Cooperative Binding of Cytosolic Type III Secretion System Proteins to the Injectisome Revealed by Live-Cell Single-Molecule Localization Microscopy

Type III secretion systems (T3SSs) are employed by many Gram-negative bacteria to translocate virulent effector proteins into host cells. Secretion occurs through the T3SS injectisome, a multi-membrane-spanning biomolecular machine. While the overall structure of the injectisome is becoming increasingly well defined, the structural dynamics that regulate secretion remain unresolved. Particularly important for the functional regulation of type 3 secretion are the cytosolic injectisome proteins which transiently associate with injectisomes and whose structural dynamics and function remain incompletely understood. Here, we use long-exposure single-molecule localization microscopy to quantify the bound times of the cytosolic components SctQ, SctL, and SctN, at the injectisome of Yersinia enterocolitica both in controllably induced secretion ON and OFF states and in the presence and absence of effector proteins. In the absence of effector proteins, each component exhibits distinct short-lived binding behavior on a timescale of seconds. However, the presence of the effector protein YopE increases bound times and synchronizes binding behavior across components, specifically without active secretion. Upon activation of secretion, a subpopulation of long-lived binding events emerges for SctQ, SctL, and SctN even within the same injectisomes, indicating that binding behavior between these proteins is secretion state-dependent and cooperative. These findings establish that the interaction kinetics between cytosolic injectisome proteins are modulated by the presence of secretion substrates and secretion activating signals, and reveal a new layer of heterogeneity in the functional regulation of type 3 secretion.