The type II secretion system (T2SS) exists widely in gram-negative bacteria and exports a variety of folded protein substrates, such as cholera toxin (CT) from Vibrio cholerae and heat- labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC). The T2SS machinery spans the entire cell envelope and consists of three subassemblies: the mysterious inner membrane platform (IMP), the dynamic pseudopilus, and the channel-forming outer membrane complex. In current mechanism models, protein substrate binding to the T2SS in the periplasm triggers the cytoplasmic ATPase (GspE) to hydrolyze ATP, which energizes the incorporation of the major pseudopilin subunit (GspG) into a pilus-like structure. This growing pseudopilus acts as a piston or Archimedes screw, pushing the folded protein substrates through the outer membrane channel to the cell surface or extracellular milieu. Despite decades of research, the structure of IMP is not yet available; mainly for this reason, it is still unknown how the inner membrane assembly platform converts energy from ATP hydrolysis in the cytoplasm to extend the pseudopilus and push the substrates across the outer membrane. It is also unknown how the inner membrane proteins regulate the GspE ATPase activity. Our proposal exploits fluorescence size exclusion chromatography and coevolution analysis to identify interactions among components of IMP. We will use an assistant-multimer strategy and unnatural amino acid crosslinking to purify stable inner membrane protein complexes, followed by single particle cryo-electron microscopy to determine complex structures. We will analysis the ATPase activity of GspE with different inner membrane protein complexes. This proposal will substantially increase the fundamental scientific knowledge about the architecture and mechanisms of the T2SS, and thereby provide a new basis for developing future therapeutic interventions against a broad range of bacterial infections.
The type II secretion system (T2SS) exists widely in Gram-negative bacterial pathogens and secretes a variety of toxins that contribute to virulence. This interdisciplinary study exploits bioinformatical, biochemical, and single particle cryo-electron microscopy (cryo-EM) approaches to dissect the structures and mechanisms of the T2SS's inner membrane platform (IMP), thereby providing a new basis for developing future therapeutic interventions against a range of bacterial infections.