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Engineering bacteria for diagnostic and therapeutic applications

Abstract

Our ability to generate bacterial strains with unique and increasingly complex functions has rapidly expanded in recent times. The capacity for DNA synthesis is increasing and costing less; new tools are being developed for fast, large-scale genetic manipulation; and more tested genetic parts are available for use, as is the knowledge of how to use them effectively. These advances promise to unlock an exciting array of 'smart' bacteria for clinical use but will also challenge scientists to better optimize preclinical testing regimes for early identification and validation of promising strains and strategies. Here, we review recent advances in the development and testing of engineered bacterial diagnostics and therapeutics. We highlight new technologies that will assist the development of more complex, robust and reliable engineered bacteria for future clinical applications, and we discuss approaches to more efficiently evaluate engineered strains throughout their preclinical development.

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Figure 1: Examples of strategies for bacterial therapeutic delivery.
Figure 2: Examples of recently developed synthetic circuits.
Figure 3: Bacterial sensing through one-component and two-component systems.
Figure 4: Control, biosafety and biocontainment strategies for therapeutic bacteria.

References

  1. 1

    Coley, W. B. The treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases. Am. J. Med. Sci. 105, 487–510 (1893).

    Google Scholar 

  2. 2

    Busch, W. Aus der Sitzung der medicinischen section vom 13. November 1867 [GERMAN]. Berl. Klin. Wochenschr. 5, 137 (1868).

    Google Scholar 

  3. 3

    Fehleisen, F. Ueber die Züchtung der Erysipelkokken auf künstlichem Nährboden und ihre Übertragbarkeit auf den Menschen [GERMAN]. Dtsch. Med. Wochenschr. 8, 553–554 (1882).

    Google Scholar 

  4. 4

    Takiishi, T. et al. Reversal of diabetes in NOD mice by clinical-grade proinsulin and IL-10-secreting Lactococcus lactisin combination with low-dose anti-CD3 depends on the induction of Foxp3-positive T cells. Diabetes 66, 448–459 (2017).

    CAS  PubMed  Google Scholar 

  5. 5

    Braat, H. et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin. Gastroenterol. Hepatol. 4, 754–759 (2006).

    CAS  PubMed  Google Scholar 

  6. 6

    Lagenaur, L. A. et al. Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus. Mucosal Immunol. 4, 648–657 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Zheng, J. H. et al. Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin. Sci. Transl Res. 9, eaak9537 (2017).

    Google Scholar 

  8. 8

    Hanson, M. L. et al. Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice. Gastroenterology 146, 210–221.e213 (2014). A nice demonstration of the unique capabilities of live bacteria over recombinant protein delivery using L. lactis expressing IL-27 to reduce colitis in the mouse.

    CAS  PubMed  Google Scholar 

  9. 9

    Hwang, I. Y. et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8, 15028 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Forkus, B., Ritter, S., Vlysidis, M., Geldart, K. & Kaznessis, Y. N. Antimicrobial probiotics reduce Salmonella enterica in turkey gastrointestinal tracts. Sci. Rep. 7, 40695 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Steidler, L. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000). A classic paper of the field, describing the use of IL-10 secreting L. lactis bacteria to mitigate colitis in mice.

    CAS  PubMed  Google Scholar 

  12. 12

    Motta, J. P. et al. Food-grade bacteria expressing elafin protect against inflammation and restore colon homeostasis. Sci. Transl Med. 4, 158ra144 (2012).

    PubMed  Google Scholar 

  13. 13

    Vandenbroucke, K. et al. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 127, 502–513 (2004).

    CAS  PubMed  Google Scholar 

  14. 14

    Vandenbroucke, K. et al. Orally administered L. lactis secreting an anti-TNF nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol. 3, 49–56 (2010).

    CAS  PubMed  Google Scholar 

  15. 15

    Steidler, L. et al. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect. Immun. 66, 3183–3189 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Sizemore, D. R., Branstrom, A. A. & Sadoff, J. C. Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 270, 299–302 (1995).

    CAS  PubMed  Google Scholar 

  17. 17

    Schafer, R., Portnoy, D. A., Brassell, S. A. & Paterson, Y. Induction of a cellular immune response to a foreign antigen by a recombinant Listeria monocytogenes vaccine. J. Immunol. 149, 53–59 (1992).

