In-vivo microscopy reveals the impact of Pseudomonas aeruginosa social interactions on host colonization


Pathogenic bacteria engage in social interactions to colonize hosts, which include quorum-sensing-mediated communication and the secretion of virulence factors that can be shared as “public goods” between individuals. While in-vitro studies demonstrated that cooperative individuals can be displaced by “cheating” mutants freeriding on social acts, we know less about social interactions in infections. Here, we developed a live imaging system to track virulence factor expression and social strain interactions in the human pathogen Pseudomonas aeruginosa colonizing the gut of Caenorhabditis elegans. We found that shareable siderophores and quorum-sensing systems are expressed during infections, affect host gut colonization, and benefit non-producers. However, non-producers were unable to successfully cheat and outcompete producers. Our results indicate that the limited success of cheats is due to a combination of the down-regulation of virulence factors over the course of the infection, the fact that each virulence factor examined contributed to but was not essential for host colonization, and the potential for negative frequency-dependent selection. Our findings shed new light on bacterial social interactions in infections and reveal potential limits of therapeutic approaches that aim to capitalize on social dynamics between strains for infection control.

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  1. 1.

    Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM. Common virulence factors for bacterial pathogenicity in plants and animals. Science. 1995;268:1899–902.

  2. 2.

    Wu HJ, Wang AHJ, Jennings MP. Discovery of virulence factors of pathogenic bacteria. Curr Opin Chem Biol. 2008;12:93–101.

  3. 3.

    Diggle SP, Griffin AS, Campbell GS, West SA. Cooperation and conflict in quorum-sensing bacterial populations. Nature. 2007;450:411–4.

  4. 4.

    Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: An emergent form of bacterial life. Nat Rev Microbiol. 2016;14:563–75.

  5. 5.

    Henkel JS, Baldwin MR, Barbieri JT. Toxins from bacteria. EXS. 2010;100:1–29.

  6. 6.

    Granato ET, Harrison F, Kümmerli R, Ross-Gillespie A. Do bacterial “Virulence Factors” always increase virulence? A meta-analysis of pyoverdine production in Pseudomonas aeruginosa as a test case. Front Microbiol. 2016;7:1952.

  7. 7.

    Köhler T, Buckling A, van Delden C. Cooperation and virulence of clinical Pseudomonas aeruginosa populations. Proc Natl Acad Sci USA. 2009;106:6339–44.

  8. 8.

    Raymond B, West SA, Griffin AS, Bonsall MB. The dynamics of cooperative bacterial virulence in the field. Science. 2012;337:85–88.

  9. 9.

    Harrison F. Bacterial cooperation in the wild and in the clinic: Are pathogen social behaviours relevant outside the laboratory? BioEssays. 2013;35:108–12.

  10. 10.

    West SA, Diggle SP, Buckling A, Gardner A, Griffin AS. The social lives of microbes. Annu Rev Ecol Evol Syst. 2007;38:53–77.

  11. 11.

    Ghoul M, Griffin AS, West SA. Toward an evolutionary definition of cheating. Evolution. 2014;68:318–31.

  12. 12.

    Özkaya Ö, Balbontín R, Gordo I, Xavier KB. Cheating on cheaters stabilizes cooperation in Pseudomonas aeruginosa. Curr Biol. 2018;26:2070–80.

  13. 13.

    Buckling A, Brockhurst MA. Kin selection and the evolution of virulence. Heredity. 2008;100:484–8.

  14. 14.

    Leggett HC, Brown SP, Reece SE. War and peace: social interactions in infections. Philos Trans R Soc Lond B. 2014;369:20130365.

  15. 15.

    Harrison F, Browning LE, Vos M, Buckling A. Cooperation and virulence in acute Pseudomonas aeruginosa infections. BMC Biol. 2006;4:21.

  16. 16.

    Rumbaugh KP, Diggle SP, Watters CM, Ross-Gillespie A, Griffin AS, West SA. Quorum sensing and the social evolution of bacterial virulence. Curr Biol. 2009;19:341–5.

