Synthetic ecosystems based on airborne inter- and intrakingdom communication

Abstract

Intercellular communication within an organism, between populations, or across species and kingdoms forms the basis of many ecosystems in which organisms coexist through symbiotic, parasitic, or predator-prey relationships. Using multistep airborne communication and signal transduction, we present synthetic ecosystems within a mammalian cell population, in mice, or across species and kingdoms. Inter- and intrakingdom communication was enabled by using sender cells that produce volatile aldehydes, small vitamin-derived molecules, or antibiotics that diffuse, by gas or liquid phase, to receiver cells and induce the expression of specific target genes. Intercellular and cross-kingdom communication was shown to enable quorum sensing between and among mammalian cells, bacteria, yeast, and plants, resulting in precise spatiotemporal control of IFN-beta production. Interconnection of bacterial, yeast, and mammalian cell signaling enabled the construction of multistep signal transduction and processing networks as well as the design of synthetic ecosystems that mimic fundamental coexistence patterns in nature, including symbiosis, parasitism, and oscillating predator-prey interactions.

Fig. 1.

AT&T. (A) Principle of airborne intra- and interkingdom signaling. Sender cells, which naturally express ADH or are transgenic for constitutive ADH expression (e.g., CHO-ADH), metabolize ethanol to volatile acetaldehyde, which diffuses by the gas phase to receiver cells (e.g., _AIRCHO-SEAP cells), which have been engineered to express a target gene such as the human placental SEAP under the control of the acetaldehyde-inducible regulation system (AIR; AlcR, acetaldehyde-dependent transactivator; P_AIR, AlcR-specific acetaldehyde-responsive promoter). Inside receiver cells, acetaldehyde triggers AlcR-dependent P_AIR-driven SEAP expression in a dose-dependent manner. _AIRCHO-SEAP receiver cells (30,000) were cultivated next to increasing numbers of sender cell populations derived from different organisms for 48 h before SEAP quantification: CHO-ADH (Chinese hamster ovary cells transgenic for mouse ADH cultivated in 1‰ ethanol-containing medium), HEK-ADH [human embryonic kidney cells (HEK293-T) transgenic for mouse ADH cultivated in 1‰ ethanol-containing medium], E. coli cells (cultivated on an LB-agar plate supplemented with 2.5% ethanol), S. cerevisiae (cultivated on a YPD-agar plate), and 5-day-old L. sativum (garden cress) plantlets cultivated in 1% ethanol. (B) Synthetic cell-to-cell communication in mice. Mice were i.p.-injected with microencapsulated _AIRCHO-SEAP cells (200 cells per capsule, 2 × 10⁴ capsules per mouse) and, after 1 h, half of the mice were further injected with CHO-ADH (2 × 10⁶ cells per mouse). Mice were kept with or without ethanol in their drinking water (uptake: 1.5 g/kg per 24 h) for 72 h before profiling of SEAP levels in the serum of both groups. Parallel assays were performed in vitro where microencapsulated _AIRCHO-SEAP (2 × 10⁴ capsules; 200 cells per capsule) populations were cultivated in 20 ml of medium in the presence and absence of CHO-ADH (2 × 10⁶ cells) and/or ethanol (1‰, vol/vol). (C) AT&T-based longimetry between mammalian sender and receiver cells. CHO-ADH (5,000) were seeded in the top-left well of a 96-well plate containing medium supplemented with 1‰ (vol/vol) ethanol. All wells were seeded with 10,000 _AIRCHO-SEAP receiver cells, and the 96-well plate was incubated for 48 h before quantification of SEAP production. (D) AT&T-based chronometry between mammalian sender and receiver cells. Increasing CHO-ADH populations were cocultivated with 20,000 cells/ml _AIRCHO-IFN in medium containing 1‰ ethanol (vol/vol), and the onset of IFN-β expression by the receiver cells was determined. (E) Timing of biopharmaceutical production by AT&T-based quorum sensing in the production culture. Serum-free suspension cultures of CHO-K1 cells transgenic for AIR-controlled expression of the multiple sclerosis therapeutic IFN-β (_AIRCHO-IFN) were cocultured with differently sized CHO-ADH populations in 10-ml cultures containing 1‰ ethanol. IFN-β production and _AIRCHO-IFN cell density were monitored for 72 h. Data are represented as mean ± SD.

Fig. 2.

AT&T-based multistep intra- and interkingdom signaling. (A) Three-step mammalian cell-based signaling and information processing. Sender cells (HEK-BTD) engineered for constitutive secretion of BTD hydrolyze biocytin [N(e)-(+)-biotinyl-l-lysine; 100 nM] and resulting biotin diffuses into processor cells (_BIOCHO-ADH-SEAP), where it heterodimerizes a synthetic transactivator (ES-Biotin-AVP16) triggering P_ETR-driven ADH and SEAP expression. ADH expression in _BIOCHO-ADH-SEAP cells results in the production of volatile acetaldehyde, which is broadcast to neighboring well 2 and induces AIR-controlled SEAP expression in the receiver cells (_AIRCHO-SEAP). (B) SEAP production profiles of the three-step mammalian cell-based signaling and information processing cascade shown in A. HEK-BTD, or native non-BTD-producing HEK293-T as control (30,000) sender and 30,000 processor cells (_BIOCHO-ADH-SEAP) were cocultivated in the presence of 100 nM biocytin and 1‰ ethanol in well 1, whereas 30,000 receiver cells (_AIRCHO-SEAP) were cultivated in well 2 for 48 h before profiling SEAP production in both wells. (C) Three-step bacteria-mammalian cell-based signaling and information processing. S. erythraea naturally produces erythromycin, which triggers erythromycin-responsive ADH and SEAP expression in the processor cells (_ECHO-ADH-SEAP) cultivated in well 1. ADH expression in the processor cells (_ECHO-ADH-SEAP) results in the production of volatile acetaldehyde, which is broadcast to the neighboring well 2 and induces AIR-controlled SEAP expression in the receiver cells (_AIRCHO-SEAP). (D) SEAP production profiles of the three-step bacteria-mammalian cell-based signaling and information processing cascade shown in C. An agar plug of an S. erythraea culture (1 mm in diameter) and 30,000 processor cells (_ECHO-ADH-SEAP) were cocultivated in the presence of 1‰ ethanol in well 1, whereas 30,000 receiver cells (_AIRCHO-SEAP) were cultivated in well 2 for 48 h before profiling SEAP production in both wells. Data are represented as mean ± SD.

Fig. 3.

Synthetic ecosystems. (A) Commensalism. Mammalian cells commensal to bacteria. When cultivating _AIRCHO-NEO-SEAP transgenic for AIR-controlled expression of the neomycin-resistance determinant (NEO) and constitutive SEAP expression in close proximity to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells survive and proliferate in neomycin-containing culture medium. _AIRCHO-NEO-SEAP (30,000) were cultivated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of LB agar supplemented with 2.5% ethanol and inoculated with 10⁵ E. coli DH5a (distance between the center of both culture dishes: 12 cm). Subsequently, the _AIRCHO-NEO-SEAP medium was supplemented with neomycin (1.6 mg/ml) for another 48 h before scoring SEAP production as an indicator of viability. (B) Amensalism. Mammalian cells amensal to bacteria. When cultivating _AIRCHO-RipDD transgenic for AIR-controlled expression of the death domain of the human receptor-interacting protein (RipDD) proximate to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells die by apoptosis. _AIRCHO-RipDD (30,000) were cultivated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of LB agar supplemented with 2.5% ethanol and inoculated with 10⁵ E. coli DH5a (distance between the center of both culture dishes: 12 cm) before scoring apoptosis. (C) Mutualism between mammalian cells and bacteria. When cultivating _AIRCHO-NEO-sBLA-SEAP transgenic for AIR-controlled neomycin resistance (NEO), sBLA, and constitutive SEAP expression proximate to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells survive and proliferate in neomycin-containing culture medium and secrete sBLA, which is transferred to the E. coli culture where it sustains bacterial growth in ampicillin-containing medium. _AIRCHO-NEO-sBLA-SEAP (30,000) were cultivated in medium supplemented with 5 μg/ml ampicillin for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of HTS medium supplemented with 2.5% ethanol and inoculated with E. coli DH5a [neo^R (0.05 OD₆₀₀)] (distance between the center of both culture dishes: 12 cm). The cell culture medium was semicontinuously transferred to the E. coli population, and _AIRCHO-NEO-sBLA-SEAP were cultured in medium supplemented with 1.6 mg/ml neomycin for another 48 h with subsequent scoring of E. coli density and SEAP production as indicator of CHO viability. (D) Parasitism. Bacteria as parasites of mammalian cells. When cocultivating CHO-sBLA engineered for constitutive sBLA expression, ampicillin in the culture medium will be degraded, and bacteria will expand, thereby exhausting nutrients and impairing growth and survival of mammalian cells. In the absence of CHO-sBLA, E. coli fail to grow. CHO-sBLA (30,000) were cocultivated with E. coli DH5a (0.05 OD₆₀₀) for 48 h in cell culture medium containing 10 μg/ml ampicillin before assessment of E. coli and CHO-sBLA cell numbers. (E) Third party-inducible parasitism. Fungi inducing bacteria-triggered parasitism of mammalian cells. When cultivating S. cerevisiae, which naturally metabolizes glucose to volatile acetaldehyde and ethanol, in close proximity to cocultures consisting of wild-type E. coli and _AIRHEK-sBLA transgenic for AIR-controlled expression of sBLA, acetaldehyde-triggered sBLA production decreases ampicillin levels and promotes E. coli proliferation, which in turn exhausts nutrients and impairs growth and survival of mammalian cells. _AIRHEK-sBLA (30,000) were cocultivated with E. coli DH5a (0.05 OD₆₀₀) in medium containing 10 μg/ml ampicillin and incubated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of YPD agar inoculated with 100 mg (dry weight) of S. cerevisiae (distance between the center of both culture dishes: 12 cm) before scoring of E. coli and _AIRHEK-sBLA cell numbers. (F) Interkingdom predator–prey-like ecosystem. Bacteria preying on mammalian cells. Cocultivation of wild-type E. coli with CHO-sBLA engineered for constitutive expression of sBLA in the presence of a continuous medium supply shows three distinct time courses; whereas the absence of ampicillin triggers rapid growth of E. coli and extinction of CHO-sBLA, high ampicillin concentrations (1 mg/ml) extinguish E. coli and enable rapid growth of CHO-sBLA. However, an intermediate ampicillin concentration (100 μg/ml) results in a predator–prey-like ecosystem in which E. coli and CHO-sBLA population size antagonistically oscillate. As the CHO-sBLA population increases and sBLA levels rise, ampicillin concentrations decrease and enable E. coli to grow more rapidly. With E. coli populations increasing, nutrients are rapidly depleted, which limits the growth of CHO-sBLA and decreases sBLA production. Continuous feeding of fresh ampicillin-containing medium results in elevated ampicillin concentrations that limit E. coli growth and promote expansion of the CHO-sBLA population, which initiates a new cycle. CHO-sBLA (150,000) were cocultivated with E. coli DH5a (starting density, 0.065 OD₆₀₀) in medium containing the indicated ampicillin concentrations with semicontinuous medium exchange (dilution rate, 0.25 day⁻¹). The E. coli population was scored by its optical density at 600 nm (OD₆₀₀), and the CHO-sBLA cell population was monitored by quantifying confluence. Data are represented as mean ± SD.

Fig. 3.

Synthetic ecosystems. (A) Commensalism. Mammalian cells commensal to bacteria. When cultivating _AIRCHO-NEO-SEAP transgenic for AIR-controlled expression of the neomycin-resistance determinant (NEO) and constitutive SEAP expression in close proximity to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells survive and proliferate in neomycin-containing culture medium. _AIRCHO-NEO-SEAP (30,000) were cultivated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of LB agar supplemented with 2.5% ethanol and inoculated with 10⁵ E. coli DH5a (distance between the center of both culture dishes: 12 cm). Subsequently, the _AIRCHO-NEO-SEAP medium was supplemented with neomycin (1.6 mg/ml) for another 48 h before scoring SEAP production as an indicator of viability. (B) Amensalism. Mammalian cells amensal to bacteria. When cultivating _AIRCHO-RipDD transgenic for AIR-controlled expression of the death domain of the human receptor-interacting protein (RipDD) proximate to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells die by apoptosis. _AIRCHO-RipDD (30,000) were cultivated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of LB agar supplemented with 2.5% ethanol and inoculated with 10⁵ E. coli DH5a (distance between the center of both culture dishes: 12 cm) before scoring apoptosis. (C) Mutualism between mammalian cells and bacteria. When cultivating _AIRCHO-NEO-sBLA-SEAP transgenic for AIR-controlled neomycin resistance (NEO), sBLA, and constitutive SEAP expression proximate to E. coli metabolizing ethanol to volatile acetaldehyde, the mammalian cells survive and proliferate in neomycin-containing culture medium and secrete sBLA, which is transferred to the E. coli culture where it sustains bacterial growth in ampicillin-containing medium. _AIRCHO-NEO-sBLA-SEAP (30,000) were cultivated in medium supplemented with 5 μg/ml ampicillin for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of HTS medium supplemented with 2.5% ethanol and inoculated with E. coli DH5a [neo^R (0.05 OD₆₀₀)] (distance between the center of both culture dishes: 12 cm). The cell culture medium was semicontinuously transferred to the E. coli population, and _AIRCHO-NEO-sBLA-SEAP were cultured in medium supplemented with 1.6 mg/ml neomycin for another 48 h with subsequent scoring of E. coli density and SEAP production as indicator of CHO viability. (D) Parasitism. Bacteria as parasites of mammalian cells. When cocultivating CHO-sBLA engineered for constitutive sBLA expression, ampicillin in the culture medium will be degraded, and bacteria will expand, thereby exhausting nutrients and impairing growth and survival of mammalian cells. In the absence of CHO-sBLA, E. coli fail to grow. CHO-sBLA (30,000) were cocultivated with E. coli DH5a (0.05 OD₆₀₀) for 48 h in cell culture medium containing 10 μg/ml ampicillin before assessment of E. coli and CHO-sBLA cell numbers. (E) Third party-inducible parasitism. Fungi inducing bacteria-triggered parasitism of mammalian cells. When cultivating S. cerevisiae, which naturally metabolizes glucose to volatile acetaldehyde and ethanol, in close proximity to cocultures consisting of wild-type E. coli and _AIRHEK-sBLA transgenic for AIR-controlled expression of sBLA, acetaldehyde-triggered sBLA production decreases ampicillin levels and promotes E. coli proliferation, which in turn exhausts nutrients and impairs growth and survival of mammalian cells. _AIRHEK-sBLA (30,000) were cocultivated with E. coli DH5a (0.05 OD₆₀₀) in medium containing 10 μg/ml ampicillin and incubated for 48 h in the presence or absence of a Petri dish (10 cm in diameter) containing 20 ml of YPD agar inoculated with 100 mg (dry weight) of S. cerevisiae (distance between the center of both culture dishes: 12 cm) before scoring of E. coli and _AIRHEK-sBLA cell numbers. (F) Interkingdom predator–prey-like ecosystem. Bacteria preying on mammalian cells. Cocultivation of wild-type E. coli with CHO-sBLA engineered for constitutive expression of sBLA in the presence of a continuous medium supply shows three distinct time courses; whereas the absence of ampicillin triggers rapid growth of E. coli and extinction of CHO-sBLA, high ampicillin concentrations (1 mg/ml) extinguish E. coli and enable rapid growth of CHO-sBLA. However, an intermediate ampicillin concentration (100 μg/ml) results in a predator–prey-like ecosystem in which E. coli and CHO-sBLA population size antagonistically oscillate. As the CHO-sBLA population increases and sBLA levels rise, ampicillin concentrations decrease and enable E. coli to grow more rapidly. With E. coli populations increasing, nutrients are rapidly depleted, which limits the growth of CHO-sBLA and decreases sBLA production. Continuous feeding of fresh ampicillin-containing medium results in elevated ampicillin concentrations that limit E. coli growth and promote expansion of the CHO-sBLA population, which initiates a new cycle. CHO-sBLA (150,000) were cocultivated with E. coli DH5a (starting density, 0.065 OD₆₀₀) in medium containing the indicated ampicillin concentrations with semicontinuous medium exchange (dilution rate, 0.25 day⁻¹). The E. coli population was scored by its optical density at 600 nm (OD₆₀₀), and the CHO-sBLA cell population was monitored by quantifying confluence. Data are represented as mean ± SD.