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Cite this:Chem. Commun., 2017,

53, 7569

Engineering multi-functional bacterial outermembrane vesicles as modular nanodevices forbiosensing and bioimaging†

Qi Chen,a Sharon Rozovsky b and Wilfred Chen *a

Outer membrane vesicles (OMVs) are proteoliposomes derived

from the outer membrane and periplasmic space of many Gram-

negative bacteria including E. coli as part of their natural growth

cycle. Inspired by the natural ability of E. coli to sort proteins to

both the exterior and interior of OMVs, we reported here a one-pot

synthesis approach to engineer multi-functionalized OMV-based

sensors for both antigen binding and signal generation. SlyB, a

native lipoprotein, was used a fusion partner to package nano-

luciferase (Nluc) within OMVs, while a previously developed INP-Scaf3

surface scaffold was fused to the Z-domain for antibody recruiting. The

multi-functionalized OMVs were used for thrombin detection with a

detection limit of 0.5 nM, comparable to other detection methods.

Using the cohesin domains inserted between the Z-domain and INP,

these engineered OMVs were further functionalized with a dockerin-

tagged GFP for cancer cell imaging.

Liposomes are frequently used in the many areas of therapyand diagnosis.1–3 Generally, liposomes are made by dissolvinglipids in organic solvents and sonication is used to ensuredispersion of liposomes of desirable sizes in aqueous solution.Different compartments of liposomes can be chemically modifiedfor added functionalities. For example, the lumen can be used toentrap molecules such as drugs or reporter dyes, and the surfacecan be decorated with ligand-specific moieties for cellular targetingand delivery.

Efforts have been made to utilize naturally occurring bacterialvesicles to achieve similar functionalities as liposomes but withsimplified production procedures. Outer membrane vesicles(OMVs) are naturally derived from the surface of Gram-negativebacteria during their growth cycles.4,5 They can self-assemble intoproteoliposomes with an average size of 20–250 nm, making

them highly compatible for drug delivery and biosensing.6,7 Asmore than 60 membrane proteins have been identified to beassociated with the OMV membranes,8 these proteins can beexploited as anchors to target fusion proteins to the desiredlocation of OMVs. Current studies mainly focus on surfacemodifications with multiple protein domains for applications asvaccines9–13 or nano-sized scaffolds for enzyme assembly.14

Recently, OMVs have been functionalized both in the lumenand on the surface for siRNA silencing. A tumor-specific affibodywas displayed on the OMVs for specific cell targeting, while electro-poration was used to encapsulate siRNA within the lumen forprotective delivery.15 However, the ability to decorate OMVs withmultiple functional protein moieties remains a major challenge.

In this work, we developed a new approach to enablesimultaneous functionalization of the interior and exterior ofOMVs as a modular platform for biosensing applications. Thenative E. coli outer membrane lipoprotein SlyB was exploited asan anchor to target proteins to the interior of OMVs. SlyB is a155 residues long protein that contributes to the integrity of thecell envelope,16 and is anchored to the periplasmic side of theouter membrane through its N-terminus lipid moiety.17,18

Nanoluciferase (Nluc) was selected as the fusion partner forSlyB based on its small size (19 kDa and monomeric) and easeof detection.19 To enable multi-targeting, an ice nucleationprotein (INP)20–22 anchor was used to simultaneous display theantibody-binding Z domain on the OMV surface. A tri-functionalscaffold, Scaf3,23 containing three orthogonal cohesin domainswas inserted between the INP and Z domain for additionaldecorations using the corresponding dockerin-tagged proteins(Fig. 1).

The SlyB gene was PCR amplified and inserted into pMal-c5xfor overexpression in the OMV-hypersecreting JC8031 cells.24

To demonstrate that SlyB can be used as a carrier to directproteins to the interior of OMVs (Fig. 2A), nanoluciferase(Nluc)19 was chosen as the fusion partner. Nluc can be easilydetected as it does not require ATP for light production and isup to 10-fold more active than luciferases from other species.The SlyB and SlyB-Nluc were engineered with a C-terminal

a Department of Chemical and Biomolecular Engineering, University of Delaware,

Newark, DE 19716, USA. E-mail: wilfred@udel.edu; Fax: (+1) 302 831 1048;

Tel: (+1) 302 831 6327b Department of Chemistry and Biochemistry, University of Delaware, Newark,

DE 19716, USA

† Electronic supplementary information (ESI) available: Detailed information of thecloning process, Nluc measurements and GFP binding. See DOI: 10.1039/c7cc04246a

Received 1st June 2017,Accepted 16th June 2017

DOI: 10.1039/c7cc04246a

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hexahistidine tag to assist in detection. Overexpression of either SlyBor SlyB-Nluc in whole-cell lysates and the membrane fraction wasfirst demonstrated by Western blot (Fig. S1, ESI†). After collectingthe resulting OMVs by ultracentrifugation, the presence of theseproteins in OMVs was also confirmed by Western blot (Fig. 2B).

