RESEARCHARTICLEAPPLIEDPHYSICALSCIENCESOPEN ACCESSEncapsulated bacteria deform lipid vesicles into flagellatedswimmersLucasLeNagarda,AidanT.Browna,AngelaDawsona,VincentA.Martineza,WilsonC.K.Poona,1,andMargaritaStaykovab,1EditedbyHowardStone,PrincetonUniversity,Princeton,NJ;receivedApril8,2022;acceptedJune29,2022We study a synthetic system of motileEscherichia colibacteria encapsulated inside giantlipid vesicles. Forces exerted by the bacteria on the inner side of the membrane aresufficient to extrude membrane tubes filled with one or several bacteria. We show thata physical coupling between the membrane tube and the flagella of the enclosed cellstransforms the tube into an effective helical flagellum propelling the vesicle. We developa simple theoretical model to estimate the propulsive force from the speed of the vesiclesand demonstrate the good efficiency of this coupling mechanism. Together, these resultspoint to design principles for conferring motility to synthetic cells.activematter|motilebacteria|Escherichiacoli|lipidvesiclesThe interaction of active and passive matter lies at the heart of biology. Active matter(1) consists of collections of entities that consume energy from their environment togenerate mechanical forces, which often result in motion. Thus, the cell membrane,whose essential component is a passive lipid bilayer, can actively remodel during cellgrowth; motility; and, ultimately, cell evolution, under the forces exerted by a hostof active agents. For example, continuous polymerization–depolymerization of actin inthe cytoskeleton deforms the eukaryotic cell membrane into two- and one-dimensionalprotrusions (lamellipodia and filopodia) that can move the whole cell (2, 3). Similar actin-supported membrane protrusions are thought to have facilitated the accidental engulfmentof bacteria that led to the emergence of eukaryotic cells (4). The biophysics of activemembranes has therefore been subjected to interdisciplinary scrutiny (5). More recently,learning how to create active membranes systems that deform, divide, and propel hasbecome a priority area in the drive to synthesize life ab initio (6).Lipid vesicles enclosing natural or artificial microswimmers are becoming a modelsystem for studying active membranes in vitro (7–15). Such composites have also directbiological relevance. For instance, from inside their eukaryotic hosts, bacterial pathogenssuch asRickettsia rickettsiiorListeria monocytogenes(16, 17) continue their life cycles byhijacking the actin polymerization–depolymerization apparatus of their hosts and pushingout a tube-like protuberance from the plasma membrane. The pathogens then contactother host cells or escape into the surrounding medium by means of these membranetubes (18).To date, research in coupling swimmers with membranes has mostly been theoreticaland numerical. Such models have predicted a range of interfacial morphological changesand, in some cases, net motion of the interface (7–13). The experimental realizationof these systems was only recently achieved by encapsulating swimmingBacillus subtilisbacteria (14) and synthetic Janus particles (15) in giant lipid vesicles. Both experimentsreported nonequilibrium membrane fluctuations and vesicle deformations, ranging fromtubular protrusions to dendritic shapes. However, net motion of the vesicles was notobserved in either case.Here we present a similar experimental design but with markedly different outcome.Escherichia coli, another common motile bacterium, also extrudes membrane tubes but inaddition sets the whole vesicle into motion. We demonstrate that such motion is due toa physical coupling between the flagella bundle of the enclosed cells and the tubes. Thetube–flagella composite functions as a helical propeller for the entire vesicle.In biology, the specificity of interactions between bacteria and the membranes of eu-karyotic hosts underlies the