Letter | Published:

Stabilization of cooperative virulence by the expression of an avirulent phenotype

Nature volume 494, pages 353356 (21 February 2013) | Download Citation

Abstract

Pathogens often infect hosts through collective actions: they secrete growth-promoting compounds or virulence factors, or evoke host reactions that fuel the colonization of the host. Such behaviours are vulnerable to the rise of mutants that benefit from the collective action without contributing to it; how these behaviours can be evolutionarily stable is not well understood1. We address this question using the intestinal pathogen Salmonella enterica serovar Typhimurium (hereafter termed S. typhimurium), which manipulates its host to induce inflammation, and thereby outcompetes the commensal microbiota2,3. Notably, the virulence factors needed for host manipulation are expressed in a bistable fashion, leading to a slow-growing subpopulation that expresses virulence genes, and a fast-growing subpopulation that is phenotypically avirulent4,5. Here we show that the expression of the genetically identical but phenotypically avirulent subpopulation is essential for the evolutionary stability of virulence in this pathogen. Using a combination of mathematical modelling, experimental evolution and competition experiments we found that within-host evolution leads to the emergence of mutants that are genetically avirulent and fast-growing. These mutants are defectors that exploit inflammation without contributing to it. In infection experiments initiated with wild-type S. typhimurium, defectors increase only slowly in frequency. In a genetically modified S. typhimurium strain in which the phenotypically avirulent subpopulation is reduced in size, defectors rise more rapidly, inflammation ceases prematurely, and S. typhimurium is quickly cleared from the gut. Our results establish that host manipulation by S. typhimurium is a cooperative trait that is vulnerable to the rise of avirulent defectors; the expression of a phenotypically avirulent subpopulation that grows as fast as defectors slows down this process, and thereby promotes the evolutionary stability of virulence. This points to a key role of bistable virulence gene expression in stabilizing cooperative virulence and may lead the way to new approaches for controlling pathogens.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & Social evolution theory for microorganisms. Nature Rev. Microbiol. 4, 597–607 (2006)

  2. 2.

    et al. Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol. 5, e244 (2007)

  3. 3.

    , , & The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen’s virulence factors, and the host’s mucosal immune response. Immunol. Rev. 245, 56–83 (2012)

  4. 4.

    et al. Self-destructive cooperation mediated by phenotypic noise. Nature 454, 987–990 (2008)

  5. 5.

    et al. The cost of virulence: retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1. PLoS Pathog. 7, e1002143 (2011)

  6. 6.

    & Host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity. Infect. Immun. 67, 3703–3713 (1999)

  7. 7.

    & The dynamics of multiple infection and the evolution of virulence. Am. Nat. 146, 881–910 (1995)

  8. 8.

    , , & Within-host competition drives selection for the capsule virulence determinant of Streptococcus pneumoniae. Curr. Biol. 20, 1222–1226 (2010)

  9. 9.

    Cooperation and conflict in host-manipulating parasites. Proc. R. Soc. Lond. B 266, 1899–1904 (1999)

  10. 10.

    , & Does multiple infection select for raised virulence? Trends Microbiol. 10, 401–405 (2002)

  11. 11.

    & Kin selection and the evolution of virulence. Heredity 100, 484–488 (2008)

  12. 12.

    , , & Cooperation and virulence in acute Pseudomonas aeruginosa infections. BMC Biol. 4, 21 (2006)

  13. 13.

    , & Cooperation and virulence of clinical Pseudomonas aeruginosa populations. Proc. Natl Acad. Sci. USA 106, 6339–6344 (2009)

  14. 14.

    et al. Quorum sensing and the social evolution of bacterial virulence. Curr. Biol. 19, 341–345 (2009)

  15. 15.

    , , & The dynamics of cooperative bacterial virulence in the field. Science 337, 85–88 (2012)

  16. 16.

    , & Evolution of virulence: triggering host inflammation allows invading pathogens to exclude competitors. Ecol. Lett. 11, 44–51 (2008)

  17. 17.

    et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog. 6, e1001097 (2010)

  18. 18.

    et al. Accelerated type III secretion system 2-dependent enteropathogenesis by a Salmonella enterica serovar enteritidis PT4/6 strain. Infect. Immun. 77, 3569–3577 (2009)

  19. 19.

    , , & Integrating global regulatory input into the Salmonella pathogenicity island 1 type III secretion system. Genetics 190, 79–90 (2012)

  20. 20.

    , , & HilE interacts with HilD and negatively regulates hilA transcription and expression of the Salmonella enterica serovar Typhimurium invasive phenotype. Infect. Immun. 71, 1295–1305 (2003)

  21. 21.