    CAS  PubMed  Google Scholar 

  18. 18

    Riglar, D. T. et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat. Biotechnol. 35, 653–658 (2017). Long-term testing of a bacterial diagnostic for inflammation in mice. Circuit function and sequence was tested during >6 months of constant colonization of the gut.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Daeffler, K. N. M. et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol. Syst. Biol. 13, 923 (2017).

    PubMed  PubMed Central  Google Scholar 

  20. 20

    Limaye, S. A. et al. Phase 1b, multicenter, single blinded, placebo-controlled, sequential dose escalation study to assess the safety and tolerability of topically applied AG013 in subjects with locally advanced head and neck cancer receiving induction chemotherapy. Cancer 119, 4268–4276 (2013).

    CAS  PubMed  Google Scholar 

  21. 21

    Le, D. T. et al. Safety and survival with GVAX pancreas prime and Listeria monocytogenes–expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer. J. Clin. Oncol. 33, 1325–1333 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Maciag, P. C., Radulovic, S.a. & Rothman, J. The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a phase i safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix. Vaccine 27, 3975–3983 (2009).

    CAS  PubMed  Google Scholar 

  23. 23

    Johnson, P. V., Blair, B. M., Zeller, S., Kotton, C. N. & Hohmann, E. L. Attenuated Listeria monocytogenes vaccine vectors expressing Influenza A nucleoprotein: preclinical evaluation and oral inoculation of volunteers. Microbiol. Immunol. 55, 304–317 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Angelakopoulos, H. et al. Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: a dose escalation study of oral inoculation. Infect. Immun. 70, 3592–3601 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Le, D. T. et al. A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction. Clin. Cancer Res. 18, 858–868 (2012).

    CAS  PubMed  Google Scholar 

  26. 26

    Le, D. T. et al. Results from a phase 2b, randomized, multicenter study of GVAX pancreas and CRS-207 compared to chemotherapy in adults with previously-treated metastatic pancreatic adenocarcinoma (ECLIPSE Study). J. Clinl Oncol. 35, 345–345 (2017).

    Google Scholar 

  27. 27

    Boles, K. S. et al. Digital-to-biological converter for on-demand production of biologics. Nat. Biotechnol. 29, 544 (2017). An insight into the possible future of automation in the construction and testing of biologics.

    Google Scholar 

  28. 28

    Nielsen, A. A. K. et al. Genetic circuit design automation. Science 352, aac734 (2016).

    Google Scholar 

  29. 29

    Xiang, S., Fruehauf, J. & Li, C. J. Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nat. Biotechnol. 24, 697–702 (2006).

    CAS  PubMed  Google Scholar 

  30. 30

    Saltzman, D. A. et al. Attenuated Salmonella typhimurium containing interleukin-2 decreases MC-38 hepatic metastases: a novel anti-tumor agent. Cancer. Biother. Radiopharm. 11, 145–153 (1996).

    CAS  PubMed  Google Scholar 

  31. 31

    Zheng, L. M. et al. Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector. Oncol. Res. 12, 127–135 (2000).

    CAS  PubMed  Google Scholar 

  32. 32

    Gunn, G. R. et al. Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16. J. Immunol. 167, 6471–6479 (2001).

    CAS  PubMed  Google Scholar 

  33. 33

    Liu, S. C., Minton, N. P., Giaccia, A. J. & Brown, J. M. Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther. 9, 291–296 (2002).

    CAS  PubMed  Google Scholar 

  34. 34

    Nishikawa, H. et al. In vivo antigen delivery by a Salmonella typhimurium type III secretion system for therapeutic cancer vaccines. J. Clin. Invest. 116, 1946–1954 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Loeffler, M., Le'Negrate, G., Krajewska, M. & Reed, J. C. Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proc. Natl Acad. Sci. USA 104, 12879–12883 (2007).

    CAS  PubMed  Google Scholar 

  36. 36

    Ryan, R. M. et al. Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumors. Gene Ther. 16, 329–339 (2009).

    CAS  PubMed  Google Scholar 

  37. 37

    Din, M. O. et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81–85 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Steidler, L. et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat. Biotechnol. 21, 785–789 (2003).

    CAS  PubMed  Google Scholar 

  39. 39

    Waeytens, A. et al. Paracellular entry of interleukin-10 producing Lactococcus lactis in inflamed intestinal mucosa in mice. Inflamm. Bowel Dis. 14, 471–479 (2008). Development and testing of several technological features that underpin several ongoing L. lactis therapeutic efforts, including the use of thiamine deficiency as a biocontainment measure.

    PubMed  Google Scholar 

  40. 40

    Ricci, S. et al. In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii. BMC Biotechnol. 3, 15 (2003).

    PubMed  PubMed Central  Google Scholar 

  41. 41

    Porzio, S., Bossù, P., Ruggiero, P., Boraschi, D. & Tagliabue, A. Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria. BMC Biotechnol. 4, 27 (2004).

    PubMed  PubMed Central  Google Scholar 

  42. 42

    Hamady, Z. Z. et al. Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-beta1 under the control of dietary xylan 1. Inflamm. Bowel Dis. 17, 1925–1935 (2011).

    PubMed  Google Scholar 

  43. 43

    Whelan, R. A. et al. A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation. Mol. Ther. 22, 1730–1740 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Caluwaerts, S. et al. AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncol. 46, 564–570 (2010).

    CAS  PubMed  Google Scholar 

  45. 45

    Kruger, C. et al. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat. Biotechnol. 20, 702–706 (2002).

    PubMed  Google Scholar 

  46. 46

    Duan, F. F., Liu, J. H. & March, J. C. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes 64, 1794–1803 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Takiishi, T. et al. Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice. J. Clin. Invest. 122, 1717–1725 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Robert, S. et al. Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice. Diabetes 63, 2876–2887 (2014).

    CAS  PubMed  Google Scholar 

  49. 49

    Agarwal, P., Khatri, P., Billack, B., Low, W.-K. & Shao, J. Oral delivery of glucagon like peptide-1 by a recombinant Lactococcus lactis. Pharm. Res. 31, 3404–3414 (2014).

    CAS  PubMed  Google Scholar 

  50. 50

    Lin, Y. et al. Oral delivery of pentameric glucagon-like peptide-1 by recombinant Lactobacillus in diabetic rats. PLOS ONE 11, e0162733 (2016).

    PubMed  PubMed Central  Google Scholar 

  51. 51

    Paton, A. W., Morona, R. & Paton, J. C. A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans. Nat. Med. 6, 265–270 (2000).

    CAS  PubMed  Google Scholar 

  52. 52

    Focareta, A., Paton, J. C., Morona, R., Cook, J. & Paton, A. W. A recombinant probiotic for treatment and prevention of cholera. Gastroenterology 130, 1688–1695 (2006).

    CAS  PubMed  Google Scholar 

  53. 53

    Duan, F. & March, J. C. Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proc. Natl Acad. Sci. USA 107, 11260–11264 (2010).

    CAS  PubMed  Google Scholar 

  54. 54

    Rao, S. et al. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc. Natl Acad. Sci. USA 102, 11993–11998 (2005).

    CAS  PubMed  Google Scholar 

  55. 55

    Liu, X. et al. Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob. Agents Chemother. 50, 3250–3259 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Chen, Z. et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Marinho, F. A. et al. An intranasal administration of Lactococcus lactis strains expressing recombinant interleukin-10 modulates acute allergic airway inflammation in a murine model. Clin. Exp. Allergy 40, 1541–1551 (2010).

    CAS  PubMed  Google Scholar 

  58. 58

    Yang, G. et al. Effective treatment of hypertension by recombinant Lactobacillus plantarum expressing angiotensin converting enzyme inhibitory peptide. Microb. Cell Fact. 14, 202 (2015).

    PubMed  PubMed Central  Google Scholar 

  59. 59

    McGregor, D. P. Discovering and improving novel peptide therapeutics. Curr. Opin. Pharmacol. 8, 616–619 (2008).

