Synthetic Cells with Chemo–Optical Signaling Enable Targeted Capture and Killing of Bacteria

Synthetic Cells with Chemo–Optical Signaling Enable Targeted Capture and Killing of Bacteria | Research Square Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synthetic Cells with Chemo–Optical Signaling Enable Targeted Capture and Killing of Bacteria Seraphine Wegner, Xiaoran Zheng, Tarek Elsayed, Yanjun Zheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8890256/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Synthetic cells are molecularly defined systems which recreate life-like behaviors, yet their capacity to autonomously engage and act on living cells remains limited. Here, we engineer synthetic cells that combine optical and chemical signaling to achieve programmable capture and killing of bacteria with high spatial precision. To enable bacteria capture, giant unilamellar vesicles (GUVs) were functionalized with the blue light responsive protein BcLOV4, allowing light activatable adhesion to bacterial membranes. Encapsulation of Nanoluciferase allows the synthetic cells to generate an internal bioluminescent light signal, which autonomously activates BcLOV4 mediated bacterial recruitment without external illumination. For subsequent bacterial elimination, galactose oxidase was used to generate hydrogen peroxide locally at the GUV membranes, resulting in the selective killing of captured bacteria. This two-step mechanism mimics a key aspect of innate immune cells, namely localized pathogen elimination, and provides a blueprint for spatially precise antimicrobial synthetic cells. Physical sciences/Chemistry/Chemical biology/Synthetic biology Physical sciences/Chemistry/Photochemistry/Photobiology Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Inspired by the fascinating complexity of natural cells and the interactions that shape their collective behavior, researchers are increasingly employing bottom-up strategies to construct artificial cell-like analogs, also referred to as synthetic cells. 1,2 These approaches have enabled the reconstitution of diverse multicellular phenomena with different social relationships between synthetic cells, including phagocytosis, 3 predation, 4 retaliatory responses, 5 immune-like response, 6,7 synthetic tissue formation, 8,9 and cooperative behaviors. 10 To coordinate activity within such systems, synthetic cells have been programmed to communicate through adhesions, 11,12 molecular diffusion, 13,14 and optical signaling, 4,15 enabling the activation of genetic circuits, 16,17 multi-step reaction cascades, 18,19 and logical computations 20 across synthetic cell populations. These advancements progressively bridge the gap between synthetic constructs and living systems, 21,22 and increasingly allow synthetic cells to interface with natural cells. To fully unlock their potential for future technological and biomedical applications, it is critical to expand communication pathways across the synthetic-natural cell boundary. 23–25 Significant progress has been made in translating internal reactions of synthetic cells into biological cues recognizable by living cells, including activation of quorum sensing in bacteria, 26,27 induction cellular differentiation, 28,29 protection of cells from toxins 30 and modulation of growth factor signaling. 31 Yet, despite these remarkable achievements, only a limited number of studies have explored the breadth of possible interactions and emergent behaviors arising from synthetic–natural cell interfaces. Synthetic cells capable of actively preying upon and eliminating living cells represent an important frontier with bottom-up synthetic immunology. 32,33 In nature, innate immune cells such as neutrophils capture bacterial pathogens at inflammatory sites and eliminate them by locally producing high concentrations of reactive oxygen species (ROS). 