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Legionella pp 221-238 | Cite as

Perturbation of Legionella Cell Infection by RNA Interference

  • Bernhard Steiner
  • A. Leoni Swart
  • Hubert HilbiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

Abstract

Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.

Key words

Atlastin Host-pathogen interactions Intracellular bacteria Large GTPase Legionella pneumophila Macrophage Epithelial cells Pathogen vacuole Type IV secretion RNA interference 

Abbreviations

ACES

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

Ago

Argonaute protein

Arf1

ADP-ribosylation factor 1

Atl3

Atlastin-3

GFP

Green fluorescent protein

Hsp90

Heat shock protein 90

Icm/Dot

Intracellular multiplication/defective organelle trafficking

LCV

Legionella-containing vacuole

MOI

Multiplicity of infection

PI

Propidium iodide

RISC

RNA-induced silencing complex

RNAi

RNA interference

siRNA

Small interfering RNA

ss/dsRNA

Single-stranded/double-stranded RNA

T4SS

Type IV secretion system

Notes

Acknowledgments

We would like to thank Stephen Weber and Daniel Strebinger for providing critical input on the manuscript. Work in the group of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_153200), the Novartis Foundation for Medical-Biological Research, and the OPO Foundation.

References

  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefGoogle Scholar
  2. 2.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498CrossRefGoogle Scholar
  3. 3.
    Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108CrossRefGoogle Scholar
  4. 4.
    Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296CrossRefGoogle Scholar
  5. 5.
    Zamore PD (2001) RNA interference: listening to the sound of silence. Nat Struct Biol 8:746–750CrossRefGoogle Scholar
  6. 6.
    Alagia A, Eritja R (2016) siRNA and RNAi optimization. Wiley Interdiscip Rev RNA 7:316–329CrossRefGoogle Scholar
  7. 7.
    Hutvagner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12:225–232CrossRefGoogle Scholar
  8. 8.
    Fischer SE (2015) RNA interference and microRNA-mediated silencing. Curr Protoc Mol Biol 112:26.1.1–26.1.5CrossRefGoogle Scholar
  9. 9.
    Sarisozen C, Salzano G, Torchilin VP (2016) Lipid-based siRNA delivery systems: challenges, promises and solutions along the long journey. Curr Pharm Biotechnol 17:728–740CrossRefGoogle Scholar
  10. 10.
    Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33CrossRefGoogle Scholar
  11. 11.
    Song MS, Rossi JJ (2017) Molecular mechanisms of Dicer: endonuclease and enzymatic activity. Biochem J 474:1603–1618CrossRefGoogle Scholar
  12. 12.
    Azlan A, Dzaki N, Azzam G (2016) Argonaute: the executor of small RNA function. J Genet Genomics 43:481–494CrossRefGoogle Scholar
  13. 13.
    Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200CrossRefGoogle Scholar
  14. 14.
    Hirsch AJ (2010) The use of RNAi-based screens to identify host proteins involved in viral replication. Future Microbiol 5:303–311CrossRefGoogle Scholar
  15. 15.
    Prudencio M, Lehmann MJ (2009) Illuminating the host – how RNAi screens shed light on host-pathogen interactions. Biotechnol J 4:826–837CrossRefGoogle Scholar
  16. 16.
    Finsel I, Hilbi H (2015) Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell Microbiol 17:935–950CrossRefGoogle Scholar
  17. 17.
    Dorer MS, Kirton D, Bader JS, Isberg RR (2006) RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics. PLoS Pathog 2:e34CrossRefGoogle Scholar
  18. 18.
    Urwyler S, Nyfeler Y, Ragaz C, Lee H et al (2009) Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases. Traffic 10:76–87CrossRefGoogle Scholar
  19. 19.
    De Jesus DA, O'Connor TJ, Isberg RR (2013) Analysis of Legionella infection using RNAi in Drosophila cells. Methods Mol Biol 954:251–264CrossRefGoogle Scholar
  20. 20.
    Vinzing M, Eitel J, Lippmann J, Hocke AC et al (2008) NAIP and Ipaf control Legionella pneumophila replication in human cells. J Immunol 180:6808–6815CrossRefGoogle Scholar
  21. 21.
    Ivanov SS, Roy CR (2013) Pathogen signatures activate a ubiquitination pathway that modulates the function of the metabolic checkpoint kinase mTOR. Nat Immunol 14:1219–1228CrossRefGoogle Scholar
  22. 22.
    Opitz B, Vinzing M, van Laak V, Schmeck B et al (2006) Legionella pneumophila induces IFNbeta in lung epithelial cells via IPS-1 and IRF3, which also control bacterial replication. J Biol Chem 281:36173–36179CrossRefGoogle Scholar
  23. 23.
    Finsel I, Ragaz C, Hoffmann C, Harrison CF et al (2013) The Legionella effector RidL inhibits retrograde trafficking to promote intracellular replication. Cell Host Microbe 14:38–50CrossRefGoogle Scholar
  24. 24.
    Rothmeier E, Pfaffinger G, Hoffmann C, Harrison CF et al (2013) Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection. PLoS Pathog 9:e1003598CrossRefGoogle Scholar
  25. 25.
    Simon S, Wagner MA, Rothmeier E, Müller-Taubenberger A et al (2014) Icm/Dot-dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator. Cell Microbiol 16:977–992PubMedGoogle Scholar
  26. 26.
    Hoffmann C, Finsel I, Otto A, Pfaffinger G et al (2014) Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages. Cell Microbiol 16:1034–1052CrossRefGoogle Scholar
  27. 27.
    Schmölders J, Manske C, Otto A, Hoffmann C et al (2017) Comparative proteomics of purified pathogen vacuoles correlates intracellular replication of Legionella pneumophila with the small GTPase Ras-related protein 1 (Rap1). Mol Cell Proteomics 16:622–641CrossRefGoogle Scholar
  28. 28.
    Steiner B, Swart AL, Welin A, Weber S et al (2017) ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila. EMBO Rep 18:1817–1836CrossRefGoogle Scholar
  29. 29.
    Segal G, Shuman HA (1998) Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010. Mol Microbiol 30:197–208CrossRefGoogle Scholar
  30. 30.
    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
  31. 31.
    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
  32. 32.
    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
  33. 33.
    Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ (2016) Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb Protoc 2016:11.  https://doi.org/10.1101/pdb.prot087288CrossRefGoogle Scholar
  34. 34.
    Tiaden AN, Kessler A, Hilbi H (2013) Analysis of Legionella infection by flow cytometry. Methods Mol Biol 954:233–249CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Bernhard Steiner
    • 1
  • A. Leoni Swart
    • 1
  • Hubert Hilbi
    • 1
    Email author
  1. 1.Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland

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