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Mesalamine serves as the gold standard in treating ulcerative colitis. However, its precise mechanism(s) of action remains unclear. Here, we show that mesalamine treatment rapidly decreases polyphosphate levels in diverse bacteria, including members of the human gut microbiome. This decrease sensitizes bacteria towards oxidative stress, reduces colonization and attenuates persister cell and biofilm formation, suggesting that mesalamine aids in diminishing the capacity of bacteria to persist within chronically inflamed environments. Mesalamine, the gold-standard ulcerative colitis treatment, rapidly decreases polyphosphate levels in bacterial members of the gut microbiome, sensitizing them towards oxidative stress and reducing colonization and persister cell and biofilm formation.

Key Points Quorum sensing is a cell–cell communication process that enables bacteria to obtain information about cell density and species composition of the vicinal community and adjust their gene expression profiles accordingly. Quorum sensing involves the production, release and detection of extracellular signalling molecules known as autoinducers. Group-wide detection of autoinducers enables bacteria to collectively execute behaviours. Autoinducers are small molecules that control quorum sensing. In Gram-negative bacteria, autoinducers are often produced from S -adenosylmethionine (SAM). Autoinducers interact with specific receptors to elicit behaviours that are controlled by quorum sensing. Quorum sensing receptors are either membrane-bound histidine sensor kinases or cytoplasmic transcription factors. Autoinduction occurs when the detection of autoinducers induces the increased production of the same autoinducer molecule, forming a feed-forward regulatory loop. Other features, such as positive and negative feedback loops and small regulatory RNAs, optimize the integration of the autoinducer-encoded information and provide ideal quorum sensing dynamics. Signal integration is a process that takes place in most Gram-negative bacteria when several autoinducers and receptors work in parallel, or in series, to synchronize functions that are controlled by quorum sensing. Processes such as bioluminescence, the production of virulence factors and the formation of biofilms are controlled by quorum sensing. Quorum sensing shapes the composition of microbial communities. For example, bacterial species in the human gut microbiota produce and respond to autoinducers. There is increasing evidence that quorum sensing controls key physiological processes in the gut and may affect the virulence programmes of invading pathogens. Host cells are also known to produce autoinducer mimics. Synthetic quorum sensing modulators are molecules that agonize or antagonize quorum sensing and they are being developed as anti-virulence medicines. Distinct from traditional antibiotics, quorum sensing modulators do not affect the growth of pathogenic bacteria, but rather, disrupt their virulence programmes. Quorum sensing is used to control the behaviour of bacterial communities. In this Review, Papenfort and Bassler highlight recent discoveries about quorum sensing in Gram-negative bacteria, such as novel autoinducers and signalling networks that promote communication that ranges from intra-species to inter-kingdom. Bacteria use quorum sensing to orchestrate gene expression programmes that underlie collective behaviours. Quorum sensing relies on the production, release, detection and group-level response to extracellular signalling molecules, which are called autoinducers. Recent work has discovered new autoinducers in Gram-negative bacteria, shown how these molecules are recognized by cognate receptors, revealed new regulatory components that are embedded in canonical signalling circuits and identified novel regulatory network designs. In this Review we examine how, together, these features of quorum sensing signal–response systems combine to control collective behaviours in Gram-negative bacteria and we discuss the implications for host–microbial associations and antibacterial therapy.

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.

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.

Antimicrobial resistance in bacteria is frightening, especially resistance in Gram-negative Bacteria (GNB). In 2017, the World Health Organization (WHO) published a list of 12 bacteria that represent a threat to human health, and among these, a majority of GNB. Antibiotic resistance is a complex and relatively old phenomenon that is the consequence of several factors. The first factor is the vertiginous drop in research and development of new antibacterials. In fact, many companies simply stop this R&D activity. The finding is simple: there are enough antibiotics to treat the different types of infection that clinicians face. The second factor is the appearance and spread of resistant or even multidrug-resistant bacteria. For a long time, this situation remained rather confidential, almost anecdotal. It was not until the end of the 1980s that awareness emerged. It was the time of Vancomycin-Resistance Enterococci (VRE), and the threat of Vancomycin-Resistant MRSA (Methicillin-Resistant Staphylococcus aureus). After this, there has been renewed interest but only in anti-Gram positive antibacterials. Today, the threat is GNB, and we have no new molecules with innovative mechanism of action to fight effectively against these bugs. However, the war against antimicrobial resistance is not lost. We must continue the fight, which requires a better knowledge of the mechanisms of action of anti-infectious agents and concomitantly the mechanisms of resistance of infectious agents.