34 While examples of artificial neutrophils producing ROS, 35,36 or predatory synthetic cells with contact dependent bacterial elimination exist, 37 a critical aspect for the desired spatial precision has not been considered. Neutrophils must first establish proximity to the pathogen so that ROS concentrations reach lethal levels only where the pathogens are, thereby preventing collateral damage in the surrounding tissue. Inspired by this principle, we engineered synthetic cells that recapitulate this two-step bacterial defense: activation dependent bacterial capture followed by localized elimination through ROS production (Fig. 1 ). Activation of the synthetic cells is achieved either through external blue light illumination or through an internal chemo-opto signaling network that generates a bioluminescence signal from the synthetic cell. In both cases, the light activates the membrane-anchored photoswitchable protein BcLOV4 to expose a positively charged amphipathic domain, which binds to negatively charged bacterial surfaces, resulting in bacterial capture. Only after bacteria are physically captured by the synthetic cell will local ROS production be sufficiently high, ensuring that lethal oxidative stress is restricted to the immediate vicinity of the adhered bacteria. This strategy empowers synthetic cells with the capacity to program bacteria capture and elimination, going beyond simple signal relays and establishing a foundation for a synthetic neutrophil-like cells preying on bacteria. RESULTS Light regulated bacterial capture using BcLOV4 functionalized synthetic cells. In the realization of light dependent bacterial capture, we first implemented blue light responsive adhesions on giant unilamellar vesicles (GUVs), used as our synthetic cell chassis. As a photoswitchable module, we identified the blue light switchable LOV-domain protein BcLOV4, 38 which undergoes a conformational transformation upon illumination due to a photoreaction at the flavin chromophore, exposing a positively charged amphipathic region that enables reversible binding to the negatively charged outer bacterial membrane. In the absence of light, BcLOV4 returns to its dark state conformation, losing affinity for the bacterial membrane. GUVs (lipid composition; DOPC with 1% DGS-NTA-Ni 2+ and 0.1% DiD for visualization), which were functionalized with His-tagged BcLOV4-mCherry, hereafter referred to as BcGUVs (Fig. 2 A). Confocal imaging confirmed a uniform distribution of BcLOV4-mCherry on the outer vesicle membrane, evidenced by colocalization of the DiD membrane dye and the mCherry signal (Fig. 2 B). We next examined if bacteria adhere to BcGUVs in a light dependent manner. For this, mCherry-expressing E. coli MG1655 bacteria (green) were incubated with BcGUVs (magenta), and their interactions were monitored over time by confocal microscopy (Fig. 2 C). Under continuous blue light illumination, bacteria progressively accumulated on the BcGUV surface, resulting in almost complete coverage within 60 min. In sharp contrast, in the dark, bacteria showed no measurable binding to BcGUVs over the same period. Quantification of bacterial fluorescence at the vesicle surface across multiple GUVs validated the single GUV observations (Fig. 2 D). In addition, control experiments confirmed that the blue light illumination used (0.2 mW·cm -2 , 60 min) induced no detectable phototoxicity (Figure S1 ) and that GUVs lacking BcLOV4 showed minimal interactions with bacteria (Figure S2). Taken together, these results demonstrate that BcGUVs capture bacteria in response to blue light, establishing a programmable mechanism for external control of bacterial adhesion. Intracellular bioluminescence signaling triggers autonomous bacterial capture . To transition from externally triggered adhesions to adhesions that arise from intercellular communication between synthetic cells and bacteria, we engineered synthetic cells capable of producing their own light signal. For this, Nanoluciferase (Nluc) was encapsulated inside BcGUVs, yielding Nluc-BcGUV (Fig. 3 A). Nluc catalyzes the oxidation of furimazine, producing bright bioluminescence with an emission peak near 480 nm, which spectrally matches to the absorption band of the flavin in BcLOV4 (Figure S3). Following the addition of the membrane permeable substrate, Nluc containing BcGUVs produced a strong bioluminescence signal, detectable both in bulk with a luminometer and inside individual GUVs with bioluminescence microscopy (Figs. 3 B and C). The bioluminescence intensity peaked immediately after substrate addition and decayed within approximately 60 min. We then investigated if this endogenous bioluminescence was