plethora of parasitic and symbiotic relations that have emergedbetween cells (18–20). Likewise, our observations illustrate the importance of small detailsin the design of active matter systems (21). Encapsulated bacteria propelled by a singlebundle of helical flagella can generate net motion of the vesicles, whereas encapsulatedswimmers propelled at similar speeds by phoresis fail to do so (15). These observationsillustrate the fact that it is dangerous to proceed from coarse-grained simulations or theorythat neglect such details to predict the behavior of particular systems. At the same time,our results point to a design principle for conferring motility to artificial cell models.SignificanceSwimmingbacterialpathogenscanpenetrateandshapethemembranesoftheirhostcells.WestudyanartificialmodelsystemofthiskindcomprisingEscherichiacolienclosedinsidevesicles,whichconsistofnothingmorethanasphericalmembranebag.Thebacteriapushoutmembranetubes,andthetubespropelthevesicles.Thisphenomenonisintriguingbecausemotioncannotbegeneratedbypushingthevesiclesfromwithin.Weexplainthemotilityofourartificialcellbyashapecouplingbetweentheflagellaofeachbacteriumandtheenclosingmembranetube.Thisconstitutesadesignprincipleforconferringmotilitytocell-sizedvesiclesanddemonstratestheuniversalityoflipidmembranesasabuildingblockinthedevelopmentofnewbiohybridsystems.Authoraffiliations:aSchoolofPhysicsandAstronomy,TheUniversityofEdinburgh,EdinburghEH93FD,UnitedKing-dom; andbDepartment of Physics, Durham University,DurhamDH13LE,UnitedKingdomAuthorcontributions:L.L.N.,W.C.K.P.,andM.S.designedresearch;A.D.contributedthebacterialstrain;L.L.N.andV.A.M. performed experiments; L.L.N. and A.T.B. per-formed the modeling; L.L.N., A.T.B., W.C.K.P., and M.S.analyzed the data and interpreted the results; L.L.N.,A.T.B.,A.D.,V.A.M.,W.C.K.P.,andM.S.discussedtheresultsandcommentedonthemanuscript;L.L.N.wrotethefirstdraft;andL.L.N.,A.T.B.,W.C.K.P.,andM.S.wrotethepaper.Theauthorsdeclarenocompetinginterest.ThisarticleisaPNASDirectSubmission.Copyright © 2022 the Author(s). Published by PNAS.This open access article is distributed underCreativeCommonsAttributionLicense4.0(CCBY).SeeonlineforrelatedcontentsuchasCommentaries.1To whom correspondence may be addressed. Email:w.poon@ed.ac.ukormargarita.staykova@durham.ac.uk.This article contains supporting information online athttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2206096119/-/DCSupplemental.PublishedAugust15,2022.PNAS2022 Vol.119 No.34 e2206096119https://doi.org/10.1073/pnas.22060961191of7
ADEFBCFig.1.Bright-fieldimagesofGUVsinthecourseofosmoticdeflationdisplayingmembranefluctuationsandtubularprotrusions.(A)TenseGUVencapsulating∼10bacteria(darkandlighterspotswithintheGUV)anddisplayingnovisiblefluctuations.(B)DeflatedGUVdisplayingbacteria-amplifiedfluctuationsillustratedby three superimposed contours. (C) GUV displaying six tubular protrusions extruded by bacteria, each containing a single cell. (D) GUV displaying a long(100μm) tube with three cells at its extremity. (E) Image sequence showing a single cell (white arrow) extruding a tube by pushing on the membrane.(F)ImagesequencerecordedonadifferentGUV,showingasecondcell(whitearrow)enteringatubepreviouslyextrudedbyasinglebacterium(blackarrow),whichleadstoelongationofthetube.(Scalebars,10μm.)ResultsEncapsulated Bacteria Extrude Lipid Tubes.We encapsulateda smooth-swimming strain of K-12–derivedE. coliin giantunilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids (POPC) and doped with afluorescent dye using the inverted emulsion method (22). Theinternal medium was lysogeny broth (LB) supplemented withsucrose, which the bacteria cannot metabolize. The externalmedium was an aqueous solution of glucose, which, likesucrose, does not diffuse across the lipid membrane. Our GUVssedimented toward the glass bottom of the sample chambers,which had been pretreated with bovine serum albumin tominimize