Journal of Molecular Biology
Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria
Introduction
Recent efforts to design and construct organisms for biotechnological applications, such as metabolic engineering and bioremediation,1 have led to developing a toolbox of modular and robust parts including biosensors and genetic circuits. The output of these systems is interfaced to control cellular behaviours such as biofilm formation,2 chemotaxis,3 and differentiation.4 Here, we link heterologous environmental sensors to the expression of a protein that enables Escherichia coli to invade mammalian cells. Reprogramming the interaction between a bacterium and mammalian cell will enable the forward engineering of bacteria for therapeutic purposes including live vaccines,5 probiotics,6 and anti-tumor agents.7
The construction of a number of synthetic biosensors and genetic circuits has been reported. Recently, Hellinga and co-workers computationally designed maltose binding protein variants that bind various unnatural ligands, including TNT.8 In addition, Buskirk et al. linked small molecules to translation by designing RNA aptamers that bind tetramethylrosamine and activate gene expression.9 To process environmental inputs, synthetic genetic circuits have been constructed that function as logic blocks,10 an oscillator,11 a bistable “toggle” switch,12 and a pulse generator.13 Synthetic eukaryotic protein circuits have also been constructed that enable the integration of multiple inputs.14
There has been an effort to link the sensors and circuits to control cellular behaviour through the activation of an “output interface”.2 For example, biofilm formation was induced by activating a toggle switch genetic circuit controlling traA. By linking this switch to quorum sensing or the activation of RecA, biofilm formation was induced at high cell densities or when exposed to UV light, respectively.2 In addition, Arnold and co-workers developed a synthetic population control circuit by linking quorum sensing to cell death by inducing the synthesis of a toxic protein.15
Emerging applications of synthetic biology are the design of bacteria to produce therapeutic agents and the use of live bacteria as targeted delivery systems.5, 6 Towards this latter goal, it will be important to control the interaction of a bacterium with a mammalian cell and to regulate this interaction in response to environmental stimuli.5, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 Bacteria have numerous systems to interact with and manipulate eukaryotic cells. Redundancies of these systems and their complex regulatory control complicate the engineering of natural bacteria. In contrast, the inv gene encoding invasin from Yersinia pseudotuberculosis represents a single-gene output interface for initiating adhesion and invasion of mammalian cells when expressed in E. coli. Invasin binds tightly to β1-integrins present on the surface of many cell lines and induces bacterial uptake by stimulating Rac-1.29 In the gut, Yersinia uses invasin to identify and invade M cells, which uniquely express β1-integrins on their apical surface.30 Transfer of inv to E. coli is sufficient to induce the invasion of mammalian cell lines that express β1-integrins.31 Moreover, the therapeutic potential of inv+ E. coli has been explored by constructing strains that can deliver proteins17, 19 and plasmids18, 16 into mammalian cells.
Here, we demonstrate that invasin-mediated internalization does not require additional known adhesion molecules. In addition, inv+ E. coli can invade a broad range of tumor cells including epithelial, hepatocarcinoma, and osteosarcoma lines. Towards the goal of engineering therapeutic bacteria, we show that bacterial internalization can be synthetically linked to cell density, hypoxia, and inducible inputs (Figure 1). This is achieved by placing inv under the control of the quorum sensing lux operon, an anaerobically induced fdhF promoter, or an arabinose-inducible araBAD promoter. Connecting the invasin output to environmental inputs required engineering beyond gene fusion. Initial construction of fdhF and araBAD-controlled invasin resulted in constitutive phenotypes. To overcome this problem, we constructed ribosome binding site libraries and developed a genetic selection to identify clones with inducible phenotypes. This combinatorial strategy readily afforded the desired phenotypes and should be broadly applicable for any output module amenable to positive selection.
Section snippets
Modularity of invasin
Invasin is a long rigid protein that is anchored in the outer membrane and extends 18 nm from the bacterial cell surface.32 Binding of β1-integrins does not require additional bacterial proteins to confer invasion since latex beads coated with invasin are taken up by mammalian cells.33 Nevertheless, bacteria use multiple strategies to interact with mammalian cells.34 E. coli MC1061 synthesize type I pili encoded by the fim operon, which bind to mammalian surface carbohydrates. This strain does
Discussion
We have described the design of bacteria able to invade mammalian cells selectively at high cell density, after anaerobic growth, or after chemical induction. This was achieved by combining an output interface module encoded by the inv gene from Y. pseudotuberculosis with the lux operon of V. fisheri, the fdhF promoter, or the arabinose operon. As an output module, inv afforded invasion of multiple cancer cell types including cervical carcinoma, hapatocarcinoma, and osteosarcoma.
Invasin presents
Molecular biology
All manipulations were performed in derivatives of E. coli strains MC1061 or EC100D™ pir-116 (Epicentre, Madison, Wis.) in 2YT liquid media or LB agar plates supplemented with antibiotics at 25 μg/ml at 37 °C. DNA-modifying enzymes were purchased from New England Biolabs. Oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, TX) and used unpurified. PCR was performed with the Roche High Fidelity PCR kit. The lux operon was derived from plasmids pKE705 and pKE555.50 Y. pseudotuberculosis
Acknowledgements
We thank Professor E. P. Greenberg for lux plasmids, J. S. Weissman for use of the anaerobic chamber, and K. R. Yamamoto for U2OS cells. J.C.A. is a Damon Runyon Cancer Research Foundation fellow. C.A.V. acknowledges support from the Sloan Foundation and a Sandler Family Award. A.P.A. acknowledges support from the Howard Hughes Medical Institute and from the National Institutes of Health (5R01GM63525-3).
References (53)
- et al.
Bacteria as tumour-targeting vectors
Lancet Oncol.
(2003) - et al.
Engineering a ligand-dependent RNA transcriptional activator
Chem. Biol.
(2004) - et al.
Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells
J. Control Release
(2004) - et al.
Elicitation of predictable immune responses by using live bacterial vectors
Biomol. Eng.
(2001) - et al.
Emerging views on integrin signaling via Rac1 during invasin-promoted bacterial uptake
Curr. Opin. Microbiol.
(2005) - et al.
Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells
Cell
(1987) - et al.
Adhesins and invasins of pathogenic bacteria: a structural view
Microbes Infect.
(2004) - et al.
Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR
J. Biol. Chem.
(2003) - et al.
Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides
Gene
(2000) - et al.
Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection
Cell
(1999)
Microbes made to order
Science
Programmable cells: interfacing natural and engineered gene networks
Proc. Natl Acad. Sci. USA
An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli
J. Bacteriol.
A synthetic multicellular system for programmed pattern formation
Nature
The use of live attenuated bacteria as a delivery system for heterologous antigens
J. Drug Target.
Engineering a mevalonate pathway in Escherichia coli for production of terpenoids
Nature Biotechnol.
Computational design of receptor and sensor proteins with novel functions
Nature
Combinatorial synthesis of genetic networks
Science
A synthetic oscillatory network of transcriptional regulators
Nature
Construction of a genetic toggle switch in Escherichia coli
Nature
Spatiotemporal control of gene expression with pulse-generating networks
Proc. Natl Acad. Sci. USA
Reprogramming control of an allosteric signaling switch through modular recombination
Science
Programmed population control by cell-cell communication and regulated killing
Nature
Functional gene transfer from intracellular bacteria to mammalian cells
Nature Biotechnol.
DNA modification and functional delivery into human cells using Escherichia coli DH10B
Nucl. Acids Res.
Potential therapeutic applications of recombinant, invasive E. coli
Gene Ther.
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