sufficient to activate BcLOV4 on the outer GUV membrane, and thereby recruit bacteria. Indeed, when mCherry-expressing E. coli were mixed with Nluc-BcGUVs, strong bacterial adhesions to the vesicle surfaces occurred following substrate addition, while no binding was observed in the absence of the bioluminescence light production (Fig. 3 D). Quantification of the mCherry signal from the bacteria on the Nluc-BcGUV surfaces revealed substantial bacterial coverage of the GUVs within 30 min, with only minor increases thereafter (Fig. 3 E). The kinetics and extent of bacterial binding induced by the bioluminescent light signal from the Nluc-BcGUVs were comparable to those achieved with external blue light illumination, whereas dark controls showed minimal interactions between bacteria and GUVs. To assess bacterial capture by Nluc-BcGUVs at the population level, we conducted flow cytometry on samples incubated for 60 min in the dark, under blue light, or bioluminescence conditions (Fig. 3 F and Figure S4). Events exhibiting high fluorescence in both the GUV and bacterial channels (GFP-expressing E. coli ), which indicate adhesions between the two, were most abundant in samples with bioluminescence producing GUVs (17.5%), followed by externally illuminated samples (11.2%), while dark controls remained below 1% (Fig. 3 F). Notably, individual GUVs in the blue light and bioluminescence conditions often exhibited substantially higher bacterial fluorescence than single bacteria alone, indicating multiple bacteria binding per vesicle. Moreover, the fraction of bacteria positive GUVs increased 160-fold under external light and 300-fold under bioluminescent conditions, compared with only 2-fold in the dark. These results suggest that endogenous bioluminescence signaling provides more efficient recruitment of bacteria than external illumination, likely due to the proximity of the photo production within the GUVs and the membrane-anchored BcLOV4. Overall, our observations demonstrate that synthetic cells can internally translate a chemical cue (substrate addition) into an optical signal that activates light responsive adhesions to bacteria, forming an integrated chemo-opto signaling circuit enabling synthetic cell-bacterial communication. Programmable two-step bacterial capture and elimination of synthetic cells. Having established synthetic cells capable of capturing bacteria with their bioluminescence, we next sought to endow them with a second, downstream function: selective killing of captured bacteria (Fig. 4 A). To this end, Nluc-BcGUVs were further functionalized with galactose oxidase (GaoA) (denoted as GaoA-Nluc-BcGUVs), which catalyzes the oxidation of galactose generating hydrogen peroxide (H 2 O 2 ) 39 , thereby generating localized reactive oxygen species at the GUV surface (Figure S5). To assess the bacterial killing efficiency these synthetic cells, E. coli MG1655 were prelabeled with a fluorescent LIVE/DEAD viability kit, in which SYTO9 (green) labels all cells, whereas propidium iodide (PI, red) stains only bacteria with compromised membranes. After bioluminescence mediated capture of bacteria on GaoA-Nluc-BcGUVs, galactose was added after 30 min to initiate H 2 O 2 production. Within 30 min, adhered bacteria displayed strong PI staining, indicative of membrane damage and cell death (Fig. 4 B). Similar results were obtained when bacterial capture was induced using external blue light rather than bioluminescence. In contrast, in the dark, where no bacterial capture occurred, bacteria remained viable even after H 2 O 2 production was induced, highlighting that proximity to the GUV surface is essential for effective killing. Importantly, distal bacteria in the medium remained viable, as H 2 O 2 generated at the membrane rapidly diluted into the bulk solution and did not reach lethal global concentrations (Figure S6). 