Letter

Synchronized cycles of bacterial lysis for in vivo delivery

Received:
Accepted:
Published online:

Abstract

The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body1,2,3. It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbour disease and thus provide a natural platform for the development of engineered therapies4,5,6. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ7,8,9,10. Here we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. As a proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies11, we orally administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    & The human microbiome: at the interface of health and disease. Nature Rev. Genet. 13, 260–270 (2012)

  2. 2.

    et al. Microbial dysbiosis is associated with human breast cancer. PLoS One 9, e83744 (2014)

  3. 3.

    , & Cell-based therapeutics: the next pillar of medicine. Sci. Transl. Med. 5, 179ps7 (2013)

  4. 4.

    , & Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res. 57, 4537–4544 (1997)

  5. 5.

    , & Synthetic biology moving into the clinic. Science 333, 1248–1252 (2011)

  6. 6.

    & Emerging biomedical applications of synthetic biology. Nature Rev. Genet. 13, 21–35 (2011)

  7. 7.

    , , , & Bacteria as vectors for gene therapy of cancer. Bioeng. Bugs 1, 385–394 (2010)

  8. 8.

    , & Dr William Coley and tumour regression: a place in history or in the future. Postgrad. Med. J. 79, 672–680 (2003)

  9. 9.

    et al. Efficacy and toxicity management of 19–28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl. Med. 6, 224ra25 (2014)

  10. 10.

    Cancer and the microbiota. Science 348, 80–86 (2015)

  11. 11.

    , , , & Combination bacteriolytic therapy for the treatment of experimental tumors. Proc. Natl Acad. Sci. USA 98, 15155–15160 (2001)

  12. 12.

    , , & A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010)

  13. 13.

    et al. A sensing array of radically coupled genetic ‘biopixels’. Nature 481, 39–44 (2011)

  14. 14.

    & Lytic action of cloned φ X174 gene E. J. Virol. 44, 993–1002 (1982)

  15. 15.

    , , , & Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology. PLoS One 5, e11909 (2010)

  16. 16.

    et al. Genetic circuits in Salmonella typhimurium. ACS Synth. Biol. 1, 458–464 (2012)

  17. 17.

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

  18. 18.

    The parB (hok/sok) locus of plasmid R1: a general purpose plasmid stabilization system. Nature Biotechnol. 6, 1402–1405 (1988)

  19. 19.

    , & Enhanced plasmid stability through post-segregational killing of plasmid-free cells. Biotechnol. Tech. 4, 39–44 (1990)

  20. 20.

    et al. Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A. Mol. Microbiol. 73, 534–552 (2009)

  21. 21.

    , , , & In vivo gene expression dynamics of tumor-targeted bacteria. ACS Synth. Biol. 1, 465–470 (2012)

  22. 22.

    et al. Programmable probiotics for detection of cancer in urine. Science Transl. Med. 7, 289ra84 (2015)

  23. 23.

    , , & Measuring growth and gene expression dynamics of tumor-targeted S. Typhimurium bacteria. JoVE e50540 (2013)

  24. 24.

    et al. Application of a proapoptotic peptide to intratumorally spreading cancer therapy. Cancer Res. 73, 1352–1361 (2013)

  25. 25.

    , , & Salmonella typhimurium engineered to produce CCL21 inhibit tumor growth. Cancer Immunol. Immunother. 58, 769–775 (2009)

  26. 26.

    Engineering the perfect (bacterial) cancer therapy. Nature Rev. Cancer 10, 785–794 (2010)

  27. 27.

    et al. Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism. Cell Host Microbe 17, 681–689 (2015)

  28. 28.

    , & Chronobiomics: the biological clock as a new principle in host–microbial interactions. PLoS Pathog. 11, e1005113 (2015)

  29. 29.

    et al. A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science 314, 1308–1311 (2006)

  30. 30.

    et al. A preclinical murine model of hepatic metastases. JoVE e51677 (2014)

Download references

Acknowledgements

The UCSD team was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R01GM069811) and the San Diego Center for Systems Biology (P50 GM085764). The MIT team was supported by a Koch Institute Support Grant (P30-CA14051) from the National Cancer Institute (Swanson Biotechnology Center), a Core Center Grant (P30-ES002109) from the National Institute of Environmental Health Sciences, the Ludwig Center for Molecular Oncology at MIT and an Amar G. Bose Research Grant. T.D. was supported by the Misrock Postdoctoral fellowship and the NIH Pathway to Independence Award NIH (K99 CA197649-01). A.P. was supported by the Department of Defense National Defense Science and Engineering Graduate Fellowship and holds a Simons Foundation Fellowship of the Helen Hay Whitney Foundation and a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. S.N.B. is an HHMI Investigator. We would like to thank R. Johnson for help with constructing microfluidic devices, H. Fleming for help with editing the manuscript, and H. Ding of The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center for help with the statistical tests carried out in this study. We would also like to thank L. You for providing the lysis gene used in this study.

