Deciphering the Mechanical Properties of Type III Secretion System EspA Protein by Single Molecule Force Spectroscopy

Langmuir. 2018 May 29;34(21):6261-6270. doi: 10.1021/acs.langmuir.8b01198. Epub 2018 May 15.


Bacterial pathogens inject virulence factors into host cells during bacterial infections using type III secretion systems. In enteropathogenic Escherichia coli, this system contains an external filament, formed by a self-oligomerizing protein called E. coli secreted protein A (EspA). The EspA filament penetrates the thick viscous mucus layer to facilitate the attachment of the bacteria to the gut-epithelium. To do that, the EspA filament requires noteworthy mechanical endurance considering the mechanical shear stresses found within the intestinal tract. To date, the mechanical properties of the EspA filament and the structural and biophysical knowledge of monomeric EspA are very limited, mostly due to the strong tendency of the protein to self-oligomerize. To overcome this limitation, we employed a single molecule force spectroscopy (SMFS) technique and studied the mechanical properties of EspA. Force extension dynamic of (I91)4-EspA-(I91)4 chimera revealed two structural unfolding events occurring at low forces during EspA unfolding, thus indicating no unique mechanical stability of the monomeric protein. SMFS examination of purified monomeric EspA protein, treated by a gradually refolding protocol, exhibited similar mechanical properties as the EspA protein within the (I91)4-EspA-(I91)4 chimera. Overall, our results suggest that the mechanical integrity of the EspA filament likely originates from the interactions between EspA monomers and not from the strength of an individual monomer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli
  • Escherichia coli Proteins / chemistry*
  • Single Molecule Imaging*
  • Type III Secretion Systems / chemistry*
  • Type III Secretion Systems / metabolism


  • Escherichia coli Proteins
  • EspA protein, E coli
  • Type III Secretion Systems