3D reconstructed confocal imaging further illustrated the spatial precision of killing, only bacteria in physical contact to the GUVs exhibited colocalization SYTO9/PI signals (yellow), while free floating bacteria remained unaffected (Fig. 4 C). Due to the precise molecular programing, it is possible to test if each component is performing as intended. Quantitative analysis of both the total attached and dead bacteria signal across experimental conditions confirmed that both functional modules, adhesion and H 2 O 2 production, are required for bacterial elimination (Fig. 4 D and E). Omission of either module results in loss of bacterial killing capacity. On the other hand, bacterial capture can either be induced light or bioluminescence. This translates into the expression: (light OR bioluminescence) AND galactose following the formalism of Boolean logic. CONCLUSION In summary, we developed a programmable synthetic cell that integrates sensing, signal transduction and actuation to achieve activity resembling native immune cells. By combining light activatable BcLOV4 mediated adhesions with either external blue light illumination or internal generated bioluminescence, we demonstrate precise optical control over synthetic cell-bacteria interactions. In a second step, localized elevation of hydrogen peroxide ensures spatially confined killing to captured bacteria. This modular and sequential response, capture followed by targeted elimination, mimics a fundamental feature of innate immune cells such as neutrophils or macrophages, which neutralize pathogens only after physically engagement, thereby minimizing the collateral damage to other cells. Our results highlight the power of light-based signaling as a modality for intracellular communication between synthetic and living cells, whereby molecular events occurring inside the synthetic cells are relayed to living cells via photons. Unlike diffusible chemical signals, light-based communication provides an orthogonal channel that is independent of diffusion, propagates at the speed of light, is unaffected by flows, and does not require specific transporters. Previous examples in synthetic cell communities have shown that light can mediate communication over micrometer length scales. 4,18,25,40 Here, we extend this concept to direct signaling between bacteria and synthetic cells. This capability establishes a general design principle for constructing synthetic cells that internally process chemical information and translate it into an optical signal using luminescence modules, which can then be relayed to living cells equipped with optogenetic response modules. 41 Beyond demonstrating a synthetic cell that captures and kills bacteria, this study represents a step towards synthetic native immune cells, which have coordinates multistage defense functions. The modularity of our platform opens possibilities of integrating alternative sensors, and effector mechanisms, with perspectives to discriminate between bacteria and mammalian cells, responding to multiple environmental cues, or cooperating with living immune cells. Collectively, our findings advance synthetic cells from passive biochemical reactors towards autonomous, adaptive agents with potential applications in antimicrobial therapy, diagnostics, and engineered living materials. EXPERIMENTAL SECTION Materials. PVA (polyvinyl alcohol, MW = 145,000 Da), glucose, sucrose, galactose, isopropyl β-D-1-thiogalactopyranoside (IPTG), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), imidazole, nickel(II) chloride (NiCl 2 ), peroxidase from horseradish (HRP), and Amplex Red were purchased from Sigma Aldrich. LB medium, LB agar, and chloroform were obtained from Carl Roth. 1,2-dioleoyl-sn-glycero-3-phosphocholine [18:1 (Δ9-Cis) PC (DOPC)], 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) [18:1 DGS-NTA-Ni 2+ ] were purchased from Avanti Polar Lipids. PureCube Ni-NTA agarose beads (#31103) were purchased from Cube Biotech. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was obtained from Thermo Fisher Scientific. LIVE/DEAD BacLight Bacterial Viability Kits was acquired from Invitrogen. Furimazine was obtained from Nano-Glo Luciferase Assay (N1110, Promega). 96-well white plate (bio-one, #655075) was acquired from Greiner. µ-Slide 18 Well glass bottom was purchased from Ibidi. The plasmid DNA coding for BcLOV4-mCherry (Addgene#114596) was a gift from Prof. Brian Chow, 38 the plasmid DNA coding for NanoLuc (Addgene#135931) was a gift from Prof. Antonio Amelio, 42 and the plasmid DNA coding for galactose oxidase (GaoA) was kindly provided by Prof. Nicholas J. Turner. 