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 ( $F_{\text{unfold}}=13.5\text{pN}$ at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state $\left(\text{Δ}{x}^{‡}=2.7\text{nm}\right)$ . In contrast, the non-effector homolog protealysin is mechanically stable ( $F_{\text{unfold}}=50.7\text{pN}$ at 100 nm/s) and brittle $\left(\text{Δ}{x}^{‡}=0.7\text{nm}\right)$ , comparable to proteins known to impair secretion ( $F_{\text{unfold}}>80\text{pN}$ ; $\text{Δ}{x}^{‡}<0.4\text{nm}$ ). 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 $\text{α}$ -helical while non-effector homologs contain $\text{β}$ -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.