    CAS  PubMed  Google Scholar 

  60. 60

    Baggio, L. L. & Drucker, D. J. Biology of Incretins: GLP-1 and GIP. Gastroenterology 132, 2131–2157 (2007).

    CAS  PubMed  Google Scholar 

  61. 61

    Suzuki, A., Nakauchi, H. & Taniguchi, H. Glucagon-like peptide 1 (1–37) converts intestinal epithelial cells into insulin-producing cells. Proc. Natl Acad. Sci. USA 100, 5034–5039 (2003).

    CAS  PubMed  Google Scholar 

  62. 62

    Creamer, B., Shorter, R. G. & Bamforth, J. The turnover and shedding of epithelial cells. I. The turnover in the gastrointestinal tract. Gut 2, 110–118 (1961).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Chen, K. & Cerutti, A. Vaccination strategies to promote mucosal antibody responses. Immunity 33, 479–491 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Atkinson, M. A., Maclaren, N. K., Riley, W. J., Sharp, D. W. & Lacey, P. E. 64 000 Mr autoantibodies as predictors of insulin-dependent diabetes. Lancet 335, 1357–1360 (1990).

    CAS  PubMed  Google Scholar 

  65. 65

    Wong, F. S. et al. Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat. Med. 5, 1026–1031 (1999).

    CAS  PubMed  Google Scholar 

  66. 66

    Akdis, C. A. & Akdis, M. Mechanisms of immune tolerance to allergens: role of IL-10 and Tregs. J. Clin. Invest. 124, 4678–4680 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    Pamer, E. G. Immune responses to Listeria monocytogenes. Nat. Rev. Immunol. 4, 812–823 (2004).

    CAS  PubMed  Google Scholar 

  68. 68

    Toso, J. F. et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J. Clin. Oncol. 20, 142–152 (2002).

    PubMed  PubMed Central  Google Scholar 

  69. 69

    Nemunaitis, J. et al. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther. 10, 737–744 (2003).

    CAS  PubMed  Google Scholar 

  70. 70

    Vacchelli, E. et al. Trial watch. Oncoimmunology 3, e29030 (2014).

    PubMed  PubMed Central  Google Scholar 

  71. 71

    Fedorak, R. N. et al. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn's disease. The Interleukin 10 Inflammatory Bowel Disease Cooperative Study Group. Gastroenterology 119, 1473–1482 (2000).

    CAS  PubMed  Google Scholar 

  72. 72

    Colombel, J. F. et al. Interleukin 10 (Tenovil) in the prevention of postoperative recurrence of Crohn's disease. Gut 49, 42–46 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73

    Tilg, H. et al. Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma. Gut 50, 191–195 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Evaluate. Press release. ActoGenix completes phase 2A clinical trial of AG011. Evaluate http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=197312 (2009).

  75. 75

    Awasthi, A. et al. A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. Nat. Immunol. 8, 1380–1389 (2007).

    CAS  PubMed  Google Scholar 

  76. 76

    Stumhofer, J. S. et al. Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat. Immunol. 8, 1363–1371 (2007).

    CAS  PubMed  Google Scholar 

  77. 77

    Billat, P.-A., Roger, E., Faure, S. & Lagarce, F. Models for drug absorption from the small intestine: where are we and where are we going? Drug Discov. Today, 22, 761–775 (2017).

    CAS  Google Scholar 

  78. 78

    Zav'yalov, V. P. et al. Specific high affinity binding of human interleukin 1β by Caf1A usher protein of Yersinia pestis. FEBS Lett. 371, 65–68 (1995).

    CAS  PubMed  Google Scholar 

  79. 79

    Luo, G., Niesel, D. W., Shaban, R. A., Grimm, E. A. & Klimpel, G. R. Tumor necrosis factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration of bacterial virulence properties. Infect. Immun. 61, 830–835 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80

    Wu, L. Recognition of host immune activation by Pseudomonas aeruginosa. Science 309, 774–777 (2005).

    CAS  PubMed  Google Scholar 

  81. 81

    Clarke, M. B., Hughes, D. T., Zhu, C., Boedeker, E. C. & Sperandio, V. The QseC sensor kinase: a bacterial adrenergic receptor. Proc. Natl Acad. Sci. USA 103, 10420–10425 (2006).

