Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms

Abstract

The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires’ disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing (“dormant”) states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption (“resuscitation”) and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The Lqs system regulates the ratio of nongrowers in L. pneumophila biofilms.
Fig. 2: Nongrowing L. pneumophila in biofilms are persisters.
Fig. 3: The Lqs system and LvbR control resuscitation of sessile L. pneumophila.
Fig. 4: Sessile L. pneumophila nongrowers are antibiotic tolerant and virulent.
Fig. 5: Sessile L. pneumophila shows temperature-dependent phenotypic heterogeneity.
Fig. 6: Heterogeneous gene expression in L. pneumophila microcolonies is controlled by quorum sensing.

Data availability

All data is available in the main text or the Supplementary Material and provided as source data files.

References

  1. 1.

    Newton HJ, Ang DK, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010;23:274–98.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Hilbi H, Hoffmann C, Harrison CF. Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep. 2011;3:286–96.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Fields BS. The molecular ecology of Legionella. Trends Microbiol. 1996;4:286–90.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev. 2004;17:413–33.

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Hoffmann C, Harrison CF, Hilbi H. The natural alternative: protozoa as cellular models for Legionella infection. Cell Microbiol. 2014;16:15–26.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Boamah DK, Zhou G, Ensminger AW, O’Connor TJ. From many hosts, one accidental pathogen: The diverse protozoan hosts of Legionella. Front Cell Infect Microbiol. 2017;7:477.

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Swart AL, Harrison CF, Eichinger L, Steinert M, Hilbi H. Acanthamoeba and Dictyostelium as cellular models for Legionella infection. Front Cell Infect Microbiol. 2018;8:61.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Sherwood RK, Roy CR. A Rab-centric perspective of bacterial pathogen-occupied vacuoles. Cell Host Microbe. 2013;14:256–68.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Asrat S, de Jesus DA, Hempstead AD, Ramabhadran V, Isberg RR. Bacterial pathogen manipulation of host membrane trafficking. Annu Rev Cell Dev Biol. 2014;30:79–109.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Finsel I, Hilbi H. Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell Microbiol. 2015;17:935–50.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Qiu J, Luo ZQ. Legionella and Coxiella effectors: strength in diversity and activity. Nat Rev Microbiol. 2017;15:591–605.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Personnic N, Bärlocher K, Finsel I, Hilbi H. Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol. 2016;24:450–62.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Steiner B, Weber S, Hilbi H. Formation of the Legionella-containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics. Int J Med Microbiol. 2018;308:49–57.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Declerck P. Biofilms: the environmental playground of Legionella pneumophila. Environ Microbiol. 2010;12:557–66.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Abdel-Nour M, Duncan C, Low DE, Guyard C. Biofilms: the stronghold of Legionella pneumophila. Int J Mol Sci. 2013;14:21660–75.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16.

    Pécastaings S, Allombert J, Lajoie B, Doublet P, Roques C, Vianney A. New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling. Biofouling. 2016;32:935–48.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  17. 17.

    Hochstrasser R, Hilbi H. Intra-species and inter-kingdom signaling of Legionella pneumophila. Front Microbiol. 2017;8:79.

    PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Mampel J, Spirig T, Weber SS, Haagensen JAJ, Molin S, Hilbi H. Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl Environ Microbiol. 2006;72:2885–95.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Hindré T, Brüggemann H, Buchrieser C, Héchard Y. Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation. Microbiology. 2008;154:30–41.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  20. 20.

    Pécastaings S, Berge M, Dubourg KM, Roques C. Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms. Biofouling. 2010;26:809–19.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  21. 21.

    Wai SN, Mizunoe Y, Yoshida S. How Vibrio cholerae survive during starvation. FEMS Microbiol Lett. 1999;180:123–31.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science. 2004;305:1622–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Harms A, Maisonneuve E, Gerdes K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science. 2016;354:aaf4268.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  24. 24.

    Claudi B, Sprote P, Chirkova A, Personnic N, Zankl J, Schurmann N, et al. Phenotypic variation of Salmonella in host tissues delays eradication by antimicrobial chemotherapy. Cell. 2014;158:722–33.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Hélaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, Holden DW. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science. 2014;343:204–8.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. 26.

    Conlon BP, Rowe SE, Gandt AB, Nuxoll AS, Donegan NP, Zalis EA, et al. Persister formation in Staphylococcus aureus is associated with ATP depletion. Nat Microbiol. 2016;1:16051.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Personnic N, Striednig B, Lezan E, Manske C, Welin A, Schmidt A, et al. Quorum sensing modulates the formation of virulent Legionella persisters within infected cells. Nat Commun. 2019;10:5216.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. 28.

    Molofsky AB, Swanson MS. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol. 2004;53:29–40.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Hammer BK, Swanson MS. Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol Microbiol. 1999;33:721–31.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Dalebroux ZD, Yagi BF, Sahr T, Buchrieser C, Swanson MS. Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation. Mol Microbiol. 2010;76:200–19.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Tiaden A, Spirig T, Hilbi H. Bacterial gene regulation by α-hydroxyketone signaling. Trends Microbiol. 2010;18:288–97.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Personnic N, Striednig B, Hilbi H. Legionella quorum sensing and its role in pathogen-host interactions. Curr Opin Microbiol. 2018;41:29–35.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Spirig T, Tiaden A, Kiefer P, Buchrieser C, Vorholt JA, Hilbi H. The Legionella autoinducer synthase LqsA produces an α-hydroxyketone signaling molecule. J Biol Chem. 2008;283:18113–23.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Tiaden A, Spirig T, Sahr T, Wälti MA, Boucke K, Buchrieser C, et al. The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila. Environ Microbiol. 2010;12:1243–59.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Kessler A, Schell U, Sahr T, Tiaden A, Harrison C, Buchrieser C, et al. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit. Environ Microbiol. 2013;15:646–62.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R, Buchrieser C, et al. The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol. 2007;9:2903–20.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Tiaden A, Spirig T, Carranza P, Brüggemann H, Riedel K, Eberl L, et al. Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions. J Bacteriol. 2008;190:7532–47.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Schell U, Simon S, Sahr T, Hager D, Albers MF, Kessler A, et al. The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila. Mol Microbiol. 2016;99:778–93.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Hochstrasser R, Hutter CAJ, Arnold FM, Bärlocher K, Seeger MA, Hilbi H. The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization. Mol Microbiol. 2020;113:1070–84.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Hochstrasser R, Kessler A, Sahr T, Simon S, Schell U, Gomez-Valero L, et al. The pleiotropic Legionella transcription factor LvbR links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and virulence. Environ Microbiol. 2019;21:1035–53.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Hochstrasser R, Hilbi H. Legionella quorum sensing meets cyclic-di-GMP signaling. Curr Opin Microbiol. 2020;55:9–16.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Simon S, Schell U, Heuer N, Hager D, Albers MF, Matthias J, et al. Inter-kingdom signaling by the Legionella quorum sensing molecule LAI-1 modulates cell migration through an IQGAP1-Cdc42-ARHGEF9-dependent pathway. PLoS Pathog. 2015;11:e1005307.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  43. 43.

    Faucher SP, Friedlander G, Livny J, Margalit H, Shuman HA. Legionella pneumophila 6S RNA optimizes intracellular multiplication. Proc Natl Acad Sci USA. 2010;107:7533–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Faucher SP, Shuman HA. Small regulatory RNA and Legionella pneumophila. Front Microbiol. 2011;2:98.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Balaban NQ, Hélaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI, et al. Definitions and guidelines for research on antibiotic persistence. Nat Rev Microbiol. 2019;17:441–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Brauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol. 2016;14:320–30.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Personnic N, Striednig B, Hilbi H. Single cell analysis of Legionella and Legionella-infected Acanthamoeba by agarose embedment. Methods Mol Biol. 2019;1921:191–204.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Byrne B, Swanson MS. Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect Immun. 1998;66:3029–34.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol. 2007;5:48–56.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol. 2008;322:107–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Hall CW, Mah TF. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev. 2017;41:276–301.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Brenzinger S, van der Aart LT, van Wezel GP, Lacroix JM, Glatter T, Briegel A. Structural and proteomic changes in viable but non-culturable Vibrio cholerae. Front Microbiol. 2019;10:793.

    PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Defraine V, Fauvart M, Michiels J. Fighting bacterial persistence: current and emerging anti-persister strategies and therapeutics. Drug Resist Updates. 2018;38:12–26.

    Article  Google Scholar 

  54. 54.

    Wu B, Liang W, Kan B. Growth phase, oxygen, temperature, and starvation affect the development of viable but non-culturable state of Vibrio cholerae. Front Microbiol. 2016;7:404.

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Ackermann M. A functional perspective on phenotypic heterogeneity in microorganisms. Nat Rev Microbiol. 2015;13:497–508.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Epstein SS. Microbial awakenings. Nature 2009;457:1083.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  57. 57.

    Sturm A, Dworkin J. Phenotypic diversity as a mechanism to exit cellular dormancy. Curr Biol. 2015;25:2272–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. 58.

    Carlson HK, Vance RE, Marletta MA. H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila. Mol Microbiol. 2010;77:930–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Loh E, Righetti F, Eichner H, Twittenhoff C, Narberhaus F. RNA thermometers in bacterial pathogens. Microbiol Spectr. 2018;6.

  60. 60.

    Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, et al. “Fluorescent timer”: protein that changes color with time. Science. 2000;290:1585–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Selina Niggli for initial cloning and Ramon Hochstrasser for comments and discussions. We would also like to thank Sébastien Faucher (McGill University, Canada) for providing the Δ6S RNA mutant and parental strains. Research of NP in the laboratory of HH was supported by the Swiss National Science Foundation (SNF) Ambizione program (PZ00P3_161492 & PZ00P3_185529) awarded to NP. Research in the laboratory of HH was supported by the SNF (31003A_153200, 31003A_175557), the Novartis Foundation for Medical-Biological Research, the OPO Foundation, and the German Research Foundation (DFG; SPP 1617). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Affiliations

Authors

Contributions

NP conceived the study with input from HH. NP designed the experiments. NP and BS performed the experiments. NP and HH wrote the paper with input from BS.

Corresponding authors

Correspondence to Nicolas Personnic or Hubert Hilbi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Personnic, N., Striednig, B. & Hilbi, H. Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms. ISME J (2020). https://doi.org/10.1038/s41396-020-00774-0

Download citation

Search