Molecular function and regulation of the bacterial injectisome

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells by translocating molecular toxins, called effector proteins. It resembles a molecular syringe, which establishes a cytosolic connection between bacteria and host cells. While the structure of the injectisome is well-defined, little is known about its molecular function and regulation. To investigate those, we focused on the adaptation of the injectisome to local pH as well as on the dynamics interactions of the cytosolic components with the effector proteins. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. We found that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. In Y. enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic injectisome components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the injectisome at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates injectisome activity in response to environmental conditions. In a next step, we study the binding of effector proteins to those dynamics of the cytosolic components, which also exchange between a free diffusing cytoplasmic and a injectisome bound state. An attempt to identify novel interaction partners of SctQ by co-IP did not yield in significantly new candidates and recent publications showed that protein dynamics are linked to the function of the injectisome. We analyzed the diffusion behavior of the cytosolic protein SctQ with single particle tracking photoactivated localization microscopy (sptPALM) in different strain backgrounds. Our data shows that SctQ diffuses in two populations and that the presence of effector proteins slows down that speed. This suggests that the cytosolic components are not only essential for secretion but participate actively in effector shuttling and recruitment to the injectisome

Supplementary Information

Supplementary Information to Direct binding of type III secretion effectors to cytosolic sorting platform complexes in live bacteria indicates an effector shuttling mechanis