To confirm the functionality of Nluc, the collected OMVswere incubated with the Nano-Glo luciferase assay reagent andthe ability to produce light was measured using a microplatereader. The photons emitted from the engineered OMVs containingSlyB-Nluc was more than 105 fold higher than the control OMVs(Fig. 2C). This highly visible level of light emission is very attractivefor both immunoassays and in vivo imaging (Fig. 2D).

To investigate whether SlyB anchored Nluc specifically to theinterior of OMVs, OMV samples were subjected to proteinase Ktreatment.25 Since proteinase K cannot penetrate the membrane,

only proteins displayed on the exterior of OMVs are degradedwhile proteins within the lumen are protected from proteolysis(Fig. 3A). For comparison, OMVs displaying INP-Scaf3 on theexternal surface was used as a positive control. As expected, whenboth engineered OMVs were exposed to proteinase K, INP-Scaf3was degraded completely while SlyB-Nluc remained intact (Fig. 3B).When a detergent (1% SDS) was added to lyse the OMVs, even theinterior SlyB-Nluc was now accessible to proteinase K and degradedas expected (Fig. 3B). The result confirms that SlyB functionscorrectly as a membrane anchor and targets the fusion partnerspecifically to the OMV’s interior. This new SlyB anchor, incombination with the INP anchor, provides a complementaryapproach for multi-functionalization of OMVs for biosensingand imaging applications.

Because Nluc is highly active and can be used to providesensitive luminescent signal, one obvious application of theSlyB-Nluc approach is to exploit the resulting engineered OMVsas a highly modular detection agent. To do that, we must firstdevise a new approach to functionalize OMVs with a target-specific antibody. Although direct fusion of IgG or scFv to INPcan be done, this often results in low yield because of foldingchallenges.26 One simple strategy to bypass this limitation andto provide universal binding to any antibody of interest is totether a small (7 kDa) antibody-capturing moiety, Z-domain,27

onto the exterior surface of OMVs. Although our initial attemptsto tether Z domain directly with INP resulted in detectableprotein expression, no IgG binding was observed probably dueto the steric hindrance from surface glycoproteins (Fig. S2, ESI†).To address this limitation, we again employed the INP-Scaf3scaffold from our previously OMV study14 to extend the accessibilityof the Z-domain (Fig. 1). This scaffold is also particular useful asthere are three orthogonal cohesin domains available for additionalprotein decorations. A new INP-Scaf3-Z scaffold was constructed byinserting the Z-domain to the C-terminus. After verifying expressionof this new scaffold by Western blot (Fig. 4A), the functionality of theZ-domain was confirmed by the ability to bind to an alkaline

Fig. 1 Schematic of multifunctionalized OMVs. The lipoprotein, SlyB, isused to direct nanoluciferase to the interior of OMVs. The ice nucleationprotein (INP) anchor is used to display the Z-domain on the exterior ofOMVs for antibody binding. A trivalent Scaf3 scaffold containing threecohesin domains from Clostridium cellulolyticum (CC), Clostridiumthermocellum (CT) and Ruminococcus flavefaciens (RF) is sandwichedbetween INP and Z-domain for additional decoration of other functionalproteins, such as dockerin-tagged GFP, using the specific interactionbetween each cohesin–dockerin pair.

Fig. 2 (A) Schematic of Nluc targeted to the interior of OMVs by SlyB.(B) Western blot analysis of SlyB (left) or SlyB-Nluc (right) expression inengineered OMVs using an anti-his tag antibody. (C) The luminescentintensity from OMVs expressing SlyB-Nluc is 105 higher than that ofcontrol OMVs. (D) The luminescence signal emitted from SlyB-Nluc inOMVs is highly visible.

Fig. 3 (A) An illustration of the proteinase K accessibility assay. (B) ProteinaseK accessibility assay was used to evaluate the localization of Nluc. (1) INP-Scaf3displayed on OMV surface was digested by proteinase K. (2) SlyB-Nluctargeted to the interior of OMVs was protected by the lipid layers fromproteinase K digestion. (3) A detergent was added to disrupt the intact OMVmembrane. SlyB-Nluc was digested by proteinase K only when the OMVintegrity was disrupted.

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phosphatase-conjugated, anti-mouse secondary antibody basedon interaction with the Fc region (Fig. 4B).

