CryoEM of bacterial secretion systems: A primer for microbiologists

Abstract

Bacterial secretion systems all tackle the challenge of secreting substrates from the cytoplasm out of the cell. Many unrelated secretion systems have evolved, strikingly diverse in their structures and mechanisms, from the small pump-like type II secretion systems to the colossal harpoon-like type VI secretion systems. What makes secretion systems intriguing also makes them difficult to study, however: they are cell envelope-associated, frequently contain moving or transiently associated components, and are very large.Cryogenic electron microscopy (CryoEM) is ideally poised to study secretion systems. CryoEM excels at determining the structures of large protein complexes, can discern different conformational states, and can image structures in situ. CryoEM has come of age in the past half-decade following a spectacular resolution revolution that changed cryoEM from low-resolution "blobology" to a methodology that can compete with X-ray crystallography and NMR for structure determination of biological molecules (Kühlbrandt, 2014). Here, we provide a primer for microbiologists on these capabilities. We describe its basic principles, its two main forms, and what the two forms can and cannot achieve. We then highlight the contributions of cryoEM to understanding how the diverse microbial secretion systems work, focusing on type III secretion systems. We end with a forward-facing overview of future prospects. | Cr yoEMCryoEM is a family of techniques that determine the three-dimensional (3-D) structures of biological molecules from two-dimensional (2-D) electron micrographs. In basic terms, this is achieved by imaging the specimen from many different angles to facilitate piecing together

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism

“…The actual mechanism behind the gatekeeper’s regulation of the secretion is either not conserved between EPEC and SPI-2 or we are describing different facets of a more complex phenomenon. Indeed, we cannot rule out the possibility that pH sensing is extrinsic to SctV, and may instead be sensed distally and transmitted to SctV such as via an allosteric mechanism through the needle ( Guo et al, 2019 ) and SctDJ ( Butan et al, 2019 , Umrekar et al, 2020 ). Alternatively, unless gated, the translocon pore being large enough to export unfolded proteins could easily permit a change in pH to transmit within the secretion channel and this might extend to the local area of SctV at the base of the secretion channel.…”

VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile

“…Nine types of microbial secretion systems are known to date, among which type 4, comprising type 4A and type 4B subfamilies, is relatively well studied and is able to deliver its substrates (DNA or proteins) to eukaryotic or prokaryotic cells (19,32). Recently, subfamily T4SS-C was found in C. difficile (14,15), though neither its biological functionality nor biochemical properties of its components have been studied.…”

How Did the Archaellum Get Its Rotation?

“…Curiously, rotation is not as frequently seen as we anticipated. Since Hendrix published his work, the phage portal protein has been shown not to rotate ( Hugel et al, 2007 ), the protease ClpAP has been shown to function without rotation ( Kim et al, 2020 ), and diverse symmetry-mismatched secretion systems have been shown to have architectures that preclude rotation ( Umrekar et al, 2021a ). Together, this suggests that strategies such as the sophisticated interlocking of different symmetries in phages ( Fang et al, 2020 ) may have evolved to preclude the potential side-effect of inter-subcomplex rotation ( Liu et al, 2014 ).…”