vesicle adhesion. Bacteria and vesicles were imagedwith an inverted microscope in phase-contrast and fluorescencemodes. We occasionally observed cell division in the GUVs,and the bacteria remained motile for at least∼8h, thanks tonutrients provided in the inner medium and/or endogenousmetabolism (23) enabled by the diffusion of dissolved O2throughthe membrane.In sample chambers sealed immediately after bacteria encap-sulation, the majority of GUVs appeared tense and spherical,with no visible sign of shape fluctuations (Fig. 1A). The∼10encapsulated bacteria typically swam just below the inner sur-face, reminiscent of previous observations ofE. coliin lecithin-stabilized spherical water-in-oil emulsion droplets (24). Amongthese GUVs, we occasionally observed GUVs with tubular pro-trusions containing one or more bacteria.To increase the number of GUVs exhibiting such protrusions,we used osmotic deflation to decrease the membrane tensionof our vesicles by keeping sample chambers open for a definedperiod of time,tw, before sealing for observation. Water evap-oration increased the osmolarity of the external solution by acontrolled factorα(Materials and Methods), leading to waterefflux. Progressive deflation yielded a higher fraction of GUVswith bacteria-containing tubes than sudden mixing with a hy-pertonic buffer. Attw=0, the internal and external media wereisotonic (α=1), giving mostly tense vesicles with only a fractionχ=0.17showing bacteria in tubular protrusions (seeSI Appendixfor details). At even a moderate deflation,α=1.05, some vesiclesexhibited pronounced fluctuations attw≈20min andχ=0.49.Further deflation,α=1.1,gaveχ=0.67,tw≈40min, withtube-bearing vesicles dominating our field of view (Fig. 1C). Thevesicles displayed either multiple tubes (Fig. 1C) or a single tubewith one or multiple bacteria inside (Fig. 1D).We were able to capture occasionally the rapid process of theinitiation of tubular protuberances by swimming bacteria. Thistakes∼1 s and requires a cell to swim perpendicular to themembrane (Fig. 1E). A second cell can enter an already formedtube (Fig. 1F), causing its extension. Tube elongation consumesmembrane and generates tension (Fig. 1F), resulting in a morespherical shape and preventing further tube growth. Interestingly,a bacterium swimming into a preexisting cell-free membrane tubewas never observed. Moreover, no bacteria-containing tubes wereseen with encapsulated dead cells, and bacteria swimming outsidevesicles do not pull off membrane tubes under our conditions(SI Appendix).BacteriainMembraneTubesPropelLipidVesicles.In strikingcontrast to GUVs encapsulating Janus particles orB. subtilisbacteria, which display large deformations but remain static (14,15), we observed that motileE. coliin membrane tubes wereable to propel GUVs at typical speedsv∼1μms−1(Fig. 2 andMovie S1). The motion is always tube-first with velocity vectorparallel to the tube (Fig. 2AandC–E). GUVs with bacteria but notubular protrusions remain static, suggesting that motile bacteriain the vesicle lumen do not contribute to vesicle propulsion.We found that vesicle trajectories varied from straight to curvedclockwise (CW) and counterclockwise (CCW) within the samesample (viewed from the fluid side) (Fig. 2C–E). Out of all2of7https://doi.org/10.1073/pnas.2206096119pnas.org
BDEACFig.2.PropulsionofGUVscontainingE.colicells.(A)Trajectory(yellowline,Δt=180s)ofaGUVpropelledbytwobacteriainamembranetube(blackarrows).Redarrowsindicatetheorientationoftheinstantaneousvelocityvectorat30-sintervals,andwhitedottedlinesindicatetheorientationofthetubeatthesametimepoints,showingthattheGUVswimstube-first.(B)SpeedasafunctionoftimeforthethreeGUVsdisplayedinC–E,withmatchingcolors.(C–E)Vesicletrajectoriesrecordedover200smayvaryfrom(C)straightline,to(D)counterclockwise,to(E)clockwiserotation.