The path and regulation of type III secretion effector export

• Applicant: Dr. Andreas Diepold
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 456615895

Bacteria that live in contact to eukaryotic cells benefit from being able to manipulate host cell behavior. One of the most direct and elegant ways to reach this aim is the type III secretion system (T3SS), a molecular syringe also known as injectisome, used by Gram-negative bacteria to inject effector proteins into host cells. The T3SS is an essential virulence factor for many important pathogens, such as Salmonella, Shigella, Yersinia and pathogenic Escherichia coli. For these bacteria, injecting effector proteins at the right time is crucial for the successful establishment of infections. Surprisingly, the events that underlie this regulated secretion – the selection and recruitment of T3SS effectors in the bacterial cytosol, and their ordered and efficient delivery to the export gate of the injectisome, are still poorly understood. We therefore aim to address this question: How are effector proteins recruited and delivered to the export gate of the T3SS, and how is this process regulated by external signals? In the proposed project, we will study the role of the three main players in substrate recruitment: the “sorting platform”, a set of essential cytosolic T3SS components that interact with export cargo, and which we recently found to shuttle between the cytosol and the injectisome; the T3SS chaperones, dedicated cytosolic effector-binding proteins; and the effectors themselves. We will visualize these core factors in T3SS protein export in real time and at the single molecule level, determine the changes in their interactions upon activation of export, and link these molecular findings to the secretion process itself. Crucially, we will perform most of these approaches in live bacteria, which will provide insight into the molecular events that underlie the function of the T3SS and its regulation by external signals such as host cell contact in vivo. Our planned experiments will reveal, where, when, and under which conditions effectors, chaperones and the sorting platform proteins interact. They will determine whether there really is a hierarchy in effector export, and which factors are involved in establishing this order, and uncover the path of effectors in single bacterial cells prior to, during, and after the activation of secretion. Addressing these questions will provide important insights into the molecular mechanism and regulation of this fascinating secretion system. As the T3SS is a conserved and often essential virulence factor in important pathogens, our study will define potential targets for infection inhibitors, especially because effector export is controlled by external signals, and therefore accessible for therapeutic interference.

• DFG Programme: Research Grants