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)


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 



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.


  1. 1.
    Costa TR, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trokter M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359CrossRefGoogle Scholar
  2. 2.
    Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14:583–594Google Scholar
  3. 3.
    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 186:5486–5495Google Scholar
  4. 4.
    Collazo CM, Galan JE (1997) The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol Microbiol 24:747–756Google Scholar
  5. 5.
    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 28:593–601Google Scholar
  6. 6.
    Schnaitman CA (1971) Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli. J Bacteriol 108:553–563Google Scholar
  7. 7.
    Schnaitman CA (1971) Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol 108:545–552Google Scholar
  8. 8.
    Birdsell DC, Cota-Robles EH (1968) Lysis of spheroplasts of Escherichia coli by a non-ionic detergent. Biochem Biophys Res Commun 31:438–446Google Scholar
  9. 9.
    Esparis-Ogando A, Zurzolo C, Rodriguez-Boulan E (1994) Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency. Am J Phys 267:C166–C176Google Scholar
  10. 10.
    Domingues L, Holden DW, Mota LJ (2014) The Salmonella effector SteA contributes to the control of membrane dynamics of Salmonella-containing vacuoles. Infect Immun 82:2923–2934Google Scholar
  11. 11.
    Sory MP, Boland A, Lambermont I, Cornelis GR (1995) Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci U S A 92:11998–12002Google Scholar
  12. 12.
    Figueira R, Watson KG, Holden DW, Helaine S (2013) Identification of S almonella pathogenicity island-2 type III secretion system effectors involved in intramacrophage replication of S. enterica serovar Typhimurium: implications for rational vaccine design. MBio 4:e00065Google Scholar
  13. 13.
    Wang RF, Kushner SR (1991) Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195–199Google Scholar
  14. 14.
    Domingues L, Ismail A, Charro N, Rodriguez-Escudero I, Holden DW, Molina M, Cid VJ, Mota LJ (2016) The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells. Cell Microbiol 18:949–969Google Scholar
  15. 15.
    Letzelter M, Sorg I, Mota LJ, Meyer S, Stalder J, Feldman M, Kuhn M, Callebaut I, Cornelis GR (2006) The discovery of SycO highlights a new function for type III secretion effector chaperones. EMBO J 25:3223–3233Google Scholar
  16. 16.
    Diepold A, Amstutz M, Abel S, Sorg I, Jenal U, Cornelis GR (2010) Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J 29:1928–1940Google Scholar
  17. 17.
    VanRheenen SM, Luo ZQ, O’Connor T, Isberg RR (2006) Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells. Infect Immun 74:3597–3606Google Scholar
  18. 18.
    Denecker G, Totemeyer S, Mota LJ, Troisfontaines P, Lambermont I, Youta C, Stainier I, Ackermann M, Cornelis GR (2002) Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells. Infect Immun 70:3510–3520Google Scholar
  19. 19.
    Drecktrah D, Knodler LA, Ireland R, Steele-Mortimer O (2006) The mechanism of Salmonella entry determines the vacuolar environment and intracellular gene expression. Traffic 7:39–51Google Scholar

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

Personalised recommendations