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Effector Translocation Assay: Differential Solubilization

  • Irina S. Franco
  • Sara V. Pais
  • Nuno Charro
  • Luís Jaime Mota
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringelike nanomachines is an important step toward understanding the mechanisms underlying the virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a nonionic detergent (Triton X-100) enables solubilization of host cell membranes but not of bacterial membranes. This allows the isolation of a Triton-soluble fraction lacking bacteria but enriched in proteins present in the host cell cytoplasm and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells.

Key words

Bacterial protein secretion system Type III secretion Effector Translocation Detergent solubilization SDS-PAGE Immunoblotting 

Notes

Acknowledgments

This work was supported by the Unidade de Ciências Biomoleculares Aplicadas—UCIBIO, which is financed by national funds from Fundação para a Ciência e a Tecnologia (FCT) (UID/Multi/04378/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728) and by FCT research grants PTDC/BIA-MIC/2821/2012 and PTDC/BIA-MIC/116780/2010. Irina Franco is recipient of apostdoctoral fellowship (SFRH/BPD/102378/2014) from FCT and Sara V. Pais holds a fellowship (PD/BD/52210/2013) within the scope of the PhD program Molecular Biosciences (PD/00133/2012) funded by FCT.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Irina S. Franco
    • 1
  • Sara V. Pais
    • 1
  • Nuno Charro
    • 1
  • Luís Jaime Mota
    • 1
  1. 1.UCIBIO-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT NOVA)CaparicaPortugal

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