The Gram-negative bacterial lipopolysaccharide (LPS) is a major component of the outer membrane that plays a key role in host–pathogen interactions with the innate immune system. During infection, bacteria are exposed to a host environment that is typically dominated by inflammatory cells and soluble factors, including antibiotics, which provide cues about regulation of gene expression. Bacterial adaptive changes including modulation of LPS synthesis and structure are a conserved theme in infections, irrespective of the type or bacteria or the site of infection. In general, these changes result in immune system evasion, persisting inflammation and increased antimicrobial resistance. Here, we review the modifications of LPS structure and biosynthetic pathways that occur upon adaptation of model opportunistic pathogens (Pseudomonas aeruginosa, Burkholderia cepacia complex bacteria, Helicobacter pylori and Salmonella enterica) to chronic infection in respiratory and gastrointestinal sites. We also discuss the molecular mechanisms of these variations and their role in the host–pathogen interaction. The authors review modifications of lipopolysaccharide structure and biosynthetic pathways that occur upon bacterial adaptation to chronic respiratory and gastrointestinal infections. Graphical Abstract Figure. The authors review modifications of lipopolysaccharide structure and biosynthetic pathways that occur upon bacterial adaptation to chronic respiratory and gastrointestinal infections.

Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies.

Lypd8 protein derived from intestinal epithelial cells binds to flagellated bacteria to reduce their motility, which limits the entry of Gram-negative bacteria into the inner colonic mucus and prevents invasion of colonic epithelia. Lypd8 separates microbiota from epithelia This paper shows that the intestinal epithelial cell derived protein Lypd8, a member of member of the Ly6/PLAUR superfamily, binds to flagellated bacteria. In doing so it reduces the bacteria's motility, limits the entry of Gram-negative bacteria into the inner colonic mucus, and prevents invasion into colonic epithelium. Colonic epithelial cells are covered by thick inner and outer mucus layers 1 , 2 . The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis 3 , 4 , 5 , 6 . In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified 7 , 8 , 9 , 10 , 11 . Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis . In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8 −/− mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8 −/− mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

Simultaneous imaging and treatment of infections remains a major challenge, with most current approaches being effective against only one specific group of bacteria or not being useful for diagnosis. Here we develop multifunctional nanoagents that can potentially be used for imaging and treatment of infections caused by diverse bacterial pathogens. The nanoagents are made of fluorescent silicon nanoparticles (SiNPs) functionalized with a glucose polymer (e.g., poly[4-O-(α-D-glucopyranosyl)-D-glucopyranose]) and loaded with chlorin e6 (Ce6). They are rapidly internalized into Gram-negative and Gram-positive bacteria by a mechanism dependent on an ATP-binding cassette (ABC) transporter pathway. The nanoagents can be used for imaging bacteria by tracking the green fluorescence of SiNPs and the red fluorescence of Ce6, allowing in vivo detection of as few as 10 5 colony-forming units. The nanoagents exhibit in vivo photodynamic antibacterial efficiencies of 98% against Staphylococcus aureus and 96% against Pseudomonas aeruginosa under 660 nm irradiation.

Key Points Porin modification is a major bacterial resistance strategy that restricts the influx of β-lactam and fluoroquinolone antibiotics. Clinical multidrug resistant enterobacterial isolates that exhibit modified membrane permeability are highly prevalent. Interactions between antibiotic molecules and porin channels govern translocation efficiency. New physico-chemical techniques are being developed to assess drug–channel interactions and to quantify translocation through porins. Computer modelling of the pathway of substrates through porins provides information on the orientation and interaction of substrates in the channel. Quantification of antibiotic translocation provides new insights into how to optimize drug molecules so that they have sufficient permeation rates to circumvent multidrug resistance mechanisms. The outer membrane of Gram-negative bacteria contains many protein channels, called porins. These channels mediate the influx of various compounds, including antibiotics. Adaptations that reduce influx contribute to the emergence and dissemination of antibiotic resistance. This Review outlines recent advances in our understanding of the physico-chemical parameters that govern antibiotic translocation through porin channels. Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.