Quorum-Quenching Human Designer Cells for Closed-Loop Control of

Jul 13, 2017 - Ferdinand Sedlmayer†, Tina Jaeger†‡, Urs Jenal‡, and Martin Fussenegger†§. † Department of Biosystems Science and Engineer...
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Letter pubs.acs.org/NanoLett

Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms Ferdinand Sedlmayer,† Tina Jaeger,†,‡ Urs Jenal,‡ and Martin Fussenegger*,†,§ †

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, CH-4058 Basel, Switzerland Focal Area of Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 46, CH-4056 Basel, Switzerland § Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducerinactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era. KEYWORDS: Synthetic gene network, Quorum Quenching, anti-infectives, Pseudomonas aeruginosa, biofilm, cell-based therapy

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devices that detect and treat pathologies such as psoriasis13 or diabetes14 have recently been established. This necessitates a cellular sensor that can eavesdrop on autoinducers, that is, hormone-like bacterial communication signals not native to the eukaryotic host.15 P. aeruginosa communicates mainly through two small diffusible homoserine lactones (HSL). The cellpermeable Pseudomonas autoinducer 1 (PAI-1, 3O-C12-HSL) assists in escaping the immune system, modulates proinflammatory pathways16,17 and controls the intertwined autoinducer PAI-2 (C4 -HSL) network involved in biofilm formation.18 An orthogonal genetic circuit for sensing PAI-1 levels that employs the PAI-1-dependent activity of the intracellular transcriptional activator LasR19 would offer a dynamically regulated input module.20 Second, antivirulent designer cells would have to be equipped with desirable therapeutic output molecules combining the following properties: (i) avoidance of bacterial resistance development by targeting nonessential metabolic pathways,21 (ii) efficient biofilm prevention and dispersal,22 (iii) compatibility with existing antibiotics,5 (iv) minimal cytotoxicity, and (v) adjustable pharmacokinetics. Fortunately, a set of biologics under preclinical investigation match these criteria: for example, inhaled lactonase enhanced survival rates in a rat pneumonia

y merging engineering principles with molecular biology, synthetic biologists have developed valuable treatment alternatives1 and diagnostic tools2 for a broad variety of human metabolic disorders. To this end, they create functions de novo through the assembly of well-characterized building blocks3 or they transfer existing desirable functions from diverse organisms into synthetic circuits and artificial hosts.4 For example, this multidisciplinary research field has fostered development of phage therapy as a novel anti-infective strategy5 and has led to resensitization of resistant bacteria to antibiotics.6 Synthetic biology has in this regard paved the way for the discovery of a compound that increases the sensitivity of multidrug-resistant Mycobacterium tuberculosis to ethionamide, a last-line-of-defense antibiotic for tuberculosis.7 Nonetheless, life-threatening wound and lung infections caused by Gram-negative Pseudomonas aeruginosa remain a major concern in the clinic, where they predominantly affect immunocompromised patients8 or infest medical devices. This is worsened by the limited repertoire of available antibiotics, increased resistance due to biofilm formation,9,10 and the release of multiple virulence factors coordinated by the cell density-dependent phenomenon of quorum sensing.11 From an engineering perspective, clearing antibiotic-resistant P. aeruginosa biofilms would require synergistic efforts of multiple standardized antivirulent components.12 We reasoned that designer cells could be programmed to autonomously detect and fight pathogenic invaders through shutting down quorum sensing (i.e., by quorum quenching). Analogous © XXXX American Chemical Society

