Fractionation Techniques to Examine Effector Translocation

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1531)

Abstract

Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular trafficking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein–protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.

Key words

Fractionation Digitonin Subcellular fractionation Translocation 

References

  1. 1.
    Buttner D (2012) Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev 76(2):262–310CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lee V, Anderson D, 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):593–601CrossRefPubMedGoogle Scholar
  3. 3.
    Palmer LE, Hobbie S, Galan JE, Bliska JB (1998) YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF-alpha production and downregulation of the MAP kinases p38 and JNK. Mol Microbiol 27(5):953–965CrossRefPubMedGoogle Scholar
  4. 4.
    Dewoody R, Merritt P, Houppert A, Marketon M (2011) YopK regulates the Yersinia pestis type III secretion system from within host cells. Mol Microbiol 79(6):1445–1461CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gauthier A, De Grado M, Finlay B (2000) Mechanical fractionation reveals structural requirements for Enteropathogenic Escherichia coli Tir insertion into host membranes. Infect Immun 68(7):4344–4348CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Linke D (2009) Detergents. Methods Enzymol 463:603–617CrossRefPubMedGoogle Scholar
  7. 7.
    Howe D, Heinzen R (2008) Fractionation of the Coxiella burnetii parasitophorous vacuole. Methods Mol Biol 445:389–406CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ramsby M, Makowski G (eds) (2005) Differential detergent fractionation of eukaryotic cells, The proteomics protocols. Humana Press, Inc, Totowa, NJGoogle Scholar
  9. 9.
    Dewoody R, Merritt P, Marketon M (2013) YopK controls both rate and fidelity of Yop translocation. Mol Microbiol 87(2):301–317CrossRefPubMedGoogle Scholar
  10. 10.
    Jeong K, Sexton J, Vogel J (2015) Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ. PLoS Pathog 11(3):e1004695CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sisko J, Spaeth K, Kumar Y, Valdivia R (2006) Multifunctional analysis of Chlamydia-specific genes in a yeast expression system. Mol Microbiol 60(1):51–66CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Veterinary PathobiologyUniversity of Missouri-ColumbiaColumbiaUSA

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