs are also formed under condi-tions where PQS is not produced. Because PQS bio-synthesis requires oxygen, its synthesis cannot occurunder anoxic conditions [41,47]. More MVs wereformed under anoxic conditions than under aerobicconditions, indicating a new route for MV biogenesis[48]. This mechanism involves a gene encoded in the

PAO1

ΔrhlI

PAO1

ΔrhlI

5-day-old biofilm

-NO3-

+NO3-

6.4 2.8

6.2 2.8

30.6 4.0

13.1 2.2

Proteins (µg / mm2)

a

b

NO3- NO2

- NO N2O N2

Low O2N-oxides PQS

C4-HSL 3oxoC12HSL

Denitrification

Figure 2. Influence of quorum sensing on denitrificationin P. aeruginosa. (a) Influence of quorum-sensing on denitrifica-tion. In addition to physiochemical factors such as low O2

tension and N-oxides, quorum-sensing signals influence deni-trification in P. aeruginosa. Modified from Ref [39]. (b)Influence of rhlI on P. aeruginosa biofilm formation. Theimage shows 5-day-old biofilms formed in flow cells with orwithout NO3

− added. ΔrhlI is a strain that does not produceC4-HSL. This strain has higher denitrifying activity comparedto the WT (PAO1) leading to increased biofilm formation.Reproduced from Ref [45].

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pyocin gene cluster. Pyocin is a type of bacteriocinproduced by P. aeruginosa that has bacteriolytic activityagainst different P. aeruginosa strains but not to thepyocin-producing strain. Pyocin production is underthe control of the stress response induced by DNAdamage. Under anoxic conditions, the DNA damagestress response is induced by self-produced nitric oxide(NO) that is an intermediate of denitrification [35].Several reports have shown that MVs produced byP. aeruginosa contain pyocin-derived proteins.

However, its role in MVs had remained unclear.When genes involved in the pyocin release were deleted,MV production decreased markedly under anoxic con-ditions [48].Meanwhile, under aerobic conditions, pyo-cin-producing genes did not affect MV formation.These results indicated that P. aeruginosa possessesMV-inducing mechanisms that differ depending onthe environmental conditions.

MV formation through explosive cell lysis inP. aeruginosa

Further study of the MV formation involving the pyo-cyanin gene, led to the discovery of a novel MV forma-tion which process is called explosive cell lysis [14]. Theconventional model forMV formation in Gram-negativebacteria supposes blebbing of the outer membrane that isinduced by intercalation of a molecule in the membraneor imbalanced synthesis of the membrane and peptido-glycan layer [10] (Figure 3). In contrast, live cell imagingof explosive cell lysis indicated that shattered membranederived from lysed cells round up and form MVs [14](Figure 4(a)). This process is triggered by a peptidoglycandegrading enzyme, endolysin, which is encoded in thepyocin gene cluster in P. aeruginosa, and generally

Blebbing Explosive cell lysis Bubbling cell death

Gram-negative bacteria Gram-positive bacteria

Figure 3. Different pathways for MV formation. Gram-negative bacteria produce MVs through blebbing of theouter membrane or through explosive cell lysis. Gram-positive bacteria produce MVs through damaging of the cellwall such as bubbling cell death. Both explosive cell lysis andbubbling cell death are induced by endolysin.

500 nm

0 min 30 min 60 min

Cell wall

MV

5µm

Explosive cell lysis

Bubbling cell death

a

b

Figure 4. Endolysin triggered MV formation. (a) Explosive cell lysis in P. aeruginosa. The shattered membrane from theexploded cell rounds up and forms MVs. Reproduced from REF [14]. (b) Bubbling cell death in B. subtilis. Cells forming MVsindicated by the white arrows die as indicated by the staining with SYTOX Green. A 3D model constructed from cryo-electronmicroscopy is shown in the lower panel. Reproduced from [15].

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involved in the release of dsDNA phage in bacteria.Under normal growth conditions, endolysin is expressedin only 1% of the total cells, that is regulated by RecA.The population of endolysin expressing cells increasesunder DNA damaging stress conditions, including bio-films and anoxic conditions.

RNA sequencing of MVs hinted the involvementof endolysin in MV formation. When MV-derivedRNA was compared with cell-derived RNA, tran-scripts of the pyocin-related gene were concentratedin MVs. Meanwhile, DNA was contained randomlyin MVs, rather than specific regions concentrated[14]. The accumulation of certain RNA supports theidea that the content of MVs reflects the physiologyof the cells. Moreover, later studies indicate the bio-logical roles of small RNAs in MVs, that is anotherexciting topic to be explored [49].

As it was previously believed that MV formation isnot accompanied by cell lysis or cell death, the find-ing of explosive cell lysis opens up new routes for MVformation. Taken together, MVs can be generatedfrom different routes either by blebbing of the mem-brane or by cell lysis and would result in the genera-tion of different types of MVs [17]. For example,MVs derived from cell lysis may contain cytoplasmicmaterials such as DNA and RNA, that are found inMVs of most bacteria. However, those cytoplasmicmaterials would not be found in an MVs raised fromblebbing of the outer membrane.

MV formation through bubbling cell death inB. subtilis

As Gram-negative and Gram-positive bacteria havedistinct cell wall structures, it was thought that MVswould form through different mechanisms betweenthese two types of cells. One of the biggest questionsin MV formation was how Gram-positive bacteriarelease MVs [12]. Gram-positive bacteria possessa thick cell wall that only allows molecules below 2nm to pass [50]. MVs are typically 40–400 nm indiameter making it difficult to understand howthese MVs pass through the cell wall and are released.Since endolysin is conserved in both Gram-negativeand Gram-positive bacteria, we assumed that endoly-sin would be involved in MV formation in Gram-positive bacteria. This was examined in Bacillus sub-tilis, a model bacterium of Gram-positive bacteria[15]. B. subtilis strain 168 contains 10 phage-likeregions, where endolysin is present in three of them(skin, SPβ, and PBSX). Analyses of mutants, andstrains of which endolysin can be induced togetherwith its transporter, holin, showed that these twoproteins in PBSX induce MV formation in B. subtilis.

While the same group of enzymes is involved, live cellimaging using confocal laser scanning microscopyrevealed different findings to that of explosive cell lysis

(Figure 3, Figure 4(b)). B. subtilis did not explode butrather, the cell died retaining their morphology whenreleasing MVs [15]. Bright field microscopy imagesrevealed that these cells are ghost cells which contenthas been released while the cell envelope remains. ThisMV formation process was named bubbling cell death[17]. Detailed analysis using cryo-electron tomographyshowed that the cell membrane is pushed out of holes inthe cell wall generated by endolysin, after which MVsform (Figure 4(b)).

Concluding remarks

Despite that bacteria have long been treated simply likebags full of enzymes, it has become evident that they aremore active than once thought. They communicatewith each other, form biofilms, and release MVs thathave a diverse function which is yet to be fully under-stood. It was previously thought that MVs were onlyreleased from blebbing of live cells, but newfindings show they can also be released asa consequence of explosive cell lysis and bubbling celldeath that is tightly regulated by RecA to occur only inthe subpopulation [17]. The finding that endolysin isa universal trigger for explosive cell lysis and bubblingcell death may explain why most bacteria form MVs asthis enzyme is highly conserved. In addition, the invol-vement of cell death and cell lysis onMV formation hasimplications on how we may treat bacteria, since anti-biotic triggered MVs can counteract certain therapy[16]. There is growing evidence indicating that thedifferent routes of MV formation would lead to thegeneration of different types of MVs [17,33]. Theunderstanding of the biogenesis of MVs will stimulatethe MV applications as they can be utilized for variouspurposes, including in vaccines as there are currently noestablished methods for systematically inducing MVformation.

Acknowledgments

I would like to express my sincere gratitude to Prof.Dr. Nobuhiko Nomura (University of Tsukuba) andProf. Dr. Leo Eberl (University of Zurich) for theircontinuing encouragements, helpful suggestions andsupport for this research. I express my deep appreciationto Prof. Emeritus Dr. Hiroo Uchiyama (University ofTsukuba), Prof. Dr. Toshiaki Nakajima (University ofTsukuba) and Prof. Dr. Michihiko Kobayashi(University of Tsukuba) for their constant suggestions.I am deeply indebted to Prof. Dr. Yosuke Tashiro(Shizuoka University), Prof. Dr. Yutaka Yawata(University of Tsukuba), Prof. Dr. Nozomu Obana(University of Tsukuba), Ms. Rie Furukawa (Universityof Tsukuba) for their helpful discussions, advices, andsupports. Finally, I thank all the laboratory membersand all the collaborators for sharing their times andefforts in exploring the unseen universe.

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Disclosure statement

No potential conflict of interest was reported by the author.

Funding

This work was supported by JSPS KAKENHI Grant#16H016189 and JST ERATO Grant #JPMJER1502.

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