Received: May 30, 2017 Revised: July 7, 2017

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Nano Letters model,23 and injected glycoside hydrolases eradicated biofilms in vivo.24 Capitalizing on these concepts, we report here the construction and validation of a cell-based anti-infective device that we call a “quorum-quencher”, which interferes with pathogenic quorum sensing, blocks biofilm formation, breaks down established biofilm, and increases sensitivity to antibiotics. We confirmed that quorum-quencher-engineered cells scored autoinducer levels during P. aeruginosa infection and autonomously coordinated the release of autoinducer-degrading lactonase and glycoside hydrolases to block the expression of bacterial virulence factors and dissolve biofilms. We envision the quorum-quencher concept as a flexible strategy that would be readily adaptable to afford a range of genetic circuits tailored to different resilient pathogens, ushering in a new era of antiinfectives. The quorum-quencher circuit was designed as a closed-loop circuit for autonomous P. aeruginosa antivirulence intervention and contains a synthetic autoinducer-responsive signaling cascade connected to a downstream quorum-quenching module (Figure 1). For monitoring the presence of bacterial

inducer-dependent gene switch thus counteracts quorum sensing-controlled pathogenicity in two ways (Figure 1): secreted glycoside hydrolases degrade key exopolysaccharide matrix components of P. aeruginosa biofilms, whereas the quorum-quenching lactonase hydrolyzes quorum-sensing signals. To demonstrate the circuit’s ability to sense the presence of PAI-1, we cotransfected human embryonic kidney (HEK)-293 cells with constitutive PAiS (VP16-NLS-LasR) expression vectors under the control of well-adjusted promoter strengths and linked them to PAI-1-dependent expression of SEAP (human placental secreted alkaline phosphatase; PPA1-SEAPpA, pFS144) or the fluorescent cytosolic protein TurboGFP (PPA1-TurboGFP-pA, pFS160) (Figure 2A). Whereas constitutive ectopic expression of either PSV40 (PSV40-PAiS-pA, pFS165) or PhEF1α-driven PAiS (PhEF1α-PAiS-pA, pFS158) resulted in individual maximum-level reporter gene expression (pFS144) upon PAI-1 exposure, carboxy-terminal fusion of the VP16 transactivation domain (PhCMV-LasR-NLS-VP16-pA, pFS145) abolished the sensor’s PAI-1-responsiveness (Figure 2B). Further characterization of the sensor network showed that PAiSSEAP-engineered (pFS165/pFS144) HEK-293 cells robustly responded to the P. aeruginosa autoinducer PAI-1 with micromolar sensitivity (EC50 = 1 μM), corresponding to levels released from P. aeruginosa isolates (Figure 2C).25 Functionality of the PAiS module was confirmed in a range of pFS158/ pFS144-co-transfected cell lines, such as human alveolar cells (A549), colorectal epithelium (Caco-2), and mesenchymal stem cells (hMSC-TERT), as well as primate and rodent cells (Figure 2D). Altered induction profiles among tested cell lines can be mainly attributed to differences in transfection efficiency. As HEK-293 cells showed greater PAI-1 sensitivity than CHOK1 cells (Figure S1, Supporting Information), we chose HEK293 cells for all follow-up experiments. To evaluate whether chromosomal integration of the human autoinducer sensor network components into HEK-293 would ensure long-term stability, functionality and sensitivity (Figure S2A, Supporting Information), we selected PAiSSEAP41 monoclonal cell populations by monitoring reporter gene expression after stimulation with 10 μM PAI-1 (Figure S2B, Supporting Information). The best-performing PAI-1-responsive transgenic cell line PAiSSEAP41(1) (PAiSSEAP41 clone 1) with the highest sensitivity (EC50 = 1.7 μM) was chosen for further experiments (Figure S2C, Supporting Information). In-depth characterization of PAiSSEAP41(1) cell populations revealed that the synthetic autoinducer sensor circuit activation was fully reversible and that the sensor could be repeatedly switched on and off by addition and withdrawal of PAI-1 (Figure 2E). The PAiS circuit could distinguish between different common autoinducers and exclusively responded to P. aeruginosa-derived 3O-C12-HSL and C. violaceum-produced C12-HSL (Figure 2F).26 High PAI-1 sensitivity of the PAiS was verified by realtime monitoring of the fast-folding intracellular TurboGFP protein in PAiStGFP (pFS165/pFS160) cell populations (Figure 2G), while fluorescence microscopy additionally confirmed PAiS-triggered cellular fluorescence 3 h after contact with PAI-1 (Figure 2E). Notably, short PAI-1 induction pulses (10 min) readily activated transgene expression in PAiSSEAP-engineered cells (Figure S3, Supporting Information). To self-sufficiently control quorum-sensing signal accumulation, we directly linked the PAiS module with expression of a quorum-quenching lactonase. To select for optimal efficacy, protein stability and secretion, we compared potential quorum-

