Continuous exchange of the inner membrane ring component SctD is required for the assembly and function of the type III secretion system

Infectious diseases continue to pose a significant burden on humanity, remaining a major cause of illness and death worldwide. Among the various mechanisms employed by infectious agents, the Type III Secretion System (T3SS) is a highly conserved and often essential virulence factor found in many Gram-negative bacterial pathogens. This molecular nanomachine assembles in the bacterial cell wall, forming a syringe-like structure with a needle-like extracellular appendage that enables pathogens to inject protein toxins directly into eukaryotic host cells. Once inside, these toxins disrupt host cellular functions to promote bacterial survival and replication. While a core of T3SS components remains static and tightly bound to the overall structure, its cytosolic components are dynamic, continuously exchanging subunits with a cytosolic pool under physiological. We recently found that unexpectedly, the core structural inner membrane (IM) ring component SctD reversibly partially dissociated from the T3SS under acidic conditions, challenging previous assumptions about its structural stability. We therefore investigated whether SctD, like the cytosolic components, also exchanges subunits at the T3SS under physiological conditions. Using functional assays and fluorescence recovery after photobleaching, we confirmed and further characterized that SctD indeed undergoes subunit exchange while the T3SS is actively secreting. To better understand the biological significance of this dynamic behavior, we engineered an SctD mutant containing two cysteine substitutions in its periplasmic domain. This allowed us to reversibly modulate SctD exchange through targeted crosslinking to neighboring SctD subunits in the IM ring, depending on the redox conditions in the periplasm. Using this engineered SctD mutant, we found that SctD subunit exchange is crucial for integrating the T3SS export apparatus within the IM ring of the T3SS basal body and for promoting protein secretion by the T3SS. Our results reveal an unexpected dynamic behavior of the central T3SS components SctD and, for the first time, directly link this exchange to the T3SS assembly and its function in secreting protein toxins.