Chemical communication at the synthetic cell/living cell interface

Abstract

Although the complexity of synthetic cells has continued to increase in recent years, chemical communication between protocell models and living organisms remains a key challenge in bottom-up synthetic biology and bioengineering. In this Review, we discuss how communication channels and modes of signal processing can be established between living cells and cytomimetic agents such as giant unilamellar lipid vesicles, proteinosomes, polysaccharidosomes, polymer-based giant vesicles and membrane-less coacervate micro-droplets. We describe three potential modes of chemical communication in consortia of synthetic and living cells based on mechanisms of distributed communication and signal processing, physical embodiment and nested communication, and network-based contact-dependent communication. We survey the potential for applying synthetic cell/living cell communication systems in biomedicine, including the in situ production of therapeutics and development of new bioreactors. Finally, we present a short summary of our findings.

Fig. 1. Schematic overview of cell-like synthetic microarchitectures.

Diverse soft micro-compartmentalized assemblages are used for synthetic cell modeling. Single and nested compartments are shown as graphical representations (left panels) and experimental microscopy images (right panels). Images were reproduced with permission from^– Copyright © 2021, American Chemical Society, Copyright © 2016, The Authors, Copyright © 2018, The Royal Society of Chemistry, Copyright © 2013, Nature Publishing Group, Copyright © 2016, John Wiley and Sons, Copyright © 2017, American Chemical Society. Copyright © 2018, The Authors^,. The central cartoon was adapted with permission from Copyright © 2018, The Royal Society Publishing.

Fig. 2. Communication pathways in synthetic cell/living cell consortia.

Three modes of chemical communication are shown involving distributed populations and through-space signal processing, nested populations, and embedded signaling, and interfacially connected populations, and contact-dependent signaling pathways. Only unidirectional modes between sender synthetic cells (red) and receiver living cells (green) are shown. Through-space diffusion in the external environment is shaded in blue. Unidirectional pathways between transmitter living cells and receiver synthetic cells as well as bilateral communication with positive or negative feedback are also possible. Adapted with permission from Copyright © 2020, Wiley-VCH GmbH.

Fig. 3. Distributed communication and signal processing.

a Communication pathway between genetically controlled sender vesicles (synthetic cell) and P. aeruginosa RepC4lux receiver cells. The transmitter system is based on the production of the signal molecule C4-HSL by the synthase RhlI and two precursors (C4-CoA and SAM). The RhlI enzyme is encoded by the rhlI gene (plasmid pWM-rhlI) and produced inside the vesicles by in vitro transcription (TX) and translation (TL) (PURE system). C4-HSL freely diffuses through the vesicle membrane into the medium containing the bacterial cells. The receiver cells contain a genetic reporter device for C4-HSL-induced bioluminescence (PrhlA::luxCDABE) and a mutation inactivating the rhlI gene, so that the living cells cannot produce C4-HSL. C4-HSL binds to receptor RhlR, which in turn triggers luxCDABE transcription by RepC4lux and bioluminescence emission. Adapted with permission from Copyright © 2018, The Royal Society of Chemistry, b communication pathway between stimuli-responsive genetically controlled liposomes and neural stem cells. Activation of the AND-gate and expression and assembly of PFO in the vesicle membrane releases BDNF, which in turn induces neural stem cell differentiation. Reproduced with permission from Copyright © 2020, Science. c Synthetic cells (circles) translate chemical signals for E. coli (oblongs). In the absence of the vesicles, E.coli cells cannot sense theophylline (top panel). The vesicles are engineered to detect theophylline and in response release IPTG, a chemical signal that induces a response in the E. coli cells (bottom panel). Reproduced with permission from © 2020 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim. d Schematic showing IPTG-induced GFP expression in E. coli by co-trapped melittin-functionalized IPTG-containing GUVs. e Schematic showing H₂O₂-induced killing of HepG2 cells by co-trapped melittin-functionalized GOx-containing GUVs. d and e are reproduced with permission from Copyright © 2019, The Royal Society of Chemistry. f Schematic showing chemical signal transduction between a melittin-functionalized GOx-containing GUV (transmitter) and peroxidase-active RBC (receiver). Reproduced with permission from © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. g Schematic illustration of the invasion-defense mutual interaction between liquid coacervate microdroplet protocells and living cells. Reproduced with permission from © 2020 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Fig. 4. Nested communication and cellular bionics.

a Inducers 3OC6-HSL (AHL) and IPTG diffuse from reservoir droplets to receiver droplets (R^*) containing engineered bacteria (left panel). The bacteria contain a genetic AND gate (center panel) with a fluorescence output. Fluorescence microscopy images arranged in a truth table (right panel); scale bars = 50 μm. Reproduced with permission from, Copyright © 2014, American Chemical Society. b Brightfield/fluorescence microscopy composite image showing a cellular bionic system comprising a single host lipid vesicle and two incarcerated BE colon carcinoma cells. Scale bar = 25 µm. Reproduced with permission from, Copyright © 2018, Springer Nature. c Schematic of a single vesicle-based synthetic cell containing colon carcinoma cells and a GOx/HRP enzyme cascade. Cell-mediated production of glucose (Glc) in the vesicle lumen switches on GOx activity to release hydrogen peroxide and D-gluconolactone (GDL) followed by HRP-mediated peroxidation of Amplex Ultra Red to give a fluorescent output (resorufin) inside the synthetic cells. Reproduced with permission from, Copyright © 2018, Springer Nature.

Fig. 5. Contact-dependent communication between the living and synthetic cells.

Summary scheme depicting ligand/receptor interactions responsible for synergistic macrophage activation in the presence of SVLP-presenting polysaccharidosomes (3). Pathways for unattached SVLPs (1) and non-functionalized polysaccharidosomes (2) are also depicted. Reproduced with permission from Elsevier.

Fig. 6. Therapeutic applications at the synthetic/living cell interface.

a Schematic showing cell encapsulation within synthetic membranes or hydrogel microcapsules (dashed circle) to produce cellular bionic systems with therapeutic potential. Therapeutic cells are shown in blue. Adapted with permission from Copyright © 2018 John Wiley and Sons. b Schematic of a therapeutic synthetic cell (AβC) with a glucose metabolism system and membrane fusion machinery that is coupled to the external release of insulin by programmed exocytosis of internalized insulin-loaded vesicles (ISVs). GOx, glucose oxidase; CAT, catalase; square brackets denote concentration. Reproduced with permission from Springer Nature. c Illustration showing in vitro and in vivo GOx/Hb cascade generation of NO at micromolar concentrations in the presence of coacervate-sequestered enzyme substrates (glucose and hydroxyurea, respectively) as a step towards protocell-mediated blood vessel vasodilation. Hb and GOx are spatially positioned on the periphery and in the interior of the protocell bioreactor, respectively. GDL, D-gluconolactone; Hu, hydroxyurea. Reproduced with permission from Springer Nature.