Type III secretion system effector proteins are mechanically labile

Multiple Gram-negative bacteria encode Type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter < 2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔG0unfold) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol respectively, typical for globular proteins and similar to published ΔG0unfold for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by AFM-...