    CAS  PubMed  Google Scholar 

  82. 82

    Guthrie, G. D., Nicholson-Guthrie, C., S. & Leary Jr., H. L. A bacterial high-affinity GABA binding protein: isolation and characterization. Biochem. Biophys. Res. Commun. 268, 65–68 (2000).

    CAS  PubMed  Google Scholar 

  83. 83

    Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A. & Shapiro, M. G. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat. Chem. Biol. 13, 75–80 (2016).

    PubMed  Google Scholar 

  84. 84

    Stirling, F. et al. Rational design of evolutionarily stable microbial kill switches. Mol. Cell 68, 686–697.e3 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Pickard, J. M. et al. Rapid fucosylation of intestinal epithelium sustains host–commensal symbiosis in sickness. Nature 514, 638–641 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86

    Winter, S. E. et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87

    Courbet, A., Endy, D., Renard, E., Molina, F. & Bonnet, J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci. Transl Med. 7, 289ra283 (2015). An interesting approach using engineered bacteria as diagnostics grown on ex vivo human samples.

    Google Scholar 

  88. 88

    Kotula, J. W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl Acad. Sci. USA 111, 4838–4843 (2014). The first test of a synthetic memory circuit in vivo in a mouse model, as well as isolation of a mouse commensal E. coli strain capable of long-term competitive growth in the mouse without selection by antibiotics.

    CAS  PubMed  Google Scholar 

  89. 89

    Danino, T. et al. Programmable probiotics for detection of cancer in urine. Sci. Transl Med. 7, 289ra284 (2015).

    Google Scholar 

  90. 90

    O'Boyle, C. J. et al. Microbiology of bacterial translocation in humans. Gut 42, 29–35 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91

    Panteli, J. T., Forkus, B. A., Van Dessel, N. & Forbes, N. S. Genetically modified bacteria as a tool to detect microscopic solidtumour masses with triggered release of a recombinant biomarker. Integr. Biol. 7, 423–434 (2015).

    CAS  Google Scholar 

  92. 92

    Bassler, B. L. & Losick, R. Bacterially speaking. Cell 125, 237–246 (2006).

    CAS  PubMed  Google Scholar 

  93. 93

    Gupta, S., Bram, E. E. & Weiss, R. Genetically programmable pathogen sense and destroy. ACS Synth. Biol. 2, 715–723 (2013).

    CAS  PubMed  Google Scholar 

  94. 94

    Hwang, I. Y. et al. Reprogramming microorganisms to be pathogen-seeking killers. ACS Synth. Biol. 3, 228–237 (2014).

    CAS  PubMed  Google Scholar 

  95. 95

    Saeidi, N. et al. Engineering microorganisms to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol. Syst. Biol. 7, 521–521 (2011).

    PubMed  PubMed Central  Google Scholar 

  96. 96

    Borrero, J., Chen, Y., Dunny, G. M. & Kaznessis, Y. N. Modified lactic acid bacteria detect and inhibit multiresistant enterococci. ACS Synth. Biol. 4, 299–306 (2015).

    CAS  PubMed  Google Scholar 

  97. 97

    Swofford, C. A., Van Dessel, N. & Forbes, N. S. Quorum-sensing Salmonella selectively trigger protein expression within tumours. Proc. Natl Acad. Sci. USA 112, 3457–3462 (2015).

    CAS  PubMed  Google Scholar 

  98. 98

    Royo, J. L. et al. In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit. Nat. Methods 4, 937–942 (2007).

    CAS  PubMed  Google Scholar 

  99. 99

    Committee for Advanced Therapies. Draft reflection paper on classification of advanced-therapy medicinal products. EMA/CAT/600280/2010 Rev.1. (European Medicines Agency, 2015).

  100. 100

    Centre for Biologics Evaluation and Research. Recommendations for Microbial Vectors use for Gene Therapy. U.S. Food & Drug Administration https://www.fda.gov/ucm/groups/fdagov-public/@fdagov-bio-gen/documents/document/ucm466625.pdf (2016). A useful set of guidelines describing the types of information necessary for regulatory approval of engineered bacteria as human therapeutics.