(Scalebars,20μm.)assessed GUV trajectories (65 in total),34%were curved CW,42%were curved CCW,20%were straight, and5%displayed areversal of their curvature during the tracking (the latter usuallytriggered by some rearrangement of the bacteria in the tube).Vesicle trajectories therefore tend to be curved, with no strongpreference between CW and CCW rotation. This contrasts withunencapsulated bacteria outside vesicles, for which only7%ofcircular trajectories near the glass slide (10 out of 146) wereCCW. Such behavior, governed by hydrodynamic interactionsbetween the bacteria and the glass substrate, agrees with previousmeasurements (25) and is consistent with a no-slip boundarycondition operating at the slide (26). It is clear that these stronghydrodynamic effects do not translate into biased rotation ofour vesicle–bacteria system, which is unsurprising given the morecomplex and variable geometry in that case.A Physical Coupling between Flagella and Membrane TubesGeneratesaPropulsiveForce.The self-propelled motion of ourbiohybrid vesicles is a surprising phenomenon because encapsu-lated bacteria are isolated from the external medium by a lipidmembrane. To get insights into its mechanisms, we turn tohigh-resolution microscopy. Imaging the membrane directly bycombining phase contrast microscopy with fluorescent imaging(Fig. 3) shows bacteria tightly wrapped in tubes withRt0.5μmalong the whole length of any tube. The portion of tube behindeach bacterial body is also noticeably thinner than the cell bodyitself. Knowing that the nearly rigid helical flagella bundle inswimmingE. colihas a helical diameterdcomparable to that ofthe cell body (27), this observation suggests that the portion oftube surrounding the flagella bundle is severely distorted from acylindrical form.Our images indeed show that the two-dimensional projectionof the membrane around the flagella bundle has a sinusoidalshape (Fig. 3A–C). We fitted the images of 10 helical tubes toa sine function (SI Appendix,Fig.S2) and determined the pitchp=2.3±0.2μmand helical diameterd=0.4±0.1μm(mean±SD), which agree with previous values for the flagella bundleofE. coli(27). High-speed imaging returned rotation frequenciesof typically 50 to 120 Hz (e.g., 90 Hz for the tube in Fig. 3B),which again falls within the range measured for the flagella bundleofE. coli(28) (Movie S2). We conclude that the lipid membranewraps closely enough around the flagella bundle to adopt its helicalshape and that the bundle retains the same geometry as in free-swimming cells.These findings suggest a propulsion mechanism that is mosteasily applied to the simplest, single-tube vesicle (Fig. 3D). Thethin membrane tube couples to the rotating helical flagella bundleof the nested bacterium, adopts its shape, and undergoes helicalmotion, which generates a thrust force. Consistent with this, weexpect the propulsive force to be proportional to the number ofbacteria in the tube if cell–cell interactions remain negligible.The Propulsive Force Scales with the Number of Bacteria inaTube.To verify this, we consider motile vesicles propelledby a single bacteria-bearing membrane tube and exclude casessuch as that in Fig. 1C. The relevant Reynolds number isRe=2Rvρos/η∼10−5,whereR∼10μmis the GUV radius,ρosis the volume mass of the outer solution, andη∼1.5 mPa sis its viscosity at 20◦C (29, 30). Inertia is therefore negligible, andthe propulsive force generated by the bacteria in a membrane tube,Fprop, is exactly balanced by the total drag force,Fdrag=ξv,acting on the composite swimmer (GUV + tube), whereξis itsfriction coefficient andvis its speed:Fdrag=ξv=Fprop.[1]We include the contribution of the GUV and of the membrane-wrapped bacteria toξbut neglect the much thinner, emptyportions of tube (SI AppendixandSI Appendix,Fig.S5). We alsoneglect wall drag because we cannot quantify the GUV–walldistance. Thus, for a GUV bearing a singleN-bacteria tube,PNAS2022 Vol.119 No.34 e2206096119https://doi.org/10.1073/pnas.22060961193of7