Figure 1. Design and mechanism of intervention of the quorumquencher device. A synthetic gene network harboring the Pseudomonas Autoinducer Sensor (PAiS) enables human HEK-293 cells to monitor levels of Pseudomonas aeruginosa-derived autoinducer PAI-1 (3O-C12HSL). PAI-1 activates ectopically expressed PAiS (PSV40-PAiS-pA; PAiS, VP16-NLS-LasR; pFS165), a protein fusion of the VP16 transactivation domain of Herpex simplex virus tagged with a nuclear localization signal (NLS) and the P. aeruginosa LasR-derived activator. PAiS binds upon activation to synthetic PPA1 promoters (PPA1-PslGhP2A-MomL-pA, pFS244/PPA1-PelAh-pA, pFS237) featuring LasRspecific operator sites upstream of a minimal promoter (PhCMVmin) set to drive expression of biofilm-destroying glycoside hydrolases (PslGh and PelAh) and autoinducer-inactivating lactonase (MomL).

communication signals, the Pseudomonas Autoinducer Sensor (PAiS) consists of a constitutively expressed synthetic fusion protein featuring the Herpes simplex-derived VP16 transactivation domain followed by a nuclear localization signal (NLS) and the autoinducer-dependent transcriptional regulator LasR, sensitive to the P. aeruginosa-derived PAI-1. Ectopically expressed PAiS exclusively binds to and modulates transcription from orthogonal chimeric promoters (PAI1) in a dosedependent manner when its cognate autoinducer PAI-1 is present and allows the quorum-quencher circuit to orchestrate the expression of an anti-infective triad. PAiS’s LasR domain attaches to the PAI1 promoter set to drive expression of the bipartite glycoside hydrolase PslGh/PelAh and the acylhomoserine lactone (AHL)-lactonase MomL. The autoB

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Figure 2. Validation of PAiS-mediated autoinducer scoring. (A) Illustration of the PAiS genetic sensor network. PAI-1 activates PAiS (PSV40-/PhEF1αPAiS-pA, pFS165/pFS158), which leads to PAiS-triggered expression of the PPA1-driven SEAP (PPA1-SEAP-pA, pFS144) or tGFP (PPA1TurboGFP:dest1-pA, pFS160) respectively. (B) HEK-293 cells were cotransfected with the pFS144 reporter and either PSV40- (pFS165) or PhEF1αdriven PAiS (pFS158), LasR-NLS-VP16 (PhCMV-LasR-NLS-VP16-pA, pFS145) or mURS (PhEF1α-URS-pA, pCK25) to produce SEAP in response to PAI-1. SEAP expression was profiled after 24 h. (C) PAiS sensitivity and adjustability. PAiSSEAP-cotransfected (pFS165/pFS144) HEK-293 cells were cultivated for 24 h in medium adjusted to increasing PAI-1 levels before SEAP levels were profiled. (D) PAI-1-induced SEAP expression in different cell lines, which were cotransfected with PAiS-encoding expression units under control of the human elongation factor 1 alpha (PhEF1α) promoter and PPA1-driven SEAP (see SI Table S1 for details) and exposed to PAI-1 for 24 h before SEAP expression was quantified. (E) Reversibility of PAI-1responsive SEAP expression was assessed by cultivating stable monoclonal PAiSSEAP41(1) HEK-293 cell populations for 72 h while alternating the PAI1 levels (5 μM) every 24 h. (F) PAiS autoinducer specificity. PAiSSEAP- or pSEAP2-control-co-transfected cell populations were cultivated for 24 h in medium containing different autoinducers (10 μM) before SEAP expression was assessed. (G) Real-time monitoring of PAI-1-triggered cellular fluorescence of PAiStGFP-engineered (pFS165/pFS160) cell populations. (H) Fluorescence microscopy of PAiStGFP cell populations exposed to PAI1. Data represent means ±SD, n = 3.

