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

Hila Nadler, Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel
Lihi Shaulov, Department Of Microbiology, Immunology And Genetics, Ben-gurion University Of The Negev, Beer Sheva, Israel
Yossi Blitsman, Department Of Chemical Engineering, Ben-gurion University Of The Negev, Beer Sheva, Israel
Moran Mordechai, Department Of Chemical Engineering, Ben-gurion University Of The Negev, Beer Sheva, Israel
Jürgen Jopp, The Ilse Katz Institute For Nanoscale Science And Technology, Ben-gurion University Of The Negev, Beer Sheva, Israel
Neta Sal-Man, Department Of Microbiology, Immunology And Genetics, Ben-gurion University Of The Negev, Beer Sheva, Israel
Ronen Berkovich, Department Of Chemical Engineering, Ben-gurion University Of The Negev, Beer Sheva, Israel

Bacterial type III secretion systems inject virulence factors by bacterial pathogens into host-cells during bacterial infection. 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 re-folded 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.