  101. 101

    Lynch, M. & Marinov, G. K. The bioenergetic costs of a gene. Proc. Natl Acad. Sci. USA 112, 15690–15695 (2015).

    CAS  PubMed  Google Scholar 

  102. 102

    Ceroni, F., Algar, R., Stan, G.-B. & Ellis, T. Quantifying cellular capacity identifies gene expression designs with reduced burden. Nat. Methods 12, 415–418 (2015).

    CAS  PubMed  Google Scholar 

  103. 103

    Danino, T., Lo, J., Prindle, A., Hasty, J. & Bhatia, S. N. In vivo gene expression dynamics of tumor-targeted bacteria. ACS Synth. Biol. 1, 465–470 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104

    Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J. & Collins, J. J. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat. Chem. Biol. 12, 82–86 (2015).

    PubMed  PubMed Central  Google Scholar 

  105. 105

    Verch, T., Pan, Z. K. & Paterson, Y. Listeria monocytogenes-based antibiotic resistance gene-free antigen delivery system applicable to other bacterial vectors and DNA vaccines. Infect. Immun. 72, 6418–6425 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106

    Sleight, S. C. & Sauro, H. M. Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multigene circuits. ACS Synth. Biol. 2, 519–528 (2013).

    CAS  PubMed  Google Scholar 

  107. 107

    Gorochowski, T. E., Avcilar-Kucukgoze, I., Bovenberg, R. A., Roubos, J. A. & Ignatova, Z. A. Minimal model of ribosome allocation dynamics captures trade-offs in expression between endogenous and synthetic genes. ACS Synth. Biol. 5, 710–720 (2016).

    CAS  PubMed  Google Scholar 

  108. 108

    Segall-Shapiro, T. H., Meyer, A. J., Ellington, A. D., Sontag, E. D. & Voigt, C. A. A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase. Mol. Syst. Biol. 10, 742 (2014).

    PubMed  PubMed Central  Google Scholar 

  109. 109

    Pasini, M. et al. Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli. N. Biotechnol. 33, 78–90 (2016).

    CAS  PubMed  Google Scholar 

  110. 110

    Ceroni, F. et al. Burden-driven feedback control of gene expression. bioRxiv http://dx.doi.org/10.1101/177030 (2017).

    Google Scholar 

  111. 111

    De Boever, P., DePlancke, B. & Verstraete, W. Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J. Nutr. 130, 2599–2606 (2000).

    CAS  PubMed  Google Scholar 

  112. 112

    Kim, H. J. & Ingber, D. E. Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integr. Biol. 5, 1130 (2013).

    CAS  Google Scholar 

  113. 113

    Yissachar, N. et al. An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell 168, 1135–1148.e12 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. 114

    U.S. Securities and Exchange Commission. Partial clinical hold lifted and enrollment resumes for Aduro Biotech LADD clinical trials. SEC https://www.sec.gov/Archives/edgar/data/1435049/000119312516772847/d280592dex991.htm (2016).

  115. 115

    Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013). A study describing removal of a codon from the entire genome of E. coli and repurposing it for the addition of non-native amino acids.

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116

    Lau, Y. H. et al. Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA. Nucl. Acids Res. 45, 6971–6980 (2017).

    CAS  PubMed  Google Scholar 

  117. 117

    Mandell, D. J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. 118

    Wang, K. et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119

    Rovner, A. J. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. 120

    Ostrov, N. et al. Design, synthesis, and testing toward a 57-codon genome. Science 353, 819–822 (2016).

    CAS  PubMed  Google Scholar 

  121. 121

    Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354–360 (2013). A landmark study describing the design and quantification of an extensive library of components to rationally modulate gene expression levels.

    CAS  PubMed  Google Scholar 

  122. 122

    Cameron, D. E. & Collins, J. J. Tunable protein degradation in bacteria. Nat. Biotechnol. 32, 1276–1281 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. 123

    Werner, S., Engler, C., Weber, E., Gruetzner, R. & Marillonnet, S. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng. Bugs 3, 38–43 (2012).

