Bacterial outer membrane vesicle nanorobot.

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Bacterial Outer Membrane Vesicles: From Discovery to Applications.

Secretion of cellular components across the plasma membrane is an essential process that enables organisms to interact with their environments. Production of extracellular vesicles in bacteria is a well-documented but poorly understood process. Outer membrane vesicles (OMVs) are produced in gram-negative bacteria by blebbing of the outer membrane. In addition to their roles in pathogenesis, cell-to-cell communication, and stress responses, OMVs play important roles in immunomodulation and the establishment and balance of the gut microbiota. In this review, we discuss the multiple roles of OMVs and the current knowledge of OMV biogenesis. We also discuss the growing and promising biotechnological applications of OMV.

Proteomic Analysis of Caco-2 Cells Disrupted by EcN 1917-Derived OMVs Reveals Molecular Information on Bacteria-Mediated Cancer Cell Migration.

Escherichia coli Nissle 1917 (EcN 1917) exhibits distinct tumor-targeting activity, and early studies demonstrated that outer membrane vesicles (OMVs) mediate bacteria-host interactions. To decipher the molecular mechanism underlying the interaction between EcN 1917 and host cells via OMV-mediated communication, we investigated the phenotypic changes in Caco-2 cells perturbed by EcN 1917-derived OMVs and constructed proteomic maps of the EcN 1917-derived OMV components and OMV-perturbed host cells. Our findings revealed that the size of the EcN 1917-derived OMV proteome increased 4-fold. Treatment with EcN 1917-derived OMVs altered the proteomic and phosphoproteomic profiles of host cells. Importantly, for the first time, we found that treatment with EcN 1917-derived OMVs inhibited cancer cell migration by suppressing the expression of ANXA9. In addition, phosphoproteomic data suggested that the ErbB pathway may be involved in OMV-mediated cell migration. Taken together, our study provides valuable data for further investigations of OMV-mediated bacteria-host interactions and offers great insights into the underlying mechanism of probiotic-assisted colorectal cancer therapy.

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling.

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.

Lysosome-Targeting Bacterial Outer Membrane Vesicles for Tumor Specific Degradation of PD-L1.

Increased expression of immune check point genes, such as PD-L1, is one of the main reasons for immunosuppression, especially for colon cancer. Development of novel therapeutic strategies is of great importance to improve the prognosis. In this study, outer membrane vesicles (OMV) derived from Gram-negative bacteria are engineered to immune checkpoint blockade nanosystem for efficient elicitation of anti-tumor immunity. Briefly, the OMVs are engineered with Lyp1-Traptavidin (S52G, R53D mutant of streptavidin) fusion protein displayed on the surface. The Lyp-1 endows the OMV with the capacity to target tumor tissues, while the Traptavidin ensures easy decoration of biotinylated anti-PD-L1 and biotinylated M6P (mannose 6-phosphate). The simultaneously anchored anti-PD-L1 and M6P (ligand for cation-independent mannose 6-phosphate receptor) on the engineered OMVs coordinately direct the membrane PD-L1 to lysosome for degradation, and thus unleash the anti-tumor immunity. With syngeneic tumor model, the engineered OMVs are confirmed to boost immunity, inhibit cancer growth, and thus prolong survival. Together, A proposed OMV-based modular nanosystem that enables assembly of biotinylated anti-PD-L1 and M6P on the surface for tumor-targeted immune checkpoint blockade.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Outer membrane vesicles as a platform for the discovery of antibodies to bacterial pathogens.

Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space, the bacterial outer membrane, and possibly other compartments. OMVs can be purified from bacterial culture supernatants, and by genetically manipulating the bacterial cells that produce them, they can be engineered to harbor cargoes and/or display molecules of interest on their surfaces including antigens that are immunogenic in mammals. Since OMV bilayer-embedded components presumably maintain their native structures, OMVs may represent highly useful tools for generating antibodies to bacterial outer membrane targets. OMVs have historically been utilized as vaccines or vaccine constituents. Antibodies that target bacterial surfaces are increasingly being explored as antimicrobial agents either in unmodified form or as targeting moieties for bactericidal compounds. Here, we review the properties of OMVs, their use as immunogens, and their ability to elicit antibody responses against bacterial antigens. We highlight antigens from bacterial pathogens that have been successfully targeted using antibodies derived from OMV-based immunization and describe opportunities and limitations for OMVs as a platform for antimicrobial antibody development. KEY POINTS: • Outer membrane vesicles (OMVs) of gram-negative bacteria bear cell-surface molecules • OMV immunization allows rapid antibody (Ab) isolation to bacterial membrane targets • Review and analysis of OMV-based immunogens for antimicrobial Ab development.

Metal ions-anchored bacterial outer membrane vesicles for enhanced ferroptosis induction and immune stimulation in targeted antitumor therapy.

The activation of ferroptosis presents a versatile strategy for enhancing the antitumor immune responses in cancer therapy. However, developing ferroptosis inducers that combine high biocompatibility and therapeutic efficiency remains challenging. In this study, we propose a novel approach using biological nanoparticles derived from outer membrane vesicles (OMVs) of Escherichia coli for tumor treatment, aiming to activate ferroptosis and stimulate the immune responses. Specifically, we functionalize the OMVs by anchoring them with ferrous ions via electrostatic interactions and loading them with the STING agonist-4, followed by tumor-targeting DSPE-PEG-FA decoration, henceforth referred to as OMV/SaFeFA. The anchoring of ferrous ions endows the OMVs with peroxidase-like activity, capable of inducing cellular lipid peroxidation by catalyzing H2O2 to •OH. Furthermore, OMV/SaFeFA exhibits pH-responsive release of ferrous ions and the agonist, along with tumor-targeting capabilities, enabling tumor-specific therapy while minimizing side effects. Notably, the concurrent activation of the STING pathway and ferroptosis elicits robust antitumor responses in colon tumor-bearing mouse models, leading to exceptional therapeutic efficacy and prolonged survival. Importantly, no acute toxicity was observed in mice receiving OMV/SaFeFA treatments, underscoring its potential for future tumor therapy and clinical translation.

Bacterial outer membrane vesicle-cancer cell hybrid membrane-coated nanoparticles for sonodynamic therapy in the treatment of breast cancer bone metastasis.

Breast cancer bone metastasis is a terminal-stage disease and is typically treated with radiotherapy and chemotherapy, which causes severe side effects and limited effectiveness. To improve this, Sonodynamic therapy may be a more safe and effective approach in the future. Bacterial outer membrane vesicles (OMV) have excellent immune-regulating properties, including modulating macrophage polarization, promoting DC cell maturation, and enhancing anti-tumor effects. Combining OMV with Sonodynamic therapy can result in synergetic anti-tumor effects. Therefore, we constructed multifunctional nanoparticles for treating breast cancer bone metastasis. We fused breast cancer cell membranes and bacterial outer membrane vesicles to form a hybrid membrane (HM) and then encapsulated IR780-loaded PLGA with HM to produce the nanoparticles, IR780@PLGA@HM, which had tumor targeting, immune regulating, and Sonodynamic abilities. Experiments showed that the IR780@PLGA@HM nanoparticles had good biocompatibility, effectively targeted to 4T1 tumors, promoted macrophage type I polarization and DC cells activation, strengthened anti-tumor inflammatory factors expression, and presented the ability to effectively kill tumors both in vitro and in vivo, which showed a promising therapeutic effect on breast cancer bone metastasis. Therefore, the nanoparticles we constructed provided a new strategy for effectively treating breast cancer bone metastasis.

Membrane-enclosed Pseudomonas quinolone signal attenuates bacterial virulence by interfering with quorum sensing.

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community.IMPORTANCEQuorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that Burkholderia cenocepacia mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that Pseudomonas quinolone signal (PQS) delivered by outer membrane vesicles from Pseudomonas aeruginosa or liposome attenuates B. cenocepacia virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in B. cenocepacia. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.

Proteomic Profiling Reveals Distinct Bacterial Extracellular Vesicle Subpopulations with Possibly Unique Functionality.

Bacterial outer membrane vesicles (OMVs) are 20- to 200-nm secreted packages of lipids, small molecules, and proteins that contribute to diverse bacterial processes. In plant systems, OMVs from pathogenic and beneficial strains elicit plant immune responses that inhibit seedling growth and protect against future pathogen challenge. Previous studies of OMV-plant interactions suggest functionally important differences in the protein composition of Pseudomonas syringae and Pseudomonas fluorescens OMVs, and that their composition and activity differ as a result of medium culture conditions. Here, we show that plant apoplast-mimicking minimal medium conditions impact OMV protein content dramatically in P. syringae but not in P. fluorescens relative to complete medium conditions. Comparative, 2-way analysis of the four conditions reveals subsets of proteins that may contribute to OMV-mediated bacterial virulence and plant immune activation as well as those involved in bacterial stress tolerance or adaptation to a beneficial relationship with plants. Additional localization enrichment analysis of these subsets suggests the presence of outer-inner membrane vesicles (OIMVs). Collectively, these results reveal distinct differences in bacterial extracellular vesicle cargo and biogenesis routes from pathogenic and beneficial plant bacteria in different medium conditions and point to distinct populations of vesicles with diverse functional roles. IMPORTANCE Recent publications have shown that bacterial vesicles play important roles in interkingdom communication between bacteria and plants. Indeed, our recently published data reveal that bacterial vesicles from pathogenic and beneficial strains elicit immune responses in plants that protect against future pathogen challenge. However, the molecules underlying these striking phenomena remain unknown. Our recent work indicated that proteins packaged in vesicles are critically important for vesicle-mediated seedling growth inhibition, often considered an indirect measure of plant immune activation. In this study, we characterize the protein cargo of vesicles from Pseudomonas syringae pathovar tomato DC3000 and Pseudomonas fluorescens from two different medium conditions and show that distinct subpopulations of vesicles contribute to bacterial virulence and stress tolerance. Furthermore, we reveal differences in how beneficial and pathogenic bacterial species respond to harsh environmental conditions through vesicle packaging. Importantly, we find that protein cargo implicates outer-inner membrane vesicles in bacterial stress responses, while outer membrane vesicles are packaged for virulence.

Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Porphyromonas gingivalis outer membrane vesicles in cerebral ventricles activate microglia in mice.

OBJECTIVE: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of Alzheimer's disease (AD). Whether Pg or its contents can reach the brain and directly affect neuropathology is, however, unknown. Here, we investigated whether outer membrane vesicles (OMVs) of Pg translocate to the brain and induce the pathogenic features of AD. MATERIAL AND METHODS: Pg OMVs were injected into the abdominal cavity of mice for 12 weeks. Pg OMV translocation to the brain was detected by immunohistochemistry using an anti-gingipain antibody. Tau protein and microglial activation in the mouse brain were examined by western blotting and immunohistochemistry. The effect of gingipains on inflammation was assessed by real-time polymerase chain reaction using human microglial HMC3 cells. RESULTS: Gingipains were detected in the region around cerebral ventricles, choroid plexus, and ventricular ependymal cells in Pg OMV-administered mice. Tau and phosphorylated Tau protein increased and microglia were activated. Pg OMVs also increased the gene expression of proinflammatory cytokines in HMC3 cells in a gingipain-dependent manner. CONCLUSION: Pg OMVs, including gingipains, can reach the cerebral ventricle and induce neuroinflammation by activating microglia. Pg OMVs may provide a better understanding of the implications of periodontal diseases in neurodegenerative conditions such as AD.

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440.

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

Outer membrane vesicles derived from heatstroke-associated intestinal microbiota promote multiple organ injury in mice.

Multiple organ injury is a common issue in heatstroke (HS); however, the underlying pathogenesis remains unclear. As an early event in HS, intestinal injury is an active participant that drives organ injury. Outer membrane vesicles (OMVs), a group of vesicles shed by unbalanced intestinal microbiota as "danger signals," mediate different functional cargo transport in cells and modulate varying biological events in distant target cells. However, the role of OMVs in HS-mediated organ damage remains unclear. Therefore, this study examined OMV production in HS and explored the effect of regulating multiple organ injury. To construct a mouse model, animals were exposed to hyperthermia. OMVs from the intestinal microbiota of HS and control mice were extracted by standardized differential ultracentrifugation. Thereafter, OMVs were characterized and infused into recipient mice via the tail vein. Cl-amidine (a pan-peptidylarginine deiminase inhibitor and OMV production inhibitor) was injected intraperitoneally (2 mg/kg) 2 h before HS treatment, and the absorption of HS OMVs by different organs was tracked. The effect of OMVs on inducing organ pathological changes, inflammatory infiltration, inflammatory cytokine expression, and serum organ injury biomarkers was demonstrated. HS increased OMV production by intestinal microbiota; OMVs were absorbed by different organs in vivo, and were especially enriched in the liver and lung. Compared to control OMVs, infusion with HS OMVs induced significant organ pathological changes, elevated inflammatory cell (macrophages and neutrophil) infiltration, inflammatory cytokine (TNF-ɑ, IL-1ß, IL-6) expression, as well as serum biomarkers of organ injury. Similarly, inhibition of endogenous OMVs alleviated these organ injury indicators induced by HS. To our knowledge, the present study is the first to illustrate that OMVs induce acute organ impairment during severe HS, offering a foundation for subsequent studies and providing novel therapeutic targets.

The effects of different thermal and chemical stresses on release of outer membrane vesicles (OMVs) by ClearColi™.

The highly immunogenic properties of outer membrane vesicles (OMVs), small spherical nanoparticles commonly released by Gram-negative bacteria, led to their application as vaccine candidate. ClearColi™ is an engineered Escherichia coli strain, which does not produce endotoxic response in humans and is useful for production of OMV-based vaccines. Therefore, producing ClearColi™ OMVs with high yield attracts particular interest. As stresses can be removed by OMVs, they may affect OMVs release. We aimed to investigate the effects of culture temperature, chemical (NaCl, ethanol, EDTA, D-cycloserine, polymyxin B, 1-octanol, and H2O2) and thermal stresses on release of ClearColi™ OMVs. Herein, the growth rate of ClearColi™ was decreased in the presence of all chemical stresses with the exception of H2O2. The optimum temperature for OMVs production was 37 â„ƒ and their release was not increased under thermal shock. The highest and lowest OMVs release was obtained in the presence of NaCl and H2O2, respectively. Electron microscopy images confirmed that the bilayer spherical-shaped OMVs were isolated under different stresses. Furthermore, SEM and DLS analysis demonstrated that OMVs released under EDTA stress are smaller than those released from untreated cultures. It can be concluded that chemical stresses have influence on the level of ClearColi™ OMVs production. However, changes in their content should be further investigated.

Collaborative Action of Microglia and Astrocytes Mediates Neutrophil Recruitment to the CNS to Defend against Escherichia coli K1 Infection.

Escherichia coli K1 is a leading cause of neonatal bacterial meningitis. Recruitment of neutrophils to the central nervous system (CNS) via local immune response plays a critical role in defense against E. coli K1 infection; however, the mechanism underlying this recruitment remains unclear. In this study, we report that microglia and astrocytes are activated in response to stimulation by E. coli K1 and/or E. coli K1-derived outer membrane vesicles (OMVs) and work collaboratively to drive neutrophil recruitment to the CNS. Microglial activation results in the release of the pro-inflammatory cytokine TNF-α, which activates astrocytes, resulting in the production of CXCL1, a chemokine critical for recruiting neutrophils. Mice lacking either microglia or TNF-α exhibit impaired production of CXCL1, impaired neutrophil recruitment, and an increased CNS bacterial burden. C-X-C chemokine receptor 2 (CXCR2)-expressing neutrophils primarily respond to CXCL1 released by astrocytes. This study provides further insights into how immune responses drive neutrophil recruitment to the brain to combat E. coli K1 infection. In addition, we show that direct recognition of E. coli K1 by microglia is prevented by the K1 capsule. This study also reveals that OMVs are sufficient to induce microglial activation.

Outer Membrane Vesicles Derived from Salmonella enterica Serotype Typhimurium Can Deliver Shigella flexneri 2a O-Polysaccharide Antigen To Prevent Shigella flexneri 2a Infection in Mice.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has been developed yet. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively biosynthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in the serum, with similar trends in IgA levels from vaginal secretions and fluid from bronchopulmonary lavage, both intranasally and intraperitoneally. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay, opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. IMPORTANCEShigella, the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. In addition, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella, from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii.

BACKGROUND: Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. RESULTS: In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97-100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. CONCLUSIONS: The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.