quenching candidates among bacterial and human AHLlactonases. We developed a cell-based screen to rank natural

and engineered lactonases according to their PAI-1 turnover. PAiSSEAP cell populations were used to measure remaining PAIC

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Figure 3. Validation of engineered lactonases for programmable quorum quenching. (A) Illustration of cell-based autoinducer inactivation assay. Degrader cells expressing AHL-lactonase candidates were treated with PAI-1 for 30 min, before their culture supernatants were transferred onto PAiSSEAP cells to quantify PAI-1. (B,C) Transiently transfected HEK-293 cells expressing synthetic or natural lactonases were exposed to (B) PAI-1 for 30 min before residual autoinducer in supernatants was quantified by scoring SEAP secretion from PAiSSEAP cells after 24 h or (C) to PAI-2 (C4− HSL), before applying the E. coli-derived PAI-2 reporter strain ECP61.5(rhlA′-lacZ) to measure PAI-2. (D,E) PAI-1 tunable FcmIgG-MomL production from PAiSMomL-engineered (pFS165/pFS227) cell population after exposure to varying PAI-1 levels during 24 h scored by (D) cell-based PAI-1 inactivation assay or (E) ELISA. (F) Transiently PAiSSEAP engineered cell populations were treated with supernatants from P. aeruginosa cultures (40%, v/v) and incubated for 24 h before measuring SEAP from culture supernatants. (G) Illustration of Transwell setup to measure P. aeruginosa cytotoxicity to lung epithelial cells in the presence of PAiSMomL-engineered cells. (H) LDH release from A-549 cells in the presence of transiently PAiSMomL-engineered cells or cell populations lacking the genetic network (control) measured after 16 h of cocultivation with P. aeruginosa PA01 or LasI− (MOI = 10). Data represent means ± SEM, n=3.

production of the virulence-associated siderophores, pyochelin (Figure S4C, Supporting Information) and pyoverdine (Figure 4D, Supporting Information). As this set of findings identified MomL as the most effective cellular quorum-quenching enzyme, its expression was placed under the control of the PAiS control network in follow-up experiments. We continued to improve this versatile lactonase in terms of protease stability, half-life, and traceability via a FcmIgG-fusion domain (PhCMV-SSIgκ-FcmIgG-MomL-pA, pFS218)30 and benchmarked the bioengineered variants against the inactive variant MomLH119S (PhCMV-SSIL2-MomLH119S-pA, pFS250) (Figure S5, Supporting Information). This allowed us to simultaneously determine PAI-1-dependent MomL potency (IC50 = 2 μM) from PAiSMomL-engineered cells (pFS165/pFS227, PPA1-FcmIgGMomL-pA) (Figure 3D) and ensured that these cells secrete

1 levels in PAI-1-spiked culture supernatants from degrader cells that ectopically overexpressed lactonase candidate genes (Figure 3A). This method confirmed that the marine-derived prokaryotic lactonase MomL27 retained extraordinary catalytic efficiency following truncation, codon optimization, and secretion engineering in human cells and outperformed both the prototypic bacterial AHL-lactonase AiiA,28 which lost activity upon secretion, and human paroxonase-129 (Figure 3B). Supernatants from degrader cells containing the best inclass AHL-lactonase MomL could additionally eliminate P. aeruginosa’s short-chain autoinducer C4-HSL, which is in contrast to all other tested candidates (Figure 3C). Quorumquenching efficacy of this enzyme was additionally inferred from the observation that MomL-engineered cells (PhCMVSSIL2-MomL-pA, pFS204) reduced PA01 swarming activity (Figures S4A and S4B, Supporting Information) and decreased D

