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Legionella pp 79-89 | Cite as

Migration of Acanthamoeba castellanii Through Legionella Biofilms

  • Ramon Hochstrasser
  • Hubert HilbiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

Abstract

The amoeba-resistant bacterium Legionella pneumophila infects humans through aerosols and thereby can cause a life-threatening pneumonia termed Legionnaires’ disease. In the environment L. pneumophila forms and colonizes biofilms, which usually comprise complex multispecies communities. In these biofilms L. pneumophila persists and replicates intracellularly in protozoa, such as the amoeba Acanthamoeba castellanii. The interactions between sessile L. pneumophila in biofilms and their natural protozoan hosts are not understood on a molecular level. Here, we describe a method to visualize by confocal microscopy the formation and architecture of mono-species L. pneumophila biofilms. Furthermore, we describe and quantify the migration or “grazing” of A. castellanii in the biofilm. This allows investigating on a molecular and cellular level L. pneumophila biofilm formation and Legionella-amoeba interactions within biofilms.

Key words

Acanthamoeba castellanii Biofilm formation Cell motility Confocal microscopy Green fluorescent protein Host-pathogen interactions Legionella pneumophila Single-cell tracking Type IV secretion 

Abbreviations

ACES

N-(2-acetamido)-2-aminoethanesulfonic acid

AYE

ACES yeast extract

Cam

Chloramphenicol

CLSM

Confocal laser scanning microscope

CYE

Charcoal yeast extract

GFP

Green fluorescent protein

OD600

Optical density at 600 nm

PYG

Peptone yeast extract glucose

Notes

Acknowledgments

This work was supported by the University of Zürich (“Forschungskredit Candoc”; K-42226-01-01) and the Swiss National Science Foundation (SNF; 31003A_153200).

