Advertisement

Evaluation of Protein Translocation into Eukaryotic Cells by Yersinia pestis

  • Yafang Tan
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Protein–protein interactions are important during bacterial pathogenesis. Bacterial proteins, whether surface bound or secreted, are crucial for mediating bacterial virulence. Many Gram-negative bacteria, including pathogenic Yersinia spp., employ type III secretion systems to translocate effector proteins into eukaryotic target cells (Du Z, Tan Y, Yang H, Qiu J, Qin L, Wang T: Int J Med Microbiol 299(5):355–66, 2009; Perry RD, Fetherston JD: Clin Microbiol Rev 10:35–66, 1997). When yersiniae are attached to host cells, virulent effectors termed Yersinia outer-membrane proteins (Yops) are delivered into the cytosol of eukaryotic cells, where they modulate the host cellular immune response (Bleves S, Marenne MN, Detry G, Cornelis GR: J Bacteriol 184(12):3214–23, 2002; Cornelis GR: Philos Trans R Soc Lond B Biol Sci 355(1397):681–93, 2000; Brubaker RR: Infect Immun 73(8):4743–4752, 2005). Thus, to elucidate the molecular mechanisms behind bacterial pathogenesis, it is important to study whether these effector proteins are translocated into eukaryotic target cells. It is also important for researchers to identify and characterize new effector proteins. The classic method for validating the cytosolic localization of secreted effector proteins is based on lysing infected cells and then using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting to identify translocated effectors. More recently, some enzymatic tags have been used to determine the cytosolic localization of secreted effector proteins. The use of β-lactamase translational fusions has proven to be a convenient and sensitive tool for detecting bacterial protein secretion. Here, we present a fluorescence resonance energy transfer-based method that can be used to identify the localization of bacterial proteins in the eukaryotic cell cytosol.

Key words

Fluorescence resonance energy transfer Interaction Protein Yersinia pestis Yops 

References

  1. 1.
    Du Z, Tan Y, Yang H, Qiu J, Qin L, Wang T, Liu H, Bi Y, Song Y, Guo Z et al (2009) Gene expression profiling of Yersinia pestis with deletion of lcrG, a known negative regulator for Yop secretion of type III secretion system. Int J Med Microbiol 299(5):355–366CrossRefPubMedGoogle Scholar
  2. 2.
    Perry RD, Fetherston JD (1997) Yersinia pestis-etiologic agent of plague. Clin Microbiol Rev 10:35–66PubMedPubMedCentralGoogle Scholar
  3. 3.
    Bleves S, Marenne MN, Detry G, Cornelis GR (2002) Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC. J Bacteriol 184(12):3214–3223CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Cornelis GR (2000) Type III secretion: a bacterial device for close combat with cells of their eukaryotic host. Philos Trans R Soc Lond Ser B Biol Sci 355(1397):681–693CrossRefGoogle Scholar
  5. 5.
    Brubaker RR (2005) Influence of Na(+), dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis. Infect Immun 73(8):4743–4752CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wolters M, Zobiak B, Nauth T, Aepfelbacher M. Analysis of Yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions. Jove-J Vis Exp 2015104  https://doi.org/10.3791/53115.
  7. 7.
    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(16):5486–5495CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Mueller KE, Fields KA (2015) Application of beta-lactamase reporter fusions as an indicator of effector protein secretion during infections with the obligate intracellular pathogen chlamydia trachomatis. PLoS One 10(8):e0135295CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    McCann JR, McDonough JA, Pavelka MS, Braunstein M (2007) Beta-lactamase can function as a reporter of bacterial protein export during mycobacterium tuberculosis infection of host cells. Microbiology 153(Pt 10):3350–3359CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sibakov M, Koivula T, von Wright A, Palva I (1991) Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol 57(2):341–348PubMedPubMedCentralGoogle Scholar
  11. 11.
    Inc. I: LiveBLAzer™ FRET – B/G Loading Kit (with CCF2-AM or CCF4-AM) Protocol. 2006.Google Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Yafang Tan
    • 1
  1. 1.Beijing Institute of Microbiology and EpidemiologyBeijingChina

Personalised recommendations