Bacterial outer membrane vesicles and the host-pathogen interaction

Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.     

外膜囊泡医学应用研究进展

外膜囊泡(OMV)是细菌自然生长的产物,呈球形双层膜纳米结构,其表面包含众多PAMPs,与免疫应答相关.OMV具有免疫原性和固有佐剂活性,因此可作为疫苗或单独的疫苗佐剂使用.OMV的中空结构可以作为药物储存载体,同时利用其外部结构实现药物的靶向作用.OMV在医学领域研究甚广,但要充分发挥其功能须清楚了解其研究进展和作用...     

Improved methods for producing outer membrane vesicles in Gram-negative bacteria

Outer membrane vesicle formation occurs during Gram-negative bacterial growth. However, natural production of large amounts of outer membrane vesicles has only been described in a few bacterial genera. The purified vesicles of some bacterial pathogens have shown potential applications in vaccinology and in antibiotic therapy. This study focused on the development of a gene expression system able to induce production of large amounts of outer membrane vesicles. The Tol-Pal system of Escherichia coli, required to maintain outer membrane integrity, is composed of five cell envelope proteins, TolA, TolB, TolQ, TolR and Pal. Tol proteins are parasitized by filamentous bacteriophages and by colicins. The phage infection process and colicin import require, respectively, the N-terminