A Type III secretion system effector evolved to be mechanically labile and initiate unfolding from the N-terminus

Many Gram-negative pathogens critically depend on the Type III secretion system (T3SS) to inject effector proteins into host cells for colonization. Because the channel of the T3SS is narrow (∼2 nm), effectors must be unfolded for secretion. However, the T3SS cannot unfold mechanically robust substrates (GFP, ubiquitin, and dihydrofolate reductase), severely impairing their secretion. Consistent with this, effectors are exceptionally mechanically labile, unfolding at low forces. Thus, secretion competency is correlated with mechanical properties. Effector sequences have significantly diverged from non-effectors, suggesting that secretion exerts evolutionary pressure selecting mechanical lability. Here, using atomic-force-microscopy–based force spectroscopy, we show that effector NleC is mechanically labile ( F unfold = 13.5 pN at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state (Δ x ‡ = 2.7 nm). In contrast, the non-effector homolog protealysin is mechanically stable ( F unfold = 50.7 pN at 100 nm/s) and brittle (Δ x ‡ = 0.7 nm), comparable to proteins known to impair secretion ( F unfold > 80 pN; Δ x ‡ < 0.4 nm). Denaturant-induced unfolding assays demonstrate that effectors exhibit rates typical of their fold, further reinforcing mechanical properties rather than fast unfolding kinetics ( k 0 ) predicts secretion. Steered molecular dynamic simulations revealed NleC unfolding initiates at the N-terminus, consistent with current secretion models, whereas protealysin unfolding initiates at the C-terminus. Notably, the NleC N-terminus is primarily α-helical while non-effector homologs contain β-sheets, which may account for the distinct unfolding pathway. Together, these results support the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion. Significance The Type III secretion system (T3SS) delivers effector proteins directly into host cells to promote bacterial colonization. Effectors must be unfolded for secretion, and this particular selective pressure is hypothesized to have driven significant sequence divergence from non-effector proteins. Here, we show that effectors are not characterized by unusually fast unfolding rates. Rather as hypothesized, effector NleC is more mechanically labile than its non-effector homolog, indicating that mechanical lability underlies both effector sequence divergence and T3SS unfolding. Simulations revealed that NleC unfolding initiates via the N-terminus consistent with the current secretion mechanism, while protealysin unfolds from the C-terminus. Together, these results strongly suggest mechanical lability is an evolved property of effectors and provide structural insight into how it is encoded.