43 The buffers used in this study were prepared with milli-Q water. Expression and purification of proteins. The plasmid DNA coding for BcLOV4-mCherry, NanoLuc, or GaoA were transformed into E. coli BL21(DE3), respectively. For each construct, a single colony from an agar plate was cultured overnight in 10 mL LB medium supplemented with the appropriate antibiotic (i.e., 50 µg/mL kanamycin for BcLOV4-mCherry, 50 µg/mL ampicillin for NanoLuc and GaoA) at 37°C, 160 rpm. The overnight culture was then diluted 1:100 into 1 L of fresh LB medium supplemented with antibiotic same as above and cultured at 37°C, 160 rpm until it reached an optical density at 600 nm (OD600) of 0.4–0.5. Next, the protein expression was induced by adding 0.5 mM IPTG at 16°C overnight. Next morning, the cells were harvested by centrifugation (6000 rpm, 12 min, 4°C) and resulting cell pellet from 1 L culture was resuspended in 30 mL buffer A (50 mM Tris, 300 mM NaCl, pH = 7.4) supplemented with 1 mM DTT and 1 mM PMSF. Afterward, the resuspended bacteria were lysed by ultrasonication on ice (for 5-minute sessions with 30% amplitude, 2.0 sec pulse on, 2.0 sec pulse off, interspersed with 1 min pauses). Finally, the cell lysate was spun down at 14000 rpm for 40 min at 4°C, and the supernatant was sequentially filtered through 0.45 µm and 0.20 µm polyethersulfone syringe filters. Subsequently, the cell lysate was purified using a column filled with 3 mL PureCube His Affinity Agarose. Similar to the manufacturer’s instructions, the column was first equilibrated with 10 column volumes (CV) of buffer A before the cell lysate was loaded at a flow rate of 2 mL/min. Following this, the column was washed with 10 CV of buffer A and then with 10 CV of wash buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH = 7.4). Subsequently, the protein was eluted with 3 CV elution buffer (50 mM Tris, 300 mM NaCl, 500 mM imidazole, pH = 7.4), and the fractions containing the protein were dialyzed against buffer A using a dialysis membrane with a molecular weight cut-off of 3.5 kDa overnight at 4°C. The purified protein solution was snap-frozen in liquid nitrogen and stored at -80°C for further experiments. Purified proteins were analyzed by SDS-PAGE to confirm their integrity and molecular weight (Figure S7). Preparation and functionalization of giant unilamellar vesicles (GUVs). GUVs were prepared by the PVA gel-assisted swelling method with slight modifications. 39 Specifically, the PVA solution was prepared by mixing 5% (w/v) PVA and 100 mM sucrose in 2 mL Milli-Q water, followed by incubation at 90°C and 400 rpm for 24 h. Subsequently, 40 µL of the PVA solution was dried as a thin film on a glass slide (60 mm × 24 mm) at 50°C for 1 hour. Next, 10 µL of the lipid mixture (dissolved in chloroform) containing DOPC, DGS-NTA-Ni 2+ , and DiD dye at a molar ratio of 98.9:1:0.1 was spread onto the PVA film and dried at 30°C for 1 h. A second glass slide was placed on top of the lipid-coated PVA slide, with a Teflon chamber (approximately 40 mm × 24 mm) used as a spacer. The lipids were then rehydrated with 1 mL of sucrose buffer (100 mM Sucrose in PBS (phosphate buffered saline)) containing 100 nM Nanoluc at 25°C for 1 h to allow GUV formation. Afterward, the glass-chamber-glass sandwich was inverted for 5 min and gently tapped three times with a pipette tip. Then 350 µL of GUVs suspension was harvested into a 1.5 mL protein low-bind Eppendorf tube, followed by adding 1 mL of glucose buffer (100 mM Glucose in PBS). The GUV suspension was allowed to settle overnight at 4°C. The next morning, 1 mL of the supernatant was removed without disturbing the bottom layer. 0.5 µM GaoA and 2 µM BcLOV4 were added to remaining GUV suspension, followed by incubation at 4°C for 1 h. The GaoA-BcLOV4-functionalized GUVs (denoted as GaoA-Nluc-BcGUVs) were then washed by adding 1 mL of glucose buffer, centrifuging at 1500 rpm for 5 min, and removing 1 mL of the supernatant after centrifugation. This step was repeated twice and the resulting GaoA-Nluc-BcGUVs were immediately utilized for further experiments. Bacterial culture. A glycerol stock of E. coli MG1655 transformed with or without plasmid, kept at − 80°C, was freshly streaked on LB-agar plates and a single colony from the agar plates was cultured overnight in LB medium containing appropriate antibiotics at 