Author information

Author notes

    • Tal Danino

    Present address: Department of Biomedical Engineering, Columbia University, New York, New York 10027, USA.These authors contributed equally to this work.

    • M. Omar Din
    •  & Tal Danino

    These authors contributed equally to this work.

    • Sangeeta N. Bhatia
    •  & Jeff Hasty

    These authors jointly supervised this work.

Affiliations

  1. Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA

    • M. Omar Din
    • , Arthur Prindle
    • , Jangir Selimkhanov
    • , Ellixis Julio
    •  & Jeff Hasty
  2. Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Tal Danino
    • , Matt Skalak
    • , Kaitlin Allen
    • , Eta Atolia
    •  & Sangeeta N. Bhatia
  3. BioCircuits Institute, University of California, San Diego, La Jolla, California 92093, USA

    • Lev S. Tsimring
    •  & Jeff Hasty
  4. Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02139, USA

    • Sangeeta N. Bhatia
  5. Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02139, USA

    • Sangeeta N. Bhatia
  6. Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Sangeeta N. Bhatia
  7. Marble Center for Cancer Nanomedicine and Ludwig Center for Molecular Oncology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Sangeeta N. Bhatia
  8. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA

    • Sangeeta N. Bhatia
  9. Molecular Biology Section, Division of Biological Science, University of California, San Diego, La Jolla, California 92093, USA

    • Jeff Hasty

Authors

  1. Search for M. Omar Din in:

  2. Search for Tal Danino in:

  3. Search for Arthur Prindle in:

  4. Search for Matt Skalak in:

  5. Search for Jangir Selimkhanov in:

  6. Search for Kaitlin Allen in:

  7. Search for Ellixis Julio in:

  8. Search for Eta Atolia in:

  9. Search for Lev S. Tsimring in:

  10. Search for Sangeeta N. Bhatia in:

  11. Search for Jeff Hasty in:

Contributions

M.O.D. and J.H. designed the synchronized lysis circuit. M.O.D., J.S., L.S.T. and J.H. developed the computational model. M.O.D. and A.P. built and tested the bacterial circuit in microfluidics and performed the co-culture experiments. M.O.D. and E.J. collected the viability data, and M.O.D. and J.H. analysed the bacterial circuit data. T.D., M.S., K.A., and E.A. designed and performed the in vivo experiments. M.O.D., T.D., A.P., S.N.B., and J.H. analysed the animal data, and wrote and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jeff Hasty.

Reviewer Information Nature thanks R. Solé, B. Vogelstein and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Text and Data and additional references.

Videos

  1. 1.

    Timelapse fluorescence microscopy of the Synchronized Lysis Circuit (SLC) in Strain 1 (S. typhimurium, no ssrA tag on LuxI) at 60X magnification.

    Images were taken every 2 min at the bottom portion of a 100x100μm chamber.

  2. 2.

    Timelapse fluorescence microscopy of the SLC in Strain 2 (S. typhimurium, ssrA tag on LuxI) at 60X magnification

    We observe a longer lysis period with a higher degradation efficiency on LuxI. The chamber size is 100x100μm and images were taken every 2 min.

  3. 3.

    Timelapse fluorescence microscopy of the SLC in Strain 13 (E. coli) at 60X magnification.

    The chamber size is 100x100μm and images were taken every 2 min.

  4. 4.

    Bacteria and cancer cell co-culture on a microfluidic device at 60X magnification.

    Strain 3 (non-motile S. typhimurium, SLC with HlyE) was loaded in the growth chambers while HeLa cells grow in the main channel of the device. Timelapse fluorescence microscopy images were taken every 3 min.

  5. 5.

    Bacteria and cancer cell co-culture on a microfluidic device at 60X magnification.

    Strain 3 (non-motile S. typhimurium, SLC with HlyE) was loaded in the growth chambers while HeLa cells grow in the main channel of the device. Timelapse fluorescence microscopy images were taken every 3 min.

  6. 6.

    Bacteria and cancer cell co-culture in a tissue culture well-plate at 20X magnification.

    Strain 4 (motile S. typhimurium, SLC with HlyE) was loaded in the well with a monolayer HeLa cells. Timelapse fluorescence microscopy images were taken every 1 min.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.