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Monitoring Effector Translocation using the TEM-1 Beta-Lactamase Reporter System

  • Julie Allombert
  • Anne Vianney
  • Xavier Charpentier
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

Among the bacterial secretion systems, the Type III, IV, and VI secretion systems enable bacteria to secrete proteins directly into a target cell. This specific form of secretion, referred to as translocation, is essential for a number of pathogens to alter or kill targeted cells. The translocated proteins, called effector proteins, can directly interfere with the normal processes of the targeted cells, preventing elimination of pathogens and promoting their multiplication. The function of effector proteins varies greatly depending on the considered pathogen and the targeted cell. In addition, there is often no magic bullet, and the number of effector proteins can range from a handful to hundreds, with, for instance, a substrate of over 300 effector proteins of the Icm/Dot Type IV secretion system in the human pathogen Legionella pneumophila. Identifying, detecting, and monitoring the translocation of each of the effector proteins represents an active field of research and is key to understanding the bacterial molecular weaponry. Translational fusion of an effector with a reporter protein of known activity remains the best method to monitor effector translocation. The development of a fluorescent substrate for the TEM-1 beta-lactamase has turned this antibiotic-resistant protein into a highly versatile reporter system for investigating protein transfer events associated with microbial infection of host cells. Here we describe a simple protocol to assay the translocation of an effector protein by the Icm/Dot system of the human pathogen Legionella pneumophila.

Key words

Effector protein Type IV secretion system β-lactamase fusion CCF4 Fluorescence Legionella pneumophila 

References

  1. 1.
    Costa TRD et al (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13(6):343–359CrossRefGoogle Scholar
  2. 2.
    Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14(3):583–594CrossRefGoogle Scholar
  3. 3.
    Day JB, Ferracci F, Plano GV (2003) Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies. Mol Microbiol 47(3):807–823CrossRefGoogle Scholar
  4. 4.
    Lee VT, Anderson DM, Schneewind O (1998) Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone. Mol Microbiol 28(3):593–601CrossRefGoogle Scholar
  5. 5.
    Charpentier X, Oswald E (2004) Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J Bacteriol 186(16):5486–5495CrossRefGoogle Scholar
  6. 6.
    Zlokarnik G et al (1998) Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279(5347):84–88CrossRefGoogle Scholar
  7. 7.
    Pechous RD, Goldman WE (2015) Illuminating targets of bacterial secretion. PLoS Pathog 11(8):e1004981CrossRefGoogle Scholar
  8. 8.
    Lodoen MB, Gerke C, Boothroyd JC (2010) A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell Microbiol 12(1):55–66CrossRefGoogle Scholar
  9. 9.
    Mills E, Baruch K, Charpentier X, Kobi S, Rosenshine I (2008) Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli. Cell Host Microbe 3(2):104–113CrossRefGoogle Scholar
  10. 10.
    Charpentier X et al (2009) Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila. PLoS Pathog 5(7):e1000501CrossRefGoogle Scholar
  11. 11.
    Harmon DE, Davis AJ, Castillo C, Mecsas J (2010) Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis. Antimicrob Agents Chemother 54(8):3241–3254CrossRefGoogle Scholar
  12. 12.
    Marketon MM, DePaolo RW, DeBord KL, Jabri B, Schneewind O (2005) Plague bacteria target immune cells during infection. Science 309(5741):1739–1741CrossRefGoogle Scholar
  13. 13.
    Geddes K, Cruz F, Heffron F (2007) Analysis of cells targeted by Salmonella Type III secretion in vivo. PLoS Pathog 3(12):e196CrossRefGoogle Scholar
  14. 14.
    de Felipe KS et al (2008) Legionella eukaryotic-like type IV substrates interfere with organelle trafficking. PLoS Pathog 4(8):e1000117CrossRefGoogle Scholar
  15. 15.
    Steinberg TH, Newman AS, Swanson JA, Silverstein SC (1987) Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix. J Cell Biol 105(6 Pt 1):2695–2702CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Julie Allombert
    • 1
  • Anne Vianney
    • 1
  • Xavier Charpentier
    • 1
  1. 1.CIRI, Centre International de Recherche en InfectiologieInserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ LyonVilleurbanneFrance

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