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Effector Translocation Assay: Differential Solubilization

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Bacterial Protein Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

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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.

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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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Authors and Affiliations

  1. UCIBIO-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT NOVA), Caparica, Portugal

    Irina S. Franco, Sara V. Pais, Nuno Charro & Luís Jaime Mota

Authors
  1. Irina S. Franco
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  2. Sara V. Pais
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  3. Nuno Charro
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  4. Luís Jaime Mota
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Corresponding author

Correspondence to Luís Jaime Mota .

Editor information

Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

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Franco, I.S., Pais, S.V., Charro, N., Mota, L.J. (2017). Effector Translocation Assay: Differential Solubilization. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_35

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  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_35

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

  • eBook Packages: Springer Protocols

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