    PubMed  Google Scholar 

  124. 124

    Sarrion-Perdigones, A. et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLOS ONE 6, e21622 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. 125

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  126. 126

    Medema, M. H. et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39, W339–346 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. 127

    Chevalier, A. et al. Massively parallel de novo protein design for targeted therapeutics. Nature 550, 74–79 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. 128

    Schreiber, H. L. et al. Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections. Sci. Transl Med. 9, eaaf1283 (2017).

    PubMed  PubMed Central  Google Scholar 

  129. 129

    Mahan, M. J., Slauch, J. M. & Mekalanos, J. J. Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259, 686–688 (1993).

    CAS  PubMed  Google Scholar 

  130. 130

    Bron, P. A., Grangette, C., Mercenier, A., De Vos, W. M. & Kleerebezem, M. Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice. J. Bacteriol. 186, 5721–5729 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. 131

    Li, H. et al. The outer mucus layer hosts a distinct intestinal microbial niche. Nat. Commun. 6, 8292 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132

    Tabor, J. J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).

    PubMed  PubMed Central  Google Scholar 

  133. 133

    Fernandez-Rodriguez, J., Moser, F., Song, M. & Voigt, C. A. Engineering RGB color vision into Escherichia coli. Nat. Chem. Biol. 13, 706–708 (2017).

    CAS  PubMed  Google Scholar 

  134. 134

    Shis, D. L., Hussain, F., Meinhardt, S., Swint-Kruse, L. & Bennett, M. R. Modular, multi-input transcriptional logic gating with orthogonal LacI/GalR family chimeras. ACS Synth. Biol. 3, 645–651 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. 135

    Plenge, R. M. Disciplined approach to drug discovery and early development. Sci. Transl Med. 8, 349ps315 (2016). Perspective discussing an approach to drug discovery and development that hopes to increase the likelihood of success through discipline early in the conceptualization and testing of a potential therapy.

    Google Scholar 

  136. 136

    Corthier, G., Delorme, C., Ehrlich, S. D. & Renault, P. Use of luciferase genes as biosensors to study bacterial physiology in the digestive tract. Appl. Environ. Microbiol. 64, 2721–2722 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. 137

    Klijn, N., Weerkamp, A. H. & de Vos, W. M. Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract. Appl. Environ. Microbiol. 61, 2771–2774 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138

    Curtiss, R. 3rd & Kelly, S. M. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect. Immun. 55, 3035–3043 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. 139

    Pawelek, J. M., Low, K. B. & Bermudes, D. Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res. 57, 4537–4544 (1997).

    CAS  PubMed  Google Scholar 

  140. 140

    Brockstedt, D. G. et al. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc. Natl Acad. Sci. USA 101, 13832–13837 (2004).

    CAS  PubMed  Google Scholar 

  141. 141

    Wallecha, A., Maciag, P. C., Rivera, S., Paterson, Y. & Shahabi, V. Construction and characterization of an attenuated Listeria monocytogenes strain for clinical use in cancer immunotherapy. Clin. Vaccine Immunol. 16, 96–103 (2009).

    CAS  PubMed  Google Scholar 

  142. 142

    Goodman, A. L. et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257 (2011).

    CAS  PubMed  Google Scholar 

  143. 143

    Geva-Zatorsky, N. et al. Mining the human gut microbiota for immunomodulatory organisms. Cell 168, 928–943.e11 (2017).

    CAS  PubMed  Google Scholar 

  144. 144

    Mimee, M., Tucker, A. C., Voigt, C. A. & Lu, T. K. Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Syst. 1, 62–71 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. 145

    Whitaker, W. R., Shepherd, E. S. & Sonnenburg, J. L. Tunable expression tools enable single-cell strain distinction in the gut microbiome. Cell 169, 538–546.e12 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. 146

    Lim, B., Zimmermann, M., Barry, N. A. & Goodman, A. L. Engineered regulatory systems modulate gene expression of human commensals in the gut. Cell 169, 547–558.e15 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. 147

    Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    CAS  PubMed  Google Scholar 

  148. 148

    Friedland, A. E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. 149

    Farzadfard, F. & Lu, T. K. Synthetic biology. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).

    PubMed  PubMed Central  Google Scholar 

  150. 150

    Yang, L. et al. Permanent genetic memory with >1-byte capacity. Nat. Methods 11, 1261–1266 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. 151

    Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl Acad. Sci. USA 109, 8884–8889 (2012).

