Advertisement

Screening for Secretion of the Type VI Secretion System Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot

  • Brent S. Weber
  • Pek Man Ly
  • Mario F. Feldman
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

The bacterial type VI secretion system (T6SS) is a secretory apparatus encoded by many Gram-negative bacteria. The T6SS facilitates the secretion and injection of toxic effector proteins into host cells, providing a competitive advantage to bacteria encoding this machinery. The activity of the T6SS can be monitored by probing for the conserved tubule component Hcp, which is secreted to the supernatants by the T6SS. Detection of Hcp in culture supernatants is indicative of an active T6SS, but this secretion system is often tightly regulated or inactive under laboratory conditions and different bacterial strains display differing Hcp secretion phenotypes. Herein, we describe an enzyme-linked immunosorbent assay (ELISA) and colony blot methods to facilitate large-scale screening of isolates for Hcp secretion and, thus, T6SS activity.

Key words

ELISA Colony blot Supernatant Hcp Effector 

References

  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 13:343–359CrossRefPubMedGoogle Scholar
  2. 2.
    Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 103:1528–1533CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S, Walz T, Joachimiak A, Mekalanos JJ (2006) A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312:1526–1530CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Russell AB, Peterson SB, Mougous JD (2014) Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol 12:137–148CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cianfanelli FR, Monlezun L, Coulthurst SJ (2016) Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol 24:51–62CrossRefPubMedGoogle Scholar
  6. 6.
    Silverman JM, Brunet YR, Cascales E, Mougous JD (2012) Structure and regulation of the type VI secretion system. Annu Rev Microbiol 66:453–472CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pukatzki S, McAuley SB, Miyata ST (2009) The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol 12:11–17CrossRefPubMedGoogle Scholar
  8. 8.
    Bernardy EE, Turnsek MA, Wilson SK, Tarr CL, Hammer BK (2016) Diversity of clinical and environmental isolates of Vibrio cholerae in natural transformation and contact-dependent bacterial killing indicative of Type VI secretion system activity. Appl Environ Microbiol 82:2833–2842CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Repizo GD, Gagne S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP (2015) Differential role of the T6SS in Acinetobacter baumannii virulence. PLoS One 10:e0138265CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Unterweger D, Kitaoka M, Miyata ST, Bachmann V, Brooks TM, Moloney J, Sosa O, Silva D, Duran-Gonzalez J, Provenzano D, Pukatzki S (2012) Constitutive type VI secretion system expression gives Vibrio cholerae intra- and interspecific competitive advantages. PLoS One 7:e48320CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tang L, Liang X, Moore R, Dong TG (2015) The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1. Front Cell Infect Microbiol 5:83CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Weber BS, Ly PM, Irwin JN, Pukatzki S, Feldman MF (2015) A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii. Proc Natl Acad Sci U S A 112:9442–9447CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Weber BS, Miyata ST, Iwashkiw JA, Mortensen BL, Skaar EP, Pukatzki S, Feldman MF (2013) Genomic and functional analysis of the type VI secretion system in Acinetobacter. PLoS One 8:e55142CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Carruthers MD, Nicholson PA, Tracy EN, Munson RS Jr (2013) Acinetobacter baumannii utilizes a type VI secretion system for bacterial competition. PLoS One 8:e59388CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Brent S. Weber
    • 1
  • Pek Man Ly
    • 1
  • Mario F. Feldman
    • 1
  1. 1.Department of Molecular MicrobiologyWashington University School of MedicineSaint LouisUSA

Personalised recommendations