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derived periplasmic glycoside hydrolase duet PslG/PelA to target different classes of P. aeruginosa. We selected the catalytic glycoside hydrolase domains from both enzymes22 and finetuned them for high expression, secretion and stability in mammalian cells. P. aeruginosa cultivation in culture supernatants from HEK-293 cells cotransfected with either MomL lactonase (pFS218) or the glycoside hydrolase domain of either PslGh (PhCMV-SSIgκ-FcmIgG-PslGh-pA, pTJ02) or PelAh (PhCMVSSIgκ-FcmIgG-PelAh-pA, pTJ01), or combinations thereof demonstrated either individual or combined prophylactic ability to reduce biofilm formation. This held especially true for the PA01 strain (Psl-dependent matrix) but was also the case for PA14 (Pel-dependent matrix), and these findings underpinned a combination treatment (MomL/PslGh/PelAh) as the most suitable biofilm prevention strategy (Figure 4A). Apart from biofilm prevention, the antivirulent triad dispersed already established P. aeruginosa biofilms within only 30 min, thus further emphasizing the rapid mode of action of the glycoside hydrolase duet (Figure 4B). After validation of the three bioengineered anti-infective components, the underlying genetic circuitry for MomL, PslGh, and PelAh was placed under the control of the autoinducerresponsive PAiS module, thereby finalizing the quorumquencher circuit. Integration of the antibiofilm extension should enable the quorum-quencher device to autonomously attenuate virulence and biofilm formation of nearby pathogens (Figure 4C). Indeed, HEK-293 cells engineered for programmable expression of MomL (PPA1-FcmIgG-MomL-pA, pFS227), PelA (PPA1-FcmIgG-PelA-pA, pFS237), as well as PslG (PPA1FcmIgG-PslG-pA, pFS239) together with the PAiS control unit (pFS158) were able to diminish the biomass of P. aeruginosa biofilms by half (Figure 4D). Programmable PAI-1-dependent biofilm disruption by PAiSPslG-engineered cell populations emphasized the functionality of the antibiofilm circuit extension (Figure S6, Supporting Information) To permanently link the infection-associated PAI-1 input to a therapeutic antivirulence output, we stably integrated the quorum-quencher components into HEK-293 cells (Figures S7A and S7D, Supporting Information). When screening monoclonal PAiSFS50 populations for optimal PAI-1-inducible expression of MomL and PslGh (Figure S7B, Supporting Information) the PAiSFS50(3) population achieved robust output titers (Figure S7C, Supporting Information) whereas the QQ50.7 cell population, which additionally harbored an inducible PelAh unit, displayed the highest sensitivity (EC50 = 0.5 μM) (Figure S7E, Supporting Information). Having proven functionality in a cell culture biofilm infection model, we last examined the ability of the antivirulence triad to enhance bactericidal efficiency and also validated the quorumquencher’s potential to eradicate bacterial biofilms. During the course of antibiotic treatment, P. aeruginosa cultivated in culture supernatants from triad-engineered (pFS218/pTJ01/pTJ02) HEK-293 cells were nine times more susceptible to killing by 2 μg/mL tobramycin as compared to bacteria grown in supernatants from mock-engineered (pEGFP-N1) cells, that is, there was an approximately 1-log greater reduction in bacterial colony-forming units (CFU) (Figure 5A). These findings were supported by a clearly visible reduction in bacterial growth (Figure S8A, Supporting Information), even at subinhibitory tobramycin concentrations (Figure S8B, Supporting Information) that have been demonstrated to promote biofilm formation.31