We next proceeded to investigate the ability to target bothSlyB-Nluc and INP-Scaf3-Z to OMVs. Presence of both proteinswas first verified by western blot (Fig. 4A). Co-expression had noimpact on targeting the individual protein, as the expressionlevels were virtually identical to those from OMVs expressingonly a single protein (Fig. 4A). Dynamic light scattering wasused to demonstrate the presence of intact vesicles with anaverage diameter of 41 � 2 nm (Fig. S3, ESI†). To assess thefunctionality of each protein, we first measured the level ofluminescence. Dual expression did not impact the level of Nlucactivity, which was on par with the single Nluc-modified OMVs(Fig. S4, ESI†). The ability of the engineered OMVs to bind IgGwas further evaluated by incubating with FITC-labeled IgG.After binding and washing twice, the level of bound IgG wasquantified by measuring the OMV-associated fluorescence. Thelevel of fluorescence was 8-fold higher when compared withunmodified OMVs, indicating specific IgG binding by the displayedZ-domain (Fig. 4C). From Fig. 4A, the ratio of INP-Scaf3-Z domainvs. SlyB-Nluc in OMVs is roughly 16 : 1, suggesting that theseengineered OMVs can provide substantial signal amplification forantigen sensing.

To evaluate the performance of OMVs as immunosensors,thrombin was used as the analyte.28–31 A control Z domain-ELP-Nlucfusion protein, which provides only a 1 : 1 signal amplification, wasused for comparison.30 As expected, both the control protein andOMVs were successful in detecting 10 nM thrombin coated directlyonto a 96-well plate (Fig. 5A). Consistent with the higher ratio ofNluc per Z-domain, the luminescence signal was more than 10-foldhigher using the OMV sensor. This improved sensitivity enabled thedetection of even 0.5 nM thrombin using this new OMV sensor(Fig. 5B), a value comparable to other reported thrombin detectionmethods.32–34 However, the key benefits are the ease of OMVpreparation by one-pot biosynthesis using simple fermentationand the flexibility to decorate with enzymes that provide eitherelectrochemical35 or colorimetric36 detection.

To further demonstrate the capability of the OMV sensor forcancer cell detection, a cancer-specific surface marker, MUC1,37

was selected. To enable fluorescence imaging, a dockerin-taggedGFP38 was first assembled onto the displayed INP-Scaf3-Z scaffold

using the high-affinity interaction between the CT cohesin anddockerin domain (Fig. 6A). For cancer cell detection, HeLa cells werefixed with formaldehyde before the addition of GFP-decoratedOMVs. Brightly fluorescent HeLa cells were detected only whenthe anti-MUC1 antibody was added (Fig. 6B), indicating lowbackground interaction between OMVs and HeLa cells. In additionto fluorescence, detection can also be done by luminescence.This is precisely the flexibility offers by this modular approachof functioning OMVs.

In summary, we presented a modular approach to engineerOMVs as a powerful biosensing platform by simultaneousdecoration of both the interior and exterior with functionalcapturing and reporting moieties. Utility of the Z-domain tocapture antibodies by binding to the Fc region provides theflexibility to adapt this sensing platform toward virtually anyantigen of interest. Encapsulation of Nluc inside OMVs usingthe lipoprotein anchor SlyB enables detection by highly sensitiveluminescence signal. As more than 60 lipoproteins have beenidentified from native OMVs,17,18 additional reporters can be easilyembedded for parallel sensing. Presence of three orthogonalcohesin domains further expands our ability to decorate otherreporting moieties such as GFP for cancer cell imaging. Sincemulti-functional OMVs can be easily customized by geneticmodifications, a virtually unlimited combination of capturing

Fig. 4 Western blot analysis of SlyB-Nluc and INP-Scaf3-Z co-expression.(A) Detection of protein expression by probing the C-terminus his-tag.(B) Detection of Z-domain by incubation with an anti-mouse secondaryantibody. Lane (1) OMV expressing SlyB-Nluc alone, lane (2) OMV expressingINP-Scaf3-Z, and lane (3) OMV expressing both SlyB-Nluc and INP-Scaf3-Z.(C) Binding of FITC-IgG to OMVs expressing SlyB-Nluc only (control) orco-expressing SlyB-Nluc and INP-Scaf3-Z.

Fig. 5 (A) Thrombin detection with Z-ELP-Nluc fusion proteins andengineered OMVs. (B) Thrombin detection using 100 nM engineeredOMVs.

Fig. 6 Phase contrast and fluorescence images of HeLa cells detectionusing GFP-decorated OMVs. (A) Binding of CT dockerin-tagged GFP toOMVs expressing SlyB-Nluc only (control) or co-expressing SlyB-Nluc andINP-Scaf3-Z. The resulting GFP fluorescence was 8-fold higher for theengineered OMVs vs. the control OMVs. (B) Detection of HeLa cells withGFP-decorated OMVs in the presence or absence of anti-MUC1 antibodies.Scale bar = 10 mm.

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and reporting moieties can be created for a wide range ofapplications including live cell imaging as demonstrated withother synthetic vesicles.3

We would like to acknowledge the funding support fromNSF (CBET1264719 and CBET1604925).

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