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In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Nature Methods volume 4, pages 937–942 (2007)Cite this article

Abstract

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20–150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine–converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

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Figure 1: Tightly regulated expression of Salmonella spp. genes by using a circuit based on the regulatory module nahR/Psal::xylS2.
Figure 2: Intracellular expression of GFP in tumor cells by recombinant S. enterica carrying the gfp coding gene under control of the regulatory module nahR/Psal::xylS2.
Figure 3: Tightly regulated in vivo expression of prokaryotic genes within tumors using an ASA or salicylate–activated control circuit based on the regulatory module nahR/Psal::xylS2.
Figure 4: Salicylate-mediated in vivo expression of cytosine deaminase in tumor cells by using the control circuit based on the nahR/Psal::xylS2 regulatory module.

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References

  1. Hohmann, E.L., Oletta, C.A., Loomis, W.P. & Miller, S.I. Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity. Proc. Natl. Acad. Sci. USA 92, 2904–2908 (1995).

    Article  CAS  Google Scholar 

  2. McKinney, J., Guerrier-Takada, C., Galan, J. & Altman, S. Tightly regulated gene expression system in Salmonella enterica serovar Typhimurium. J. Bacteriol. 184, 6056–6059 (2002).

    Article  CAS  Google Scholar 

  3. Bumann, D. Regulated antigen expression in live recombinant Salmonella enterica serovar Typhimurium strongly affects colonization capabilities and specific CD4(+)-T-cell responses. Infect. Immun. 69, 7493–7500 (2001).

    Article  CAS  Google Scholar 

  4. Ward, S.J., Douce, G., Figueiredo, D., Dougan, G. & Wren, B.W. Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A. Infect. Immun. 67, 2145–2152 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Huang, Y., Hajishengallis, G. & Michalek, S.M. Construction and characterization of a Salmonella enterica serovar typhimurium clone expressing a salivary adhesin of Streptococcus mutans under control of the anaerobically inducible nirB promoter. Infect. Immun. 68, 1549–1556 (2000).

    Article  CAS  Google Scholar 

  6. Medina, E., Paglia, P., Rohde, M., Colombo, M.P. & Guzman, C.A. Modulation of host immune responses stimulated by Salmonella vaccine carrier strains by using different promoters to drive the expression of the recombinant antigen. Eur. J. Immunol. 30, 768–777 (2000).

    Article  CAS  Google Scholar 

  7. Bateman, B.T., Donegan, N.P., Jarry, T.M., Palma, M. & Cheung, A.L. Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation. Infect. Immun. 69, 7851–7857 (2001).

    Article  CAS  Google Scholar 

  8. Qian, F. & Pan, W. Construction of a tetR-integrated Salmonella enterica serovar Typhi CVD908 strain that tightly controls expression of the major merozoite surface protein of Plasmodium falciparum for applications in human vaccine production. Infect. Immun. 70, 2029–2038 (2002).

    Article  CAS  Google Scholar 

  9. Pawelek, J.M., Low, K.B. & Bermudes, D. Bacteria as tumour-targeting vectors. Lancet Oncol. 4, 548–556 (2003).

    Article  Google Scholar 

  10. Weissmann, G. Aspirin. Sci. Am. 264, 84–90 (1991).

    Article  CAS  Google Scholar 

  11. Hennekens, C.H. Update on aspirin in the treatment and prevention of cardiovascular disease. Am. J. Manag. Care 8, S691–S700 (2002).

    PubMed  Google Scholar 

  12. Yin, M.J., Yamamoto, Y. & Gaynor, R.B. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396, 77–80 (1998).

    Article  CAS  Google Scholar 

  13. Cebolla, A., Sousa, C. & de Lorenzo, V. Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity. Nucleic Acids Res. 29, 759–766 (2001).

    Article  CAS  Google Scholar 

  14. Suarez, A. et al. Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter. Appl. Environ. Microbiol. 63, 122–127 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Cebolla, A., Sousa, C. & de Lorenzo, V. Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers. J. Biol. Chem. 272, 3986–3992 (1997).

    Article  CAS  Google Scholar 

  16. Cebolla, A., Royo, J.L., De Lorenzo, V. & Santero, E. Improvement of recombinant protein yield by a combination of transcriptional amplification and stabilization of gene expression. Appl. Environ. Microbiol. 68, 5034–5041 (2002).

    Article  CAS  Google Scholar 

  17. Clairmont, C. et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J. Infect. Dis. 181, 1996–2002 (2000).

    Article  CAS  Google Scholar 

  18. Yu, Y.A. et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat. Biotechnol. 22, 313–320 (2004).

    Article  CAS  Google Scholar 

  19. Low, K.B. et al. Lipid A mutant Salmonella with suppressed virulence and TNFα induction retain tumor-targeting in vivo. Nat. Biotechnol. 17, 37–41 (1999).

    Article  CAS  Google Scholar 

  20. Wei, M.Q. et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur. J. Cancer 43, 490–496 (2007).

    Article  Google Scholar 

  21. 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).

    Article  Google Scholar 

  22. Medina, E. et al. Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses. Infect. Immun. 67, 1093–1099 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Tzschaschel, B.D., Guzman, C.A., Timmis, K.N. & de Lorenzo, V. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat. Biotechnol. 14, 765–769 (1996).

    Article  CAS  Google Scholar 

  24. Medina, E., Guzman, C.A., Staendner, L.H., Colombo, M.P. & Paglia, P. Salmonella vaccine carrier strains: effective delivery system to trigger anti-tumor immunity by oral route. Eur. J. Immunol. 29, 693–699 (1999).

    Article  CAS  Google Scholar 

  25. 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 

  26. 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).

    Article  CAS  Google Scholar 

  27. Burns-Keliher, L., Nickerson, C.A., Morrow, B.J. & Curtiss, R., III. Cell-specific proteins synthesized by Salmonella typhimurium. Infect. Immun. 66, 856–861 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Miller, J.H. Experiments in molecular genetics. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1972).

    Google Scholar 

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Author notes

  1. José Luis Royo and Pablo Daniel Becker: These authors contributed equally to this work.

Authors and Affiliations

  1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide–Consejo Superior de Investigaciones Científicas, Carretera, Utrera, Km 1, Sevilla, E-41013, Spain

    José Luis Royo, Eva María Camacho & Eduardo Santero

  2. Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, Braunschweig, D-38124, Germany

    José Luis Royo, Pablo Daniel Becker, Claudia Link & Carlos Alberto Guzmán

  3. Biomedal SL, Avda. Américo Vespucio 5, Blq E 1a planta, Sevilla, E-41092, Spain

    Angel Cebolla

Authors
  1. José Luis Royo
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  3. Eva María Camacho
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Contributions

J.L.R. and P.D.B. performed most of the experimental work and analysis of data, and contributed to experimental design. E.M.C. mapped the insertion of the regulatory module and constructed the strains bearing the expression module integrated into the aroC locus. A.C. initially designed the cascade expression system for use in eukaryotic cells. C.L. contributed to the in vivo work. E.S. and C.A.G. were responsible for experimental design, participated in the analysis of raw data and wrote the paper.

Corresponding author

Correspondence to Eduardo Santero.

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Competing interests

A.C. is an employee of Biomedal S.L.

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Supplementary Figures 1–2, Supplementary Table 1, Supplementary Methods (PDF 381 kb)

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Royo, J., Becker, P., Camacho, E. et al. In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit. Nat Methods 4, 937–942 (2007). https://doi.org/10.1038/nmeth1107

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