  17. 17.

    Rumbaugh KP, Trivedi U, Watters C, Burton-Chellew MN, Diggle SP, West SA. Kin selection, quorum sensing and virulence in pathogenic bacteria. Proc R Soc B. 2012;279:3584–8.

  18. 18.

    Diard M, Garcia V, Maier L, Remus-Emsermann MNP, Regoes RR, Ackermann M, et al. Stabilization of cooperative virulence by the expression of an avirulent phenotype. Nature. 2013;494:353–6.

  19. 19.

    Pollitt EJG, West SA, Crusz SA, Burton-Chellew MN, Diggle SP. Cooperation, quorum sensing, and evolution of virulence in Staphylococcus aureus. Infect Immun. 2014;82:1045–51.

  20. 20.

    Zhou L, Slamti L, Nielsen-LeRoux C, Lereclus D, Raymond B. The social biology of quorum sensing in a naturalistic host pathogen system. Curr Biol. 2014;24:2417–22.

  21. 21.

    Harrison F, McNally A, Da Silva AC, Heeb S, Diggle SP. Optimised chronic infection models demonstrate that siderophore ‘cheating’ in Pseudomonas aeruginosa is context specific. ISME J. 2017;11:2492–509.

  22. 22.

    Andersen SB, Marvig RL, Molin S, Krogh Johansen H, Griffin AS. Long-term social dynamics drive loss of function in pathogenic bacteria. Proc Natl Acad Sci USA. 2015;112:10756–61.

  23. 23.

    Andersen SB, Ghoul M, Marvig RL, Bin LeeZ, Molin S, Johansen HK, et al. Privatisation rescues function following loss of cooperation. Elife. 2018;7:e38594.

  24. 24.

    Brown SP, West Sa, Diggle SP, Griffin AS. Social evolution in micro-organisms and a Trojan horse approach to medical intervention strategies. Philos Trans R Soc Lond B. 2009;364:3157–68.

  25. 25.

    Allen RC, Popat R, Diggle SP, Brown SP. Targeting virulence: can we make evolution-proof drugs? Nat Rev Microbiol. 2014;12:300–8.

  26. 26.

    Granato ET, Ziegenhain C, Marvig RL, Kümmerli R. Low spatial structure and selection against secreted virulence factors attenuates pathogenicity in Pseudomonas aeruginosa. ISME J. 2018;12:2907–18.

  27. 27.

    André JB, Godelle B. Multicellular organization in bacteria as a target for drug therapy. Ecol Lett. 2005;8:800–10.

  28. 28.

    Clatworthy AE, Pierson E, Hung DT. Targeting virulence: a new paradigm for antimicrobial therapy. Nat Chem Biol. 2007;3:541–8.

  29. 29.

    Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug Discov. 2010;9:117–28.

  30. 30.

    Pepper JW. Drugs that target pathogen public goods are robust against evolved drug resistance. Evol Appl. 2012;5:757–61.

  31. 31.

    Rezzoagli C, Wilson D, Weigert M, Wyder S, Kümmerli R. Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa. Evol Med Public Heal. 2018;1:246–59.

  32. 32.

    Tan MW, Ausubel FM. Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 2000;3:29–34.

  33. 33.

    Ewbank JJ. Tackling both sides of the host–pathogen equation with Caenorhabditis elegans. Microbes Infect. 2002;4:247–56.

  34. 34.

    Papaioannou E, Utari P, Quax W. Choosing an appropriate infection model to study quorum sensing inhibition in Pseudomonas infections. Int J Mol Sci. 2013;14:19309–40.

  35. 35.

    Félix M-A, Braendle C. The natural history of Caenorhabditis elegans. Curr Biol. 2010;20:R965–R969.

  36. 36.

    Portal-Celhay C, Bradley ER, Blaser MJ. Control of intestinal bacterial proliferation in regulation of lifespan in Caenorhabditis elegans. BMC Microbiol. 2012;12:49.

