Calcium controls type III secretion switch through an SctV-SctW interplay

Type III secretion (T3S) is employed by many Gram-negative pathogens to inject toxins into eukaryotic cells via a syringe-like nanomachine, the injectisome. Secretion of T3 substrates follows a hierarchical order: early substrates and translocators are secreted first to assemble the injectisome, followed by effectors that initiate infection. Gatekeeper proteins and environmental signals, such as calcium, are known to regulate the secretion switch from translocators to effectors; however, the underlying molecular mechanism remains elusive. In this study, we demonstrate that the gatekeeper SctW and the export apparatus component SctV bind Ca²⁺ in solution with high affinity. Ca-binding is not required for anchoring SctW to the export apparatus but modulates the affinity of this interaction. High-affinity, Ca-dependent association engages the SctV C-domain, promoting high-affinity targeting and secretion of translocators. Low Ca or loss of Ca-binding weakens the SctW-SctV association, leaving the SctV C-domain available for high-affinity effector targeting and secretion. Mutations in either component freeze the bipartite receptor in high or low-affinity states, thereby disrupting Ca-dependent crosstalk and blocking secretion. These findings indicate that calcium acts as a ruling factor of T3 secretion.