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

Abstract

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)-EspA-(I91) 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)-EspA-(I91) 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.

A single filament biomechanical study of the enteropathogenic Escherichia coli Type III secretion system reveals a high elastic aspect ratio

“…43 Accordingly, the interpretation that the filaments remain intact after their detachment from the AFM tip is highly reasonable as EspA, the T3SS filament building block, unfolds at forces lower than ∼80 pN. 17…”

“…Despite these mechanical stresses, the ability to maintain the structural integrity increases the possibility that the long and thin T3SS filament possesses noteworthy mechanical endurance. Although roughly estimated, 17 the hypothesis that the T3SS can sustain its functional-structural properties when subjected to mechanical loads was not yet verified. We have recently studied the biomechanical response of a single EspA subunit to an external load and found that a single EspA protein exhibits relatively low mechanical stability.…”

Interplay between Viscoelasticity and Force Rate Affects Sequential Unfolding in Polyproteins Pulled at Constant Velocity

“…The proteins were purified using HEPES buffer (1.0 mM EDTA, 150 mM NaCl, 20 mM HEPES, pH 7.5) as described elsewhere. 47 …”

“…Poly­(I91) octamers were expressed and purified in the laboratory of Dr. Neta Sal-Man at Ben-Gurion University of the Negev, using plasmids that were kindly provided by Prof. J. M. Fernandez from Columbia University. The proteins were purified using HEPES buffer (1.0 mM EDTA, 150 mM NaCl, 20 mM HEPES, pH 7.5) as described elsewhere …”

“…The proteins were purified using HEPES buffer (1.0 mM EDTA, 150 mM NaCl, 20 mM HEPES, pH 7.5) as described elsewhere. 47 Atomic Force Spectroscopy Measurements. Pulling experiments were carried out with a Luigs & Neumann LTD AFM.…”