    CAS  PubMed  Google Scholar 

  152. 152

    Guet, C. C., Elowitz, M. B., Hsing, W. & Leibler, S. Combinatorial synthesis of genetic networks. Science 296, 1466–1470 (2002).

    CAS  PubMed  Google Scholar 

  153. 153

    Green, A. A. et al. Complex cellular logic computation using ribocomputing devices. Nature 548, 117–121 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. 154

    Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016). Development of state machines using DNA recombinase recognition sites. Circuits based on this concept could be used to integrate both the presence and order of several signals for diagnosis or control of therapeutic expression in the future.

    PubMed  Google Scholar 

  155. 155

    Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

    CAS  PubMed  Google Scholar 

  156. 156

    Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008).

    CAS  PubMed  Google Scholar 

  157. 157

    Potvin-Trottier, L., Lord, N. D., Vinnicombe, G. & Paulsson, J. Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016).

    PubMed  PubMed Central  Google Scholar 

  158. 158

    Peccoud, J. et al. Essential information for synthetic DNA sequences. Nat. Biotechnol. 29, 22; discussion 22–23 (2011). Details the importance of sharing full DNA sequence information about all published synthetic circuits.

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank A. Naydich for critical review of the manuscript. D.T.R. is supported by a Human Frontier Science Program Long-Term Fellowship and a National Health and Medical Research Council (NHMRC) RG Menzies Early Career Fellowship from the Menzies Foundation through the Australian NHMRC.

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D.T.R. researched data for the article and wrote the article. D.T.R. and P.A.S. substantially contributed to the discussion of content and reviewed and edited the manuscript before submission.

Corresponding author

Correspondence to Pamela A. Silver.

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Supplementary information

Supplementary information S1 (table)

Completed, Active and Proposed clinical trials involving engineered live bacteria as therapeutics. (DOCX 30 kb)

Supplementary information S2 (table)

Examples of pre-clinical studies testing therapeutic engineered bacteria in animal disease models. (DOCX 27 kb)

PowerPoint slides

Glossary

Prodrug

An inactive form of a drug that requires activation, often by enzymatic cleavage, before adopting its therapeutic form.

Toll-like receptor

(TLR). A class of membrane receptors used by the innate immune system to recognize microbial molecules.

Metastasis

The spread of cancer cells from the original tumour to secondary sites around the body.

Colitis

Inflammation of the colon.

Trefoil factors

(TFFs). A family of peptides that are expressed at mucous membranes, including the gastrointestinal mucosa, and may have a protective role.

Recombinases

Enzymes that catalyse the excision, insertion, inversion or translocation of DNA between sites of specific DNA sequence.

Memory circuit

A genetic circuit that is designed to encode an extended response, or memory, following a transient cellular event.

Burden

The combined resources required by a cell to operate a given synthetic genetic pathway.

Quorum sensing

A common mechanism by which bacteria naturally sense the local population density of their own or other bacterial species to enable density- dependent cellular responses. Bacteria produce and sense a specific quorum sensing molecule. Constant secretion ensures that concentrations only reach threshold levels and change downstream transcriptional profiles when many bacteria are present in the population.

Kill switches

Circuits used as safety mechanisms to prevent incorrect activity of an engineered bacterial strain, usually by attempting to kill it or to prevent engineered functions.

Probiotics

Live microorganisms that are beneficial to health.

Genetic firewalls

Changes to the underlying genetic code of an organism in an attempt to prevent the possibility of effective genetic exchange between the engineered strain and other bacteria in the environment.

Log reduction

Measure of reduced bacterial growth based on the logarithm (base 10). Every additional log reduction therefore corresponds to tenfold lower growth or survival.

Logic gates

Circuits that use Boolean logic to activate an output only when a given combination of inputs is present.

State machines

Devices able to exist in one ofseveral unique states depending on the history of its inputs.

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Riglar, D., Silver, P. Engineering bacteria for diagnostic and therapeutic applications. Nat Rev Microbiol 16, 214–225 (2018). https://doi.org/10.1038/nrmicro.2017.172

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