  37. 37.

    Tan MW, Mahajan-Miklos S, Ausubel FM. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl Acad Sci USA. 1999;96:715–20.

  38. 38.

    Jimenez PN, Koch G, Thompson JA, Xavier KB, Cool RH, Quax WJ. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol Mol Biol Rev. 2012;76:46–65.

  39. 39.

    Meyer JM, Neely A, Stintzi A, Georges C, Holder IA. Pyoverdin is essential for virulence of Pseudomonas aeruginosa. Infect Immun. 1996;64:518–23.

  40. 40.

    Takase H, Nitanai H, Hoshino K, Otani T. Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice. Infect Immun. 2000;68:1834–9.

  41. 41.

    Cornelis P, Dingemans J. Pseudomonas aeruginosa adapts its iron uptake strategies in function of the type of infections. Front Cell Infect Microbiol. 2013;3:1–7.

  42. 42.

    Smith RS, Iglewski BH. P. aeruginosa quorum-sensing systems and virulence. Curr Opin Microbiol. 2003;6:56–60.

  43. 43.

    Alibaud L, Köhler T, Coudray A, Prigent-Combaret C, Bergeret E, Perrin J, et al. Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model. Cell Microbiol. 2008;10:729–40.

  44. 44.

    Lee J, Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell. 2015;6:26–41.

  45. 45.

    Zaborin A, Romanowski K, Gerdes S, Holbrook C, Lepine F, Long J, et al. Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1. Proc Natl Acad Sci USA. 2009;106:6327–32.

  46. 46.

    Kirienko NV, Kirienko DR, Larkins-Ford J, Wahlby C, Ruvkun G, Ausubel FM. Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death. Cell Host Microbe. 2013;13:406–16.

  47. 47.

    Cezairliyan B, Vinayavekhin N, Grenfell-Lee D, Yuen GJ, Saghatelian A, Ausubel FM. Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans. PLOS Pathog. 2013;9:e1003101.

  48. 48.

    Zhu J, Cai X, Harris TL, Gooyit M, Wood M, Lardy M, et al. Disarming Pseudomonas aeruginosa virulence factor LasB by leveraging a Caenorhabditis elegans infection model. Chem Biol. 2015;22:483–91.

  49. 49.

    Stiernagle T. Maintenance of C. elegans. WormBook: the online review of C. elegans biology. WormBook. 2006. p 1–11.

  50. 50.

    Portman DS. Profiling C. elegans gene expression with DNA microarrays. WormBook: the online review of C. elegans biology. WormBook. 2006. p 1–11.

  51. 51.

    Sommer C, Strähle C, Köthe U, Hamprecht FA. ilastik: interactive learning and segmentation toolkit. Eighth IEEE International Symposium on Biomedical Imaging (ISBI). Proceedings. 2011. p 230–3.

  52. 52.

    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

  53. 53.

    Preibisch S, Saalfeld S, Tomancak P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics. 2009;25:1463–5.

  54. 54.

    Vega NM, Gore J. Stochastic assembly produces heterogeneous communities in the Caenorhabditis elegans intestine. PLOS Biol. 2017;15:e2000633.

  55. 55.

    Ross-Gillespie A, Gardner A, West SA, Griffin AS. Frequency dependence and cooperation: theory and a test with bacteria. Am Nat. 2007;170:331–42.

  56. 56.

    Kocsis E, Trus BL, Steer CJ, Bisher ME, Steven AC. Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment. J Struct Biol. 1991;107:6–14.

  57. 57.

    R Development Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2013.

  58. 58.

    Dumas Z, Ross-Gillespie A, Kümmerli R. Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments. Proc Biol Sci. 2013;280:20131055.

  59. 59.

    Van Gestel J, Weissing FJ, Kuipers OP, Kovács ÁT. Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms. ISME J. 2014;8:2069–79.

  60. 60.

    Weigert M, Kümmerli R. The physical boundaries of public goods cooperation between surface-attached bacterial cells. Proc R Soc B. 2017;284:20170631.

  61. 61.

    Griffin AS, West SA, Buckling A. Cooperation and competition in pathogenic bacteria. Nature. 2004;430:1024–7.

  62. 62.

    Sandoz KM, Mitzimberg SM, Schuster M. Social cheating in Pseudomonas aeruginosa quorum sensing. Proc Natl Acad Sci USA. 2007;104:15876–81.

  63. 63.

    Kümmerli R, Griffin AS, West Sa, Buckling A, Harrison F. Viscous medium promotes cooperation in the pathogenic bacterium Pseudomonas aeruginosa. Proc Biol Sci. 2009;276:3531–8.

  64. 64.

    Popat R, Crusz SA, Messina M, Williams P, West SA, Diggle SP. Quorum-sensing and cheating in bacterial biofilms. Proc R Soc B. 2012;279:4765–71.

  65. 65.

    O’Brien S, Luján AM, Paterson S, Cant MA, Buckling A. Adaptation to public goods cheats in Pseudomonas aeruginosa. Proc R Soc B. 2017;284:20171089.

  66. 66.

    van Leeuwen E, O’Neill S, Matthews A, Raymond B. Making pathogens sociable: The emergence of high relatedness through limited host invasibility. ISME J. 2015;9:2315–23.

  67. 67.

    Ross-Gillespie A, Gardner A, Buckling A, West SA, Griffin AS. Density dependence and cooperation: theory and a test with bacteria. Evolution. 2009;63:2315–25.

  68. 68.

    Scholz RL, Greenberg EP. Sociality in Escherichia coli: enterochelin is a private good at low cell density and can be shared at high cell density. J Bacteriol. 2015;197:2122–8.

  69. 69.

    Pukkila-Worley R, Ausubel FM. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Curr Opin Immunol. 2012;24:3–9.

  70. 70.

    Imperi F, Tiburzi F, Visca P. Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 2009;106:20440–5.

  71. 71.

    Kümmerli R, Brown SP. Molecular and regulatory properties of a public good shape the evolution of cooperation. Proc Natl Acad Sci USA. 2010;107:18921–6.

  72. 72.

    Lindsay RJ, Kershaw MJ, Pawlowska BJ, Talbot NJ, Gudelj I. Harbouring public good mutants within a pathogen population can increase both fitness and virulence. Elife. 2016;5:1–25.

  73. 73.

    dos Santos M, Ghoul M, West SA. Pleiotropy, cooperation and the social evolution of genetic architecture. PLOS Biol. 2018;16:e2006671.

  74. 74.

    Ross-Gillespie A, Dumas Z, Kümmerli R. Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa. J Evol Biol. 2015;28:29–39.

  75. 75.

    Dandekar AA, Chugani S, Greenberg PE. Bacterial quorum sensing and metabolic incentives to cooperate. Science. 2012;338:264–6.

  76. 76.

    Mellbye B, Schuster M. The sociomicrobiology of antivirulence drug resistance: a proof of concept. MBio. 2011;2:e00131–11.

  77. 77.

    Gerdt JP, Blackwell HE. Competition studies confirm two major barriers that can preclude the spread of resistance to quorum-sensing inhibitors in bacteria. ACS Chem Biol. 2014;9:2291–9.

  78. 78.

    Ross-Gillespie A, Weigert M, Brown SP, Kümmerli R. Gallium-mediated siderophore quenching as an evolutionarily robust antibacterial treatment. Evol Med Public Heal. 2014;2014:18–29.

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We thank two anonymous reviewers for constructive comments and the Center of Microscopy and Image Analysis (University of Zürich) for support with image acquisition and advice on image analysis.


This project has received funding from the Swiss National Science Foundation (grant no. PP00P3_165835 and 31003A_182499 to RK and no. P2ZHP3_174751 to ETG), and the European Research Council under the grant agreement no. 681295 (to RK).

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Correspondence to Chiara Rezzoagli or Rolf Kümmerli.

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