Effector Translocation Assay: Differential Solubilization

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

doi:10.1007/978-1-0716-3445-5_34

Effector Translocation Assay: Differential Solubilization

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

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

Abstract

The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringe-like nanomachines is an important step towards an understanding of the mechanisms underlying virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a non-ionic 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, nucleus, and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells.

Original language	English
Title of host publication	Bacterial Secretion Systems
Subtitle of host publication	Methods and Protocols
Editors	Laure Journet, Eric Cascales
Place of Publication	New York
Publisher	Springer
Pages	547-561
Number of pages	15
ISBN (Electronic)	978-1-0716-3445-5
ISBN (Print)	978-1-0716-3444-8, 978-1-0716-3447-9
DOIs	https://doi.org/10.1007/978-1-0716-3445-5_34
Publication status	Published - 2024

Publication series

Name	Methods in Molecular Biology
Publisher	Springer
Volume	2715
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

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

Access to Document

10.1007/978-1-0716-3445-5_34

Cite this

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abstract = "The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringe-like nanomachines is an important step towards an understanding of the mechanisms underlying virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a non-ionic 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, nucleus, and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells.",

keywords = "Bacterial protein secretion system, Type III secretion, Effector; translocation, Detergent solubilization, SDS-PAGE, Immunoblotting",

author = "Franco, {Irina S.} and Pais, {Sara V.} and Nuno Charro and Mota, {Lu{\'i}s Jaime}",

note = "Publisher Copyright: {\textcopyright} 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. This work was supported by Funda{\c c}{\~a}o para a Ci{\^e}ncia e Tecnologia (FCT) in the scope of the projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO, and LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB., and by FCT research grants PTDC/BIA-MIC/2821/2012 and PTDC/ BIA-MIC/116780/2010. Irina Franco and Sara V. Pais were recipients of post-doctoral (SFRH/BPD/102378/2014) and PhD fellowships (SFRH/BD/52210/2013) from FCT.",

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Effector Translocation Assay: Differential Solubilization. / Franco, Irina S.; Pais, Sara V.; Charro, Nuno et al.
Bacterial Secretion Systems: Methods and Protocols. ed. / Laure Journet; Eric Cascales. New York: Springer, 2024. p. 547-561 (Methods in Molecular Biology; Vol. 2715).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

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AU - Pais, Sara V.

AU - Charro, Nuno

AU - Mota, Luís Jaime

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AB - The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringe-like nanomachines is an important step towards an understanding of the mechanisms underlying virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a non-ionic 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, nucleus, 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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Effector Translocation Assay: Differential Solubilization

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