Cryo-EM structure of a conjugative type IV secretion system suggests a molecular switch regulating pilus biogenesis.

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

Structural and functional diversity of type IV secretion systems.

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.

Substrate recruitment mechanism by gram-negative type III, IV, and VI bacterial injectisomes.

Bacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems.

Cryo-EM structure of a type IV secretion system.

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.

Proteins DotY and DotZ modulate the dynamics and localization of the type IVB coupling complex of Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen infecting alveolar macrophages and protozoa species. Legionella utilizes a Type IV Secretion System (T4SS) to translocate over 300 effector proteins into its host cell. In a recent study, we have isolated and solved the cryo-EM structure of the Type IV Coupling Complex (T4CC), a large cytoplasmic determinant associated with the inner membrane that recruits effector proteins for delivery to the T4SS for translocation. The T4CC is composed of a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module flexibly protrudes. The DotY and DotZ proteins were newly reported members of this complex and their role remained elusive. In this study, we observed the effect of deleting DotY and DotZ on T4CC stability and localization. Furthermore, we found these two proteins are co-dependent, whereby the deletion of DotY resulted in DotZ absence from the coupling complex, and vice versa. Additional cryo-EM data analysis revealed the dynamic movement of the IcmSW module is modified by the DotY/Z proteins. We therefore determined the likely function of DotY and DotZ and revealed their importance on T4CC function.

Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525.

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties.

Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila.

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively.

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.

The Remarkable Biomechanical Properties of the Type 1 Chaperone-Usher Pilus: A Structural and Molecular Perspective.

Chaperone-usher (CU) pili are long, supramolecular protein fibers tethered to the surface of numerous bacterial pathogens. These virulence factors function primarily in bacterial adhesion to host tissues, but they also mediate biofilm formation. Type 1 and P pili of uropathogenic Escherichia coli (UPEC) are the two best-studied CU pilus examples, and here we primarily focus on the former. UPEC can be transmitted to the urinary tract by fecal shedding. It can then ascend up the urinary tract and cause disease by invading and colonizing host tissues of the bladder, causing cystitis, and the kidneys, causing pyelonephritis. FimH is the subunit displayed at the tip of type 1 pili and mediates adhesion to mannosylated host cells via a unique catch-bond mechanism. In response to shear forces caused by urine flow, FimH can transition from a low-affinity to high-affinity binding mode. This clever allosteric mechanism allows UPEC cells to remain tightly attached during periods of urine flow, while loosening their grip to allow dissemination through the urinary tract during urine stasis. Moreover, the bulk of a CU pilus is made up of the rod, which can reversibly uncoil in response to urine flow to evenly spread the tensile forces over the entire pilus length. We here explore the novel structural and mechanistic findings relating to the type 1 pilus FimH catch-bond and rod uncoiling and explain how they function together to enable successful attachment, spread, and persistence in the hostile urinary tract.

From conjugation to T4S systems in Gram-negative bacteria: a mechanistic biology perspective.

Conjugation is the process by which bacteria exchange genetic materials in a unidirectional manner from a donor cell to a recipient cell. The discovery of conjugation signalled the dawn of genetics and molecular biology. In Gram-negative bacteria, the process of conjugation is mediated by a large membrane-embedded machinery termed "conjugative type IV secretion (T4S) system", a large injection nanomachine, which together with a DNA-processing machinery termed "the relaxosome" and a large extracellular tube termed "pilus" orchestrates directional DNA transfer. Here, the focus is on past and latest research in the field of conjugation and T4S systems in Gram-negative bacteria, with an emphasis on the various questions and debates that permeate the field from a mechanistic perspective.

Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri.

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.

X-ray crystal structures of the type IVb secretion system DotB ATPases.

Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and ß, with an αßαßαß configuration. The existence of α and ß conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααßααß configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease. PDB Code(s): Will; be; provided.

Protein-Injection Machines in Bacteria.

Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases.

Legionella DotM structure reveals a role in effector recruiting to the Type 4B secretion system.

Legionella pneumophila, a causative agent of pneumonia, utilizes the Type 4B secretion (T4BS) system to translocate over 300 effectors into the host cell during infection. T4BS systems are encoded by a large gene cluster termed dot/icm, three components of which, DotL, DotM, and DotN, form the "coupling complex", which serves as a platform for recruitment of effector proteins. One class of effectors includes proteins containing Glu-rich/E-block sequences at their C terminus. However, the protein or region of the coupling complex mediating recruitment of such effectors is unknown. Here we present the crystal structure of DotM. This all alpha-helical structure exhibits patches of positively charged residues. We show that these regions form binding sites for acidic Glu-rich peptides and that mutants targeting these patches are defective in vivo in the translocation of acidic Glu-rich motif-containing effectors. We conclude that DotM forms the interacting surface for recruitment of acidic Glu-rich motif-containing Legionella effectors.

Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate.

Bacterial conjugation, a mechanism of horizontal gene transfer, is the major means by which antibiotic resistance spreads among bacteria (1, 2). Conjugative plasmids are transferred from one bacterium to another through a type IV secretion system (T4SS) in the form of single-stranded DNA covalently attached to a protein called relaxase. The relaxase is fully functional both in a donor cell (prior to conjugation) and recipient cell (after conjugation). Here, we demonstrate that the protein substrate has to unfold for efficient translocation through the conjugative T4SS. Furthermore, we present various relaxase modifications that preserve the function of the relaxase but block substrate translocation. This study brings us a step closer to deciphering the complete mechanism of T4SS substrate translocation, which is vital for the development of new therapies against multidrug-resistant pathogenic bacteria.IMPORTANCE Conjugation is the principal means by which antibiotic resistance genes spread from one bacterium to another (1, 2). During conjugation, a covalent complex of single-stranded DNA and a protein termed relaxase is transported by a type IV secretion system. To date, it is not known whether the relaxase requires unfolding prior to transport. In this report, we use functional assays to monitor the transport of wild-type relaxase and variants containing unfolding-resistant domains and show that these domains reduce conjugation and protein transport dramatically. Mutations that lower the free energy of unfolding in these domains do not block translocation and can even promote it. We thus conclude that the unfolding of the protein substrate is required during transport.

Type IV secretion in Gram-negative and Gram-positive bacteria.

Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram-negative and Gram-positive bacteria. They play important roles through the contact-dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact-independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F-pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of 'paradigmatic' and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.

The Cryoelectron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod.

Adhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable "spring-like" properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro.