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Extracellular vesicles are produced by species across all domains of life, suggesting that vesiculation represents a fundamental principle of living matter. In Gram-negative bacteria, membrane vesicles (MVs) can originate either from blebs of the outer membrane or from endolysin-triggered explosive cell lysis, which is often induced by genotoxic stress. Although less is known about the mechanisms of vesiculation in Gram-positive and Gram-neutral bacteria, recent research has shown that both lysis and blebbing mechanisms also exist in these organisms. Evidence has accumulated over the past years that different biogenesis routes lead to distinct types of MV with varied structure and composition. In this Review, we discuss the different types of MV and their potential cargo packaging mechanisms. We summarize current knowledge regarding how MV composition determines their various functions including support of bacterial growth via the disposal of waste material, nutrient scavenging, export of bioactive molecules, DNA transfer, neutralization of phages, antibiotics and bactericidal functions, delivery of virulence factors and toxins to host cells and inflammatory and immunomodulatory effects. We also discuss the advantages of MV-mediated secretion compared with classic bacterial secretion systems and we introduce the concept of quantal secretion.In this Review, Toyofuku, Schild, Kaparakis-Liaskos and Eberl discuss the different types of bacterial membrane vesicle, how they are formed, their structure and composition and their diverse functions.

Iron is an essential trace element for most organisms. A common way for bacteria to acquire this nutrient is through the secretion of siderophores, which are secondary metabolites that scavenge iron from environmental stocks and deliver it to cells via specific receptors. While there has been tremendous interest in understanding the molecular basis of siderophore synthesis, uptake and regulation, questions about the ecological and evolutionary consequences of siderophore secretion have only recently received increasing attention. In this Review, we outline how eco-evolutionary questions can complement the mechanistic perspective and help to obtain a more integrated view of siderophores. In particular, we explain how secreted diffusible siderophores can affect other community members, leading to cooperative, exploitative and competitive interactions between individuals. These social interactions in turn can spur co-evolutionary arms races between strains and species, lead to ecological dependencies between them and potentially contribute to the formation of stable communities. In brief, this Review shows that siderophores are much more than just iron carriers: they are important mediators of interactions between members of microbial assemblies and the eukaryotic hosts they inhabit.Secreted siderophores help bacteria to take up iron from the environment. In this Review, Kramer, Özkaya and Kümmerli discuss the functions and implications that siderophores have for social interactions between bacterial cells and the resulting consequences for communities and hosts.

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.In this Review, Costa and colleagues summarize the current knowledge of type IV secretion system functioning in Gram-negative bacteria, with a focus on their architectures and adaptations for specialized functions. They also explore the biogenesis pathways and spatial localization of type IV secretion systems.

Gut microbiota plays a key role in insulin resistance (IR). Here we perform a case-control study of Chinese adults (ChiCTR2200065715) and identify that Parabacteroides distasonis is inversely correlated with IR. Treatment with P. distasonis improves IR, strengthens intestinal integrity, and reduces systemic inflammation in mice. We further demonstrate that P. distasonis- derived nicotinic acid (NA) is a vital bioactive molecule that fortifies intestinal barrier function via activating intestinal G-protein-coupled receptor 109a (GPR109a), leading to ameliorating IR. We also conduct a bioactive dietary fiber screening to induce P. distasonis growth. Dendrobium officinale polysaccharide (DOP) shows favorable growth-promoting effects on P. distasonis and protects against IR in mice simultaneously. Finally, the reduced P. distasonis and NA levels were also validated in another human type 2 diabetes mellitus cohort. These findings reveal the unique mechanisms of P. distasonis on IR and provide viable strategies for the treatment and prevention of IR by bioactive dietary fiber. Here, the authors show that the gut commensal Parabacteroides distasonis alleviates insulin resistance via nicotinic acid-intestinal GPR109a axis activation, a process promoted by Dendrobium officinale polysaccharide.

Key Points Vesicles derived from the outer membrane of Gram-negative bacteria, or outer-membrane vesicles (OMVs), are heterogeneous in size and composition, encapsulate soluble periplasmic content and are ubiquitously produced. The difficulty in finding a single molecular or genetic basis for OMV production is probably due to species-dependent differences in envelope architecture, environmental influences on envelope composition and redundancy of OMV-producing pathways. Mutations that subtly affect envelope crosslinking affect OMV production, whereas bacterial mutants that are unable to crosslink the envelope are typically unstable and form lysis products instead of OMVs. Lipopolysaccharide (LPS) subtypes also affect the levels of OMV production, as well as OMV cargo recruitment. OMV cargo may be enriched or excluded compared with its abundance in the bacterial envelope, suggesting that cargo recruitment is a regulated rather than stochastic process. Well-characterized cargoes include virulence factors, antibiotic-degrading enzymes, surface adherence factors, proteases and enzymes that are important for nutrient acquisition. OMVs can serve in bacterial communities as 'public goods' by distributing enzymes that break down extracellular material into nutrients, by recruiting iron, by acting as decoys for bacteriophages or antibiotics and by transferring DNA between cells. The versatile characteristics of OMVs and their immunomodulatory properties can be exploited for bioengineering applications and vaccine development. In this Review, Schwechheimer and Kuehn describe recent developments in elucidating the mechanisms of biogenesis and cargo selection of the outer-membrane vesicles (OMVs) produced by Gram-negative bacteria. They also discuss the functions of OMVs in bacterial physiology and during pathogenesis. Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Key Points More than one-third of the bacterial proteome is destined for the cell envelope. The general secretory (Sec) pathway is the ubiquitous, central and essential protein export pathway into, and through, the plasma membrane. Nascent exported chains that emerge from the ribosome are scanned by chaperones and export-specific proteins that select them from cytoplasmic residents and direct them for either co-translational or post-translational export through the SecYEG channel. Kinetic competition between the scanning factors that recognize exported protein signals selects for the export route. SecA, the ATPase motor of post-translational export, 'proof-reads' its substrates, which are possibly already at the ribosome, and assembles with SecYEG into the translocase holoenzyme. SecA is allosterically activated by amino-terminal signals that are present on exported proteins and uses its highly regulated quaternary and intraprotomeric dynamics for chemo–mechanical coupling that drives protein translocation. SecYEG is regulated by its cytoplasmic partners, ribosomes and SecA, and is activated by exported proteins for either vectorial or lateral translocation. In this Review, Tsirigotaki et al . discuss recent biochemical, structural and mechanistic insights that have been gained into the consecutive steps of the general secretory (Sec) pathway. They focus on the architecture and dynamics of SecYEG and its regulation by ribosomes and SecA, and present current models of the mechanisms and energetics of the Sec-pathway-dependent secretion process in bacteria. The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway — sorting and targeting, translocation and release — in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.

Key Points Type III secretion systems (T3SSs) are protein transport nanomachines that are used by numerous important Gram-negative bacterial pathogens and symbionts to establish trans -kingdom interactions with different hosts. They are essential virulence factors for many notorious bacterial pathogens, including the agents of plague and typhoid fever. T3SSs evolved from the flagellum, which is a key organelle for bacterial motility, and the core components that are involved in the assembly of these complex nanomachines are highly conserved. Although the flagellar systems are largely inherited vertically, the non-flagellar T3SSs can be transmitted through horizontal gene transfer. T3SSs are syringe-shaped, multi-megadalton complexes that are composed of more than 20 proteins and a series of ring structures embedded in the bacterial inner and outer membranes, as well as translocation pores in the host cell membrane. Encircled by these rings is a hollowed conduit that enables the delivery of partially unfolded virulence effector proteins into the host cell. Integrative imaging technologies that combine cryo-electron microscopy (cryo-EM), X-ray crystallography, NMR and computer modelling have enabled the high-resolution visualization of key substructures of the T3SSs and the nanomachine in action. Assembly of the T3SS apparatus and substrate secretion occur in a defined temporal order and hierarchy, and genetic analyses and molecular biology have enabled the identification and functional characterization of the key regulators that control these processes. Effector proteins that are secreted through T3SSs carry out various functions within the host cell, including the manipulation of host immune responses and actin cytoskeletal dynamics, subverting gene expression and post-translational modifications, hijacking signal transduction pathways, and interrupting vesicle transport and endocytic trafficking, all of which can promote bacterial colonization, survival and replication. T3SSs are attractive targets for vaccines and therapeutics owing to their essential roles in bacterial virulence and pathogenicity. By targeting bacterial virulence mechanisms instead of growth, inhibitors of T3SSs may exert less selective pressure on pathogens to develop drug resistance. Structural and functional characterization of T3SSs should facilitate mechanism-based drug design. Type III secretion systems (T3SSs) are protein transport nanomachines that resemble molecular syringes and are found in numerous Gram-negative bacterial species. This Review summarizes our current understanding of the structure and function of these important protein secretion systems, incorporating new advances from cryo-electron microscopy and integrative imaging studies. Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans -kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

Key Points Gram-negative bacteria have evolved a wide array of secretion systems to transport small molecules, proteins and DNA into the extracellular space or target cells. In this Review, we describe insights into the structural and mechanistic features of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli biogenesis machinery. These systems are remarkably varied in size, composition and architecture. Double-membrane-spanning secretion systems are composed of many tens of protein subunits and can reach multi-megadalton sizes, whereas outer-membrane-spanning systems are relatively simple and are usually composed of only one type of subunit. These systems can transport folded or unfolded substrates and use various energy sources to power transport, from ATP to proton or entropy gradients. Recent structural and molecular advances have uncovered remarkable structural and functional similarities between secretion systems that have the potential to be exploited for the development of novel antibacterial compounds. In this Review, Waksman and colleagues describe the structural and mechanistic details of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli biogenesis machinery. They discuss both conserved and divergent properties of these systems and their potential as targets of novel antibacterial compounds. Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.

Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects. Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.