Figure 4. Implementation of catalytic Pseudomonas biofilm reduction. (A) Prophylaxis of P. aeruginosa biofilms by combined action of glycoside hydrolases and lactonase. HEK-293 cells engineered for constitutive secretion of MomL (PhCMV-SSIgκ-FcmIgG-MomL-pA, pFS218), PslGh (PhCMV-SSIgκ-FcmIgG-PslGh-pA, pTJ02), or/and PelAh (PhCMV-SSIgκ-FcmIgG-PelAh pA, pTJ01) were grown for 48 h before supernatants (50% v/v) were mixed with P. aeruginosa (PA01/PA14) cultures. Bacterial attachment was quantified by crystal violet staining after 24 h of incubation. (B) Treatment of established P. aeruginosa biofilms. Preformed P. aeruginosa biofilms (24 h) were challenged for 30 min with supernatants (50% v/v) from pFS218-/pTJ01- and/or pTJ02-engineered cells grown for 48 h and remaining biofilms were quantified with crystal violet. (C) Experimental schematic of biofilm control by the quorum-quencher device. (D) Quantification of PA01 biofilms after cocultivation with quorum-quencher-transgenic (pFS165/pFS227/pFS237/pFS239) cell populations or with EGFPtransgenic (pEGFP-N1) cells (control) for 24 h. Data presented represent absolute attachment and are means ±SD, n = 3.

high doses of FcmIgG-MomL when triggered with PAI-1 (EC50 = 0.7 μM) (Figure 3E). Next, after successful implementation of an autoinducerresponsive sensor circuit and the downstream quorumquenching output, we tested PAiS’s ability to sense and respond to a complex mixture of virulence-inducing quorumsensing signals released from P. aeruginosa. The observation that reporter expression levels were exclusively increased by the PAI-1-secreting P. aeruginosa strain PA01 (Figure 3F) suggested that the PAiS is sufficiently sensitive to detect PAI1 released from this clinical isolate. On the basis of this finding, we continued to verify programmable autoinducer destruction by the closed-loop control circuit from PAiSMomL-engineered cells. We chose a coculture model featuring PAiSMomL-cells on Transwell permeable supports and scored their protective influence on lung epithelial cells (A549) cultivated in close proximity in the bottom compartment during infection with P. aeruginosa (Figure 3G). Indeed, PAiSMomL-engineered cells attenuated Pseudomonas-mediated killing of lung epithelial cells by more than one-third (35%) through autoinducer 1 and 2 degradation, which was only paralleled by cells engineered for constitutive MomL expression (Figure 3H). To augment antivirulence treatment efficacy, we subsequently implemented the second stage of the synthetic quorum-quencher cascade that targets intractable biofilms. To this end, we capitalized on the ability of the P. aeruginosaE

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recalcitrant Pseudomonas infections. Accordingly, in this work we designed and implemented a synthetic human quorumquencher device with an integrated autoinducer input module connected to an anti-infective output. Validation studies indicated that this quorum-quencher is the first cell-based infection interface that can successfully block quorum sensing of treatment-refractory P. aeruginosa. Because the designer cells were programmed to disarm rather than kill the pathogen, they should exert very little selective pressure on essential metabolic pathways and are thus expected to avoid or at least delay the evolution of new antibiotic resistance. In the case of P. aeruginosa, the quorum-quencher cascade controlling the triad of MomL, PslGh, and PelAh yielded the most promising results by simultaneous dismantling the biofilm matrix and ablating both PAI-1 and PAI-2. It is particularly noteworthy that the experimental outcome underlined the impact of PAI-2 on virulence, which is currently seen as the most crucial in vivo target.35 Many bacteria exploit quorum sensing for virulence control, and we believe this prototypic cell-based anti-infective device could be readily extended to work on other Grampositive and Gram-negative pathogens, including Staphylococcus aureus36 or Vibrio cholerae. The modular nature of the quorumquencher circuit allows for quick refinement of the input molecule. Species-specific biofilm-degrading enzymes,37 and extra building blocks such as bacteriocins38 or antivirulent autoinducers39 can likewise be envisaged as outputs and might ultimately make conventional antibiotics obsolete. To date, treatment of recalcitrant biofilms relies solely on the administration of small-molecule antibiotics,31 although antibiotic penetration and killing inside biofilms remains controversial.40,41 The cellular device described here provides a new option to treat biofilms and should improve the outcome for chronic infections, as well as preventing medical deviceassociated P. aeruginosa infections. This antibiofilm technology would be particularly useful for hindering biofilm formation around cell implants in future cell-based therapies. The most encouraging current approaches to anti-infectives involve the combination of antibiofilm agents with minimal risk of bacterial resistance,42 quorum-sensing inhibitors,43 and reactivation of established antibiotics.44 Metabolic stimulation was key for targeted antibiotic resensitization in the case of P. aeruginosa.45 Although the development of new antibiotics is lagging far behind demand, we suggest that synthetic biologyinspired designer cells rather than pills could in the not-toodistant future open new avenues to treat extensively drugresistant bacteria.

Figure 5. Quorum-quencher-based control of antibiotic-treated biofilms. (A) Potentiation of antibiotics by combination of catalytic effector molecules. HEK-293 cells engineered for constitutive secretion of MomL (PhCMV-SSIgκ-FcmIgG-MomL-pA, pFS218), PslGh (PhCMVSSIgκ-FcmIgG-PslGh-pA, pTJ02), and PelAh (PhCMV-SSIgκ-FcmIgG-PelAh pA, pTJ01) were grown for 48 h before their supernatants (50% v/v) were mixed with P. aeruginosa (PA01) cultures for 24 h in the presence or absence of tobramycin. PA01 CFUs in biofilms were counted from LB agar plates. (B) Experimental schematic of dual PA01 treatment with the quorum-quencher device and antibiotic. (C,D) Biofilm prevention of tobramycin-treated PA01 in the presence of quorumquencher-transgenic cells or cells lacking the MomL/PslGh/PelAh expression components (control) during 24 h. (C) Total biofilm biomass quantified by crystal violet staining. Data are means from ±SD, n = 3. (D) Representative image of PA01 biofilms stained with crystal violet after 24 h of cocultivation.

To assess whether a quorum-quencher−antibiotic combination can surpass conventional antibiotic treatment of P. aeruginosa infections, we designed a cocultivation setup in which self-sufficient antibiofilm intervention can be monitored (Figure 5B). We found that the quorum-quencher device indeed accomplished elimination of P. aeruginosa biofilms during 24 h, accompanied by a total lack of bacterial attachment (Figure 5C) and the absence of any apparent bacterial biomass. In marked contrast, cells lacking the quorum-quencher circuit failed to prevent biofilm formation and did not enhance tobramycin sensitivity (Figure 5D). These results indicate that the quorum-quencher circuit successfully processed pathogenic communication signals and enhanced antibiotic susceptibility by releasing an antivirulent triad that interfered with quorumsensing-related virulence and biofilm formation. According to the Centers for Disease Control and Prevention (CDC), the nosocomial pathogen P. aeruginosa infects more than 51 000 patients in the United States each year and increasingly escapes traditional treatment strategies due to development of potent drug resistance, including multidrug resistance. Although P. aeruginosa is already naturally endowed with tolerance to a variety of antibiotics,8 established Pseudomonas biofilms are even more difficult to eradicate because they are shielded from the host immune defense32 and are recalcitrant to traditional antibiotics.33,34 We considered that a strategy of interfering with pathogen coordination would be an effective approach to overcome

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b02270. Detailed experimental procedures for cell line development, genetic circuit characterization, autoinducer and lactonase activity determination, biofilm inhibition assay, antibiotic activity quantification, two supplemental tables and eight supplemental figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +41 61 387 31 60. Fax: +41 61 387 39 88. E-mail: [email protected]. F

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Martin Fussenegger: 0000-0001-8545-667X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a European Research Council advanced grant (ProNet, No. 321381). T.J. was in part financed by the Swiss BNF program. We thank Tania Roberts, Dennis Hell, and David Fuchs for generous advice and experimental support.

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ABBREVIATIONS AHL, acyl-homoserine lactone; CFU, colony forming unit; GFP, green fluorescent protein; PAI-1, Pseudomonas autoinducer 1 (3O-C12-HSL); PAI-2, Pseudomonas autoinducer 2 (C4-HSL); PAiS, Pseudomonas autoinducer sensor; SEAP, human placental secreted alkaline phosphatase

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DOI: 10.1021/acs.nanolett.7b02270 Nano Lett. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.nanolett.7b02270 Nano Lett. XXXX, XXX, XXX−XXX