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-based force spectroscopy. SptP and SopE2 unfolded at low force
(Funfold ≤ 17 pN @ 100 nm/s), making them among the most mechanically
labile proteins studied to date by AFM. Moreover, their mechanical
compliance is large, as measured by the distance to the transition state
(Δx‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast,
prior measurements of GFP, ubiquitin, and DHFR show them to be
mechanically robust (Funfold > 80 pN) and brittle (Δx‡ < 0.4
nm). These results suggest that effector protein unfolding by T3SS is a
mechanical process and that mechanical lability facilitates efficient
effector protein secretion.