Cellular Electron Cryotomography: Toward Structural Biology In Situ.

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

Type 9 secretion system structures reveal a new protein transport mechanism.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane ß-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome.

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Using Cryo-EM to Investigate Bacterial Secretion Systems.

Bacterial secretion systems are responsible for releasing macromolecules to the extracellular milieu or directly into other cells. These membrane complexes are associated with pathogenicity and bacterial fitness. Understanding of these large assemblies has exponentially increased in the last few years thanks to electron microscopy. In fact, a revolution in this field has led to breakthroughs in characterizing the structures of secretion systems and other macromolecular machineries so as to obtain high-resolution images of complexes that could not be crystallized. In this review, we give a brief overview of structural advancements in the understanding of secretion systems, focusing in particular on cryo-electron microscopy, whether tomography or single-particle analysis. We describe how such techniques have contributed to knowledge of the mechanism of macromolecule secretion in bacteria and the impact they will have in the future.

Multiple Structures Disclose the Secretins' Secrets.

Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homo-oligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Vancomycin-induced biofilm formation by methicillin-resistant Staphylococcus aureus is associated with the secretion of membrane vesicles.

Chronic burn wound infections caused by Stapyhylococcus aureus (S. aureus) are largely associated with biofilm formation. However, the mechanism by which S. aureus form biofilm in clinical environments is far less understood. In the present study we addressed the association between biofilm formation and membrane vesicle (MV) secretion of S. aureus during vancomycin treatment. A representative methicillin-resistant S. aureus (MRSA) strain BWMR22 obtained from a chronic burn wound was used in this study. Transmission electron microscope was used to observe the MV secretion. Fourier transform infrared spectroscopy was used to analyze the chemical component of MV. Biofilm formation was assayed under conditions of sub-inhibitory concentrations of vancomycin. Functional potencies of MV in surface adhesion and auto-aggregation were assayed in the presence of additional purified MVs. Biofilm formation by S. aureus BWMR22 was enhanced in the presence of sub-inhibitory concentration of vancomycin. Vancomycin treatment caused an increase in the chemical composition of protein relative to carbohydrates of secreted MVs, a property which was highly associated with bacterial hydrophobicity, surface adhesion, and intercellular aggregation. These findings suggest that MV secretion is correlated with biofilm formation by MRSA especially under clinical conditions with improper vancomycin chemotherapy. This study first demonstrates a potential role of MVs in the biofilm formation by S. aureus, stresses on the importance of avoiding low dose of antibiotic therapy in controlling of S. aureus infections, and provides further information to reveal the mechanisms behind MRSA infections.

Super-resolution imaging of bacterial pathogens and visualization of their secreted effectors.

Recent advances in super-resolution imaging techniques, together with new fluorescent probes have enhanced our understanding of bacterial pathogenesis and their interplay within the host. In this review, we provide an overview of what these techniques have taught us about the bacterial lifestyle, the nucleoid organization, its complex protein secretion systems, as well as the secreted virulence factors.

CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae.

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.

The Rich Tapestry of Bacterial Protein Translocation Systems.

The translocation of proteins across membranes is a fundamental cellular function. Bacteria have evolved a striking array of pathways for delivering proteins into or across cytoplasmic membranes and, when present, outer membranes. Translocated proteins can form part of the membrane landscape, reside in the periplasmic space situated between the inner and outer membranes of Gram-negative bacteria, deposit on the cell surface, or be released to the extracellular milieu or injected directly into target cells. One protein translocation system, the general secretory pathway, is conserved in all domains of life. A second, the twin-arginine translocation pathway, is also phylogenetically distributed among most bacteria and plant chloroplasts. While all cell types have evolved additional systems dedicated to the translocation of protein cargoes, the number of such systems in bacteria is now known to exceed nine. These dedicated protein translocation systems, which include the types 1 through 9 secretion systems (T1SSs-T9SSs), the chaperone-usher pathway, and type IV pilus system, are the subject of this review. Most of these systems were originally identified and have been extensively characterized in Gram-negative or diderm (two-membrane) species. It is now known that several of these systems also have been adapted to function in Gram-positive or monoderm (single-membrane) species, and at least one pathway is found only in monoderms. This review briefly summarizes the distinctive mechanistic and structural features of each dedicated pathway, as well as the shared properties, that together account for the broad biological diversity of protein translocation in bacteria.