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

Type III secretion systems (T3SSs) are syringe-like protein complexes used by some of the most harmful bacterial pathogens to infect host cells. While the T3SS filament, a long hollow conduit that bridges between bacteria and host cells, has been characterized structurally, very little is known about its physical properties. These filaments should endure shear and normal stresses imposed by the viscous mucosal flow during infection within the intestinal tract. We used atomic force microscopy (AFM) to probe the longitudinal and radial mechanical response of individual T3SS filaments by pulling on filaments extending directly from bacterial surfaces and later pressing into filaments that were detached from the bacteria. The measured longitudinal elastic moduli were higher by about two orders of magnitude than the radial elastic moduli. These proportions are commensurate with the role of the T3SS filament, which requires horizontal flexibility while maintaining its structural integrity to withstand intense stresses during infection.

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

Polyproteins are unique constructs, comprised of folded protein domains in tandem and polymeric linkers. These macromolecules perform under biological stresses by modulating their response through partial unfolding and extending. Although these unfolding events are considered independent, a history dependence of forced unfolding within polyproteins was reported. Here we measure the unfolding of single poly(I91) octamers, complemented with Brownian dynamics simulations, displaying increasing hierarchy in unfolding-foces, accompanied by a decrease in the effective stiffness. This counters the existing understanding that relates stiffness with variations in domain size and probe stiffness, which is expected to reduce the unfolding forces with every consecutive unfolding event. We utilize a simple mechanistic viscoelastic model to show that two effects are combined within a sequential forced unfolding process: the viscoelastic properties of the growing linker chain lead to a hierarchy of the unfolding events, and force-rate application governs the unfolding kinetics.

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

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.