    , & Growth dynamics and the evolution of cooperation in microbial populations. Sci. Rep. 2, 281 (2012)

  22. 22.

    et al. Host transmission of Salmonella enterica serovar Typhimurium is controlled by virulence factors and indigenous intestinal microbiota. Infect. Immun. 76, 403–416 (2008)

  23. 23.

    et al. Tackling antibiotic resistance. Nature Rev. Microbiol. 9, 894–896 (2011)

  24. 24.

    R Devlopment Core Team. A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2010)

  25. 25.

    , & Solving differential equations in R: Package deSolve. J. Stat. Softw. 33, 1–25 (2010)

  26. 26.

    & Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981)

  27. 27.

    , & The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol. Microbiol. 13, 555–568 (1994)

  28. 28.

    & One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

  29. 29.

    et al. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog. 5, e1000477 (2009)

  30. 30.

    , , , & Identification of Salmonella SPI-2 secretion system components required for SpvB-mediated cytotoxicity in macrophages and virulence in mice. FEMS Immunol. Med. Microbiol. 52, 194–201 (2008)

  31. 31.

    et al. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol. Microbiol. 30, 163–174 (1998)

  32. 32.

    et al. Accelerated type III secretion system 2-dependent enteropathogenesis by a Salmonella enterica serovar enteritidis PT4/6 strain. Infect. Immun. 77, 3569–3577 (2009)

  33. 33.

    & AraC/XylS family members, HilC and HilD, directly bind and derepress the Salmonella typhimurium hilA promoter. Mol. Microbiol. 40, 1289–1299 (2001)

  34. 34.

    , , & Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995)

  35. 35.

    et al. The cost of virulence: retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1. PLoS Pathog. 7, e1002143 (2011)

  36. 36.

    et al. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun. 72, 4138–4150 (2004)

  37. 37.

    , & Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells. Appl. Environ. Microbiol. 69, 7480–7491 (2003)

  38. 38.

    & ColE1-type vectors with fully repressible replication. Gene 105, 17–22 (1991)

  39. 39.

    et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhoea. PLoS Pathog. 6, e1001097 (2010)

  40. 40.

    , & Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears. J. Clin. Microbiol. 35, 2642–2648 (1997)

  41. 41.

    et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676–682 (2012)

  42. 42.

    et al. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun. 71, 2839–2858 (2003)

  43. 43.

    et al. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 325, 617–620 (2009)

Download references

Acknowledgements

We thank E. Slack, R. Kümmerli, M. Sellin and L. Robert for comments on the manuscript, G. Paul for input on the modelling, and Hardt laboratory members for discussions. M.D. was supported in part by the Fondation pour la Recherche Médicale. M.A., R.R.R. and W.-D.H. were supported by grants from the Swiss National Science Foundation.

Author information

Affiliations

  1. Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland

    • Médéric Diard
    • , Lisa Maier
    • , Mitja N. P. Remus-Emsermann
    •  & Wolf-Dietrich Hardt
  2. Institute of Integrative Biology, ETH Zurich, Universitaetsstr. 16, 8092 Zurich, Switzerland

    • Victor Garcia
    •  & Roland R. Regoes
  3. Department of Environmental Systems Science, ETH Zurich, and Department of Environmental Microbiology Eawag, Ueberlandstr. 133PO Box 611, 8600 Duebendorf, Switzerland

    • Martin Ackermann

Authors

  1. Search for Médéric Diard in:

  2. Search for Victor Garcia in:

  3. Search for Lisa Maier in:

  4. Search for Mitja N. P. Remus-Emsermann in:

  5. Search for Roland R. Regoes in:

  6. Search for Martin Ackermann in:

  7. Search for Wolf-Dietrich Hardt in:

Contributions

M.D., V.G. and R.R.R. conceived and analysed the mathematical simulations. M.D., V.G. and R.R.R. wrote the theoretical part of the paper. M.D., W.-D.H., L.M. (Supplementary Figs 14 and 20), M.N.P.R.-E (Supplementary Fig. 8) and M.A. designed the experiments and analysed the data. M.D., W.-D.H. and M.A. wrote the paper. M.D. and L.M. (Supplementary Figs 14 and 20) performed the experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Roland R. Regoes or Martin Ackermann or Wolf-Dietrich Hardt.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains Supplementary Text and Data, Supplementary Table 1, Supplementary Figures 1-20 and Supplementary References.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature11913

Further reading

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.