37°C, 160 rpm. The next morning, the overnight culture was diluted 1:100 in fresh LB medium supplemented with the same antibiotics. The bacterial culture was then incubated at 37°C under shaking conditions (150 rpm) until it reached OD600 of 0.4–0.5 and harvested for further experiments (denoted as starting culture). For the E. coli MG1655 that without any plasmid, the addition of antibiotics to LB medium was skipped. Phototoxicity test. The starting culture of E. coli MG1655 obtained above was centrifuged (5 min, 4000 rpm), the supernatant was discarded and the pellet was then resuspended in glucose buffer to an OD600 = 0.3 in a protein low-bind Eppendorf tube. Subsequently, 6 µL of Live/Dead assay kit was added into 1 mL of bacterial suspension, which was then placed on an orbital shaker at 50 rpm under different light conditions (0-0.8 mW·cm -2 ). After 1 h, 100 µL of bacterial suspension was transferred to an 18-well Ibidi dish and images were acquired using Leica SP8 confocal laser scanning microscope (CLSM) through a 63× water objective. Live bacteria were excited with a 488 nm laser and emission was detected at 500–560 nm, and dead bacteria were excited with a 552 nm laser and emission was detected at 580–650 nm. Bioluminescence inside the GUVs. 100 µL settled Nluc-BcGUVs and 100 µL of the buffer solution above were transferred to a 96-well white plate. Afterward, 1 µL of furimazine was added, and measurements were started immediately. The bioluminescence intensities between 400–500 nm were measured every 5 minutes for 1 h at room temperature using a SPARK 20M microplate reader. Before each 5-minute measurement interval, the 96-well white plate was shaken for 3 s in the plate reader. The bioluminescence microscopy images were obtained by an Olympus spinning disk microscope (IXplore IX85, Japan) using a 10x objective. Bioluminescence images were acquired with a 30 s exposure time. For fluorescence imaging, Nluc-BcGUVs stained with DiD dye were excited with a 561 nm laser with the emission recorded after passing through either 617/73 nm filter cubes. Light-activated bacterial capture. The starting culture of E. coli MG1655 transformed with pTrc-mCherry was centrifuged (5 min, 4000 rpm), the supernatant was discarded and the pellet was resuspended in glucose buffer to an OD600 = 2. Subsequently, aliquots of 50 µL of Nluc-BcGUVs were mixed with 50 µL of bacteria (OD600 = 2) in a 0.5 mL protein low-bind Eppendorf tube. The resulting mixture was placed on orbital shaker, at 50 rpm with different treatment as follows: illuminated with 0.2 mW·cm⁻² blue light, kept in the dark after the addition of 1 µL of furimazine for bioluminescence production, or kept in the dark without addition of furimazine. At pre-designed time points (t = 0, 30, 60 min), the 100 µL mixture was diluted with 200 µL glucose buffer and afterward, 100 µL of the diluted mixture was transferred into an 18-well Ibidi dish. CLSM images were taken after 5 min, which allows Nluc-BcGUVs and bacteria to settle down, using a 63× water objective. The mCherry-expressing bacteria were excited with a 552 nm laser and emission was detected at 580–640 nm, and Nluc-BcGUVs with DiD in their membrane were excited with a 638 nm laser and emission was detected at 655–780 nm. To determine the efficiency of bacterial capture, the mCherry signal from bacteria on the GUV surfaces were measured. In brief, GUVs were segmented using the DiD channel, followed by morphological dilation to expand the region of interest (ROI) around the GUVs using a dilation diameter of 2 µm. Subsequently, the dilated ROI of GUVs was overlaid onto the mCherry channel of the bacteria and the mCherry intensity in these ROIs was measured. Flow cytometry analysis of GUV-bacterial capture. To analyze the adhesion of GUVs and bacteria, the starting culture of E. coli MG1655 transformed with pTrc-GFP was centrifuged (5 min, 4000 rpm), the supernatant was discarded and the pellet was resuspended in glucose buffer to an OD600 = 2. Subsequently, the same steps as described in the for the light-activated bacterial capture were followed to prepare 400 µL of bacteria-Nluc-BcGUV mixtures following 60-min incubation under blue light, in the dark and under bioluminescence conditions, respectively. For flow cytometer analysis, 400 µL of the bacteria-Nluc-BcGUV mixture was diluted into 800 µL PBS and then, assayed using the Cyflow Cube 6 (Sysmex, Germany). For each sample at least 50 000 gated events were recorded. The gates for bacteria (expressing GFP) and Nluc-BcGUVs (DiD stained) were set in the fluorescein isothiocyanate (488 nm laser and 536 nm detector) and allophycocyanin (640 nm laser and 675 nm detector) channels, respectively, using single population samples. FCS files were exported and processed with FlowJo 10.9. Forward scatter area and side scatter area were used to specify GUVs and bacteria. Subsequently quadrant gating was established using fluorescence and nonfluorescent control bacteria and GUVs for establishing the thresholds for the GFP and DiD channels, respectively (Figure S4). Amplex red assay. 100 µL of GaoA-Nluc-BcGUVs was added to an 18-well Ibidi dish. Subsequently, 0.5 µL of 20 µM HRP, 1 µL of 100 mM galactose, and 5 µL of 1 mM Amplex Red were mixed together, and were added to the imaging dish. Images were acquired immediately afterwards on a CLSM. Resorufin forming as a product of Amplex Red and hydrogen peroxide was excited with a 552 nm laser and emission was detected at 565–640 nm, and GaoA-Nluc-BcGUVs were excited with a 638 nm laser and emission was detected at 655–780 nm. Bacterial elimination via chemo-optical signaling. The starting culture of E. coli MG1655 without any plasmid was centrifuged (5 min, 4000 rpm), the supernatant was discarded and the pellet was resuspended in glucose buffer to an OD600 = 2. Then 6 µL Live/Dead assay kit was added to 1 mL of bacterial suspension, followed by incubation for 10 min. Subsequently, 50 µL of GaoA-Nluc-BcGUVs was mixed with 50 µL of stained bacteria (OD600 = 2) in a 0.5 mL protein low-bind Eppendorf tube. The resulting mixture was placed on an orbital shaker, at 50 rpm with different treatment as follows: illuminated with 0.2 mW·cm -2 blue light, kept in the dark after the addition of 1 µL of furimazine for bioluminescence production, or kept in the dark without addition of furimazine. After 30 min of incubation, 1 mM galactose was added to the above mixtures, and the samples were incubated under the same conditions for another 30 min. Parallel samples of 100 µL from different groups at different time points (t = 0, 30, 60 min) were diluted with 200 µL of glucose buffer. Subsequently, 100 µL of diluted samples were transferred into an 18-well Ibidi dish. CLSM images were acquired after 5 min of settling time using a 63× water objective. Live bacteria were excited with a 488 nm laser and emission was detected at 495–570 nm, dead bacteria were excited with a 552 nm laser and emission was detected at 580–640 nm, and GaoA-Nluc-BcGUVs were excited with a 638 nm laser and emission was detected at 655–780 nm. To determine the efficiency of bacterial killing, signals from live and dead bacteria on the GUV surface were measured using the same analysis method as described above for bacterial capture. Statistical analysis. All data were presented as the mean ± standard deviations. The statistical analyses were performed using the Origin 2018 (OriginLab, USA). Comparisons among three or more groups were analyzed using one-way ANOVA. Declarations CONFLICT OF INTEREST The author declares no conflict of interest. ACKNOWLEDGMENTS This work has been funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the Project 433682494 − SFB 1459 − Intelligent Matter and the European Research Council ERC Consolidator Grant Lighthouse (#101169813). XZ and YZ would like to thank the Chinese Scholarship Council for a doctoral fellowship. References Buddingh, B. C. & van Hest, J. C. M. Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity. Acc Chem Res 50 , 769–777 (2017). Schwille, P. et al. MaxSynBio: Avenues Towards Creating Cells from the Bottom Up. Angew Chem Int Ed Engl 57 , 13382–13392 (2018). Rodriguez-Arco, L., Li, M. & Mann, S. Phagocytosis-inspired behaviour in synthetic protocell communities of compartmentalized colloidal objects. Nat Mater 16 , 857–863 (2017). Chakraborty, T. & Wegner, S. V. Cell to Cell Signaling through Light in Artificial Cell Communities: Glowing Predator Lures Prey. ACS Nano 15 , 9434–9444 (2021). 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