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In Situ Investigations of the Bacterial Type II Secretion System

Secretion systems, which are cell envelope located protein complexes, are used by bacteria to secrete various virulence factor proteins that are important for bacteria’s survival and pathogenicity 1 . One of the secretion systems is the type II secretion system (T2SS), which is widely used by gram-negative bacteria to secrete virulence-related proteins that can intoxicate target cells, absorb nutrients, help adhesion to host cells, and so on, causing various diseases 2 . The T2SS is made up of 12-15 protein components, including the outer membrane channel protein GspD (the secretin), which constitutes the last step of substrate transportation of T2SS. The in vitro structure of the GspD secretin has previously been revealed by cryo-EM single particle analysis (SPA), which provided its detailed architecture information 3 . However, its in situ structural information under the original physiological and biochemical conditions is still lacking, and macromolecules may require their native biological environment to achieve their functional state, through interactions with its surroundings such as the cell membrane and the peptidoglycan cell wall. In this study, we investigate the in situ structure of the GspD secretin through cryo-electron tomography (cryo-ET) and subtomogram averaging. We present that, when overexpressed in E. coli cells, the GspD secretin locates to the bacterial inner membrane where it can be clearly recognized from the tomogram. After subtomogram averaging, we solved a subnanometer resolution structure of the in situ GspD. This result identified the central gate region, the N3 constriction site and the N0 domain which is missing in the previous high-resolution in vitro structure. Also, our structure shows the transmembrane