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Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo- OMVs a promising vaccine candidate to be tested in clinics.

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet 1 – 3 . The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface 4 . LPS transport and assembly requires seven conserved and essential LPS transport components 5 (LptA–G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface 6 , 7 , but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5–10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly. As well as being the substrate for the lipopolysaccharide transport protein complex comprising LptA–G, lipopolysaccharide binding to Lpt proteins promotes their assembly into a bridge linking the inner and outer membranes of Gram-negative bacteria.

Outer membrane vesicles (OMVs), shed by Gram-negative bacteria, are spherical nanostructures that play a pivotal role in bacterial communication and host-pathogen interactions. Comprising an outer membrane envelope and encapsulating a variety of bioactive molecules from their progenitor bacteria, OMVs facilitate material and informational exchange. This review delves into the recent advancements in OMV research, providing a comprehensive overview of their structure, biogenesis, and mechanisms of vesicle formation. It also explores their role in pathogenicity and the techniques for their enrichment and isolation. Furthermore, the review highlights the burgeoning applications of OMVs in the field of biomedicine, emphasizing their potential as diagnostic tools, vaccine candidates, and drug delivery vectors.

Outer membrane vesicles (OMVs) are spherical, bilayered, and nanosized membrane vesicles that are secreted from gram-negative bacteria. OMVs play a pivotal role in delivering lipopolysaccharide, proteins and other virulence factors to target cells. Multiple studies have found that OMVs participate in various inflammatory diseases, including periodontal disease, gastrointestinal inflammation, pulmonary inflammation and sepsis, by triggering pattern recognition receptors, activating inflammasomes and inducing mitochondrial dysfunction. OMVs also affect inflammation in distant organs or tissues via long-distance cargo transport in various diseases, including atherosclerosis and Alzheimer’s disease. In this review, we primarily summarize the role of OMVs in inflammatory diseases, describe the mechanism through which OMVs participate in inflammatory signal cascades, and discuss the effects of OMVs on pathogenic processes in distant organs or tissues with the aim of providing novel insights into the role and mechanism of OMVs in inflammatory diseases and the prevention and treatment of OMV-mediated inflammatory diseases.

Bacterial secretion of extracellular polysaccharides is essential for surface colonization, biofilm formation, and pathogenesis. In diderm bacteria, such polymers traverse the periplasm and outer membrane (OM) through outer-membrane polysaccharide export (OPX) proteins that form secretion pores. Among them, Class-3 OPX proteins are the most widespread but lack an OM-spanning pore domain, leaving their mechanisms poorly understood. Here, we characterize WzaB from Myxococcus xanthus as a model for Class-3 OPX-mediated secretion. Structural and molecular dynamics analyses reveal that WzaB exists as a rigid monomer in solution, in contrast to the constitutive octamerization observed in Class-1 OPX proteins. Biochemical, biophysical, and in vivo analyses show that WzaB oligomerizes in a lipidation-dependent manner and directly interacts with the OM porin WzpB and the inner-membrane co-polymerase WzcB, with binding determinants mapped for both partners. Together, these proteins assemble into a trans-envelope polysaccharide secretion complex, redefining OPX function and revealing a distinct translocon architecture for Class-3 OPX systems. Bacteria secrete polysaccharides essential for colonization and infections. Here, the authors reveal the structure and mechanism of WzaB, a Class-3 OPX protein, uncovering a distinct trans-envelope secretion complex driving critical polysaccharide export in diderm bacteria.

Virtually all integral outer membrane proteins (OMPs) produced by Gram-negative bacteria contain a unique ‘β barrel’ structure that serves as a membrane spanning domain. The universal b arrel a ssembly m achine (BAM) catalyzes OMP assembly (folding and membrane insertion) in vivo, and purified Escherichia coli BAM that is reconstituted into proteoliposomes catalyzes OMP assembly in vitro. Here we show that BAM also catalyzes the assembly of OMPs into outer membrane fractions (‘native OMs’) that are purified by optimized conventional methods. Interestingly, we found that OMP assembly was moderately impaired when native OMs were isolated from a mlaA - strain that is deficient in maintaining OM lipid homeostasis but was strongly reduced when native OMs were isolated from a pldA - strain that is deficient in a parallel pathway. We also found that the mlaA and pldA deletions altered the OM phospholipid profile to different degrees that correlated with the degree to which the mutations impaired OMP assembly. Taken together, our results provide direct evidence that the mla and pldA pathways play distinct roles in maintaining OM homeostasis and strongly suggest that OM phospholipids play a more significant role in OMP biogenesis than previously appreciated. The BAM protein complex catalyzes the integration of newly made proteins into the outer membrane of Gram-negative bacteria. Here, Nilaweera et al. provide evidence that outer-membrane lipids also play an important role in this process.

Exclusive  to Firmicutes, endospore formation is a complex process that results in the release of a mature spore after the mother cell lyses. The spore is protected by multiple layers, including inner and outer spore membranes (IsM and OsM), both originating from the mother cell’s inner membrane (IM). While well understood in monoderm bacteria like Bacillus subtilis , less is known about diderm sporulators such as Acetonema longum , which retain and remodel their OsM into a functional outer membrane (OM) during germination. Here, we demonstrate that outer membrane proteins (OMPs), including the essential BAM complex component BamA and the LPS translocon protein LptD, are present in vegetative and germinating cells but absent from mature spores, indicating that OM biogenesis occurs without pre-existing OMPs. Growth-stage proteomics and expression profiling identify two previously uncharacterized proteins, SonA and SonB, co-expressed with BamA and in a conserved operon among diderm Firmicutes. SonA is a β-barrel OMP with three POTRA domains, while SonB is a predicted OM lipoprotein. In vitro, BamA and SonA spontaneously fold and insert into liposomes, supporting a model where BamA and/or SonA initiate self-insertion into the OsM, driving its stepwise transformation into a fully functional OM. Sporulation in diderm Firmicutes requires the remodeling of an inner membrane into a functional outer membrane. Uncharacterized to date, this process involves the complete replacement of all proteins and lipids within a membrane. Here, the authors present a model in which the self-insertion of BamA triggers the inner-to-outer membrane remodeling.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins. The pathogen Salmonella Typhimurium increases production of outer membrane vesicles (OMVs) inside acidic vacuoles of host cells, but the mechanisms are unclear. Here, Dehinwal et al. show that acidic pH induces conformational changes in an outer membrane protein that affect its interaction with membrane lipids, thus modulating OMV formation.