References

  1. 1.
    Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23:274–298CrossRefGoogle Scholar
  2. 2.
    Hilbi H, Hoffmann C, Harrison CF (2011) Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep 3:286–296CrossRefGoogle Scholar
  3. 3.
    Murga R, Forster TS, Brown E, Pruckler JM et al (2001) Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147:3121–3126CrossRefGoogle Scholar
  4. 4.
    Taylor M, Ross K, Bentham R (2009) Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb Ecol 58:538–547CrossRefGoogle Scholar
  5. 5.
    Kwon S, Moon E, Kim TS, Hong S et al (2011) Pyrosequencing demonstrated complex microbial communities in a membrane filtration system for a drinking water treatment plant. Microbes Environ 26:149–155CrossRefGoogle Scholar
  6. 6.
    Stewart CR, Muthye V, Cianciotto NP (2012) Legionella pneumophila persists within biofilms formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under dynamic flow conditions. PLoS One 7:e50560CrossRefGoogle Scholar
  7. 7.
    van der Kooij D, Bakker GL, Italiaander R, Veenendaal HR et al (2017) Biofilm composition and threshold concentration for growth of Legionella pneumophila on surfaces exposed to flowing warm tap water without disinfectant. Appl Environ Microbiol 83:e02737–e02716PubMedPubMedCentralGoogle Scholar
  8. 8.
    Rowbotham TJ (1981) Pontiac fever, amoebae, and legionellae. Lancet 1:40–41CrossRefGoogle Scholar
  9. 9.
    Hilbi H, Weber SS, Ragaz C, Nyfeler Y et al (2007) Environmental predators as models for bacterial pathogenesis. Environ Microbiol 9:563–575CrossRefGoogle Scholar
  10. 10.
    Kuiper MW, Wullings BA, Akkermans AD, Beumer RR et al (2004) Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl Environ Microbiol 70:6826–6833CrossRefGoogle Scholar
  11. 11.
    Valster RM, Wullings BA, van der Kooij D (2010) Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test. Appl Environ Microbiol 76:7144–7153CrossRefGoogle Scholar
  12. 12.
    Thomas JM, Thomas T, Stuetz RM, Ashbolt NJ (2014) Your garden hose: a potential health risk due to Legionella spp. growth facilitated by free-living amoebae. Environ Sci Technol 48:10456–10464CrossRefGoogle Scholar
  13. 13.
    Bigot R, Bertaux J, Frere J, Berjeaud JM (2013) Intra-amoeba multiplication induces chemotaxis and biofilm colonization and formation for Legionella. PLoS One 8:e77875CrossRefGoogle Scholar
  14. 14.
    Piao Z, Sze CC, Barysheva O, Iida K et al (2006) Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila. Appl Environ Microbiol 72:1613–1622CrossRefGoogle Scholar
  15. 15.
    Koubar M, Rodier MH, Frere J (2013) Involvement of minerals in adherence of Legionella pneumophila to surfaces. Curr Microbiol 66:437–442CrossRefGoogle Scholar
  16. 16.
    Portier E, Bertaux J, Labanowski J, Héchard Y (2016) Iron availability modulates the persistence of Legionella pneumophila in complex biofilms. Microbes Environ 31:387–394CrossRefGoogle Scholar
  17. 17.
    Liu Z, Lin YE, Stout JE, Hwang CC et al (2006) Effect of flow regimes on the presence of Legionella within the biofilm of a model plumbing system. J Appl Microbiol 101:437–442CrossRefGoogle Scholar
  18. 18.
    Oder M, Fink R, Bohinc K, Torkar KG (2017) The influence of shear stress on the adhesion capacity of Legionella pneumophila. Arh Hig Rada Toksikol 68:109–115CrossRefGoogle Scholar
  19. 19.
    Rogers J, Dowsett AB, Dennis PJ, Lee JV et al (1994) Influence of plumbing materials on biofilm formation and growth of Legionella pneumophila in potable water systems. Appl Environ Microbiol 60:1842–1851PubMedPubMedCentralGoogle Scholar
  20. 20.
    Moritz MM, Flemming HC, Wingender J (2010) Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials. Int J Hyg Environ Health 213:190–197CrossRefGoogle Scholar
  21. 21.
    Mallegol J, Duncan C, Prashar A, So J et al (2012) Essential roles and regulation of the Legionella pneumophila collagen-like adhesin during biofilm formation. PLoS One 7:e46462CrossRefGoogle Scholar
  22. 22.
    Lucas CE, Brown E, Fields BS (2006) Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention. Microbiology 152:3569–3573CrossRefGoogle Scholar
  23. 23.
    De Buck E, Maes L, Meyen E, Van Mellaert L et al (2005) Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation. Biochem Biophys Res Commun 331:1413–1420CrossRefGoogle Scholar
  24. 24.
    Mampel J, Spirig T, Weber SS, Haagensen JAJ et al (2006) Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl Environ Microbiol 72:2885–2895CrossRefGoogle Scholar
  25. 25.
    Carlson HK, Vance RE, Marletta MA (2010) H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila. Mol Microbiol 77:930–942PubMedPubMedCentralGoogle Scholar
  26. 26.
    Pécastaings S, Allombert J, Lajoie B, Doublet P et al (2016) New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling. Biofouling 32:935–948CrossRefGoogle Scholar
  27. 27.
    Segal G (2013) The Legionella pneumophila two-component regulatory systems that participate in the regulation of Icm/Dot effectors. Curr Top Microbiol Immunol 376:35–52PubMedGoogle Scholar
  28. 28.
    Hochstrasser R, Hilbi H (2017) Intra-species and inter-kingdom signaling of Legionella pneumophila. Front Microbiol 8:79CrossRefGoogle Scholar
  29. 29.
    Personnic N, Striednig B, Hilbi H (2017) Legionella quorum sensing and its role in pathogen-host interactions. Curr Opin Microbiol 41:29–35CrossRefGoogle Scholar
  30. 30.
    Hindré T, Brüggemann H, Buchrieser C, Héchard Y (2008) Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation. Microbiology 154:30–41CrossRefGoogle Scholar
  31. 31.
    Pécastaings S, Berge M, Dubourg KM, Roques C (2010) Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms. Biofouling 26:809–819CrossRefGoogle Scholar
  32. 32.
    Sadosky AB, Wiater LA, Shuman HA (1993) Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun 61:5361–5373Google Scholar
  33. 33.
    Tiaden A, Spirig T, Weber SS, Brüggemann H et al (2007) 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 9:2903–2920CrossRefGoogle Scholar
  34. 34.
    Horwitz MA (1983) Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 158:1319–1331CrossRefGoogle Scholar
  35. 35.
    Feeley JC, Gibson RJ, Gorman GW, Langford NC et al (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10:437–441PubMedPubMedCentralGoogle Scholar
  36. 36.
    Moffat JF, Tompkins LS (1992) A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect Immun 60:296–301PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland

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