Assay for Type III Secretion in Escherichia coli

Bosko Mitrovic, Neta Sal-Man

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


The type III secretion system (T3SS) is crucial for the virulence of several pathogenic Escherichia coli species as well as for other gram-negative bacterial strains. Therefore, the ability to monitor this system constitutes a valuable tool for assessing the involvement of different proteins in bacterial virulence, for identifying critical domains and specific mutations, and for evaluating the antivirulence activities of various drugs. The major advantage of the T3SS secretion assay for E. coli over assays for other gram-negative pathogens is that it does not necessarily require specific antibodies. Here, we describe how to grow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains under T3SS-inducing conditions, separate the supernatant fraction from the bacterial pellet, analyze this fraction on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and evaluate the level of T3SS activity. We describe a qualitative analysis using Coomassie staining and a quantitative assay using western blotting.

Original languageEnglish
Pages (from-to)37-46
Number of pages10
JournalMethods in molecular biology (Clifton, N.J.)
StatePublished - 27 May 2022


  • Attaching and effacing E. coli
  • Bacterial secretion system
  • Bacterial virulence
  • EHEC
  • EPEC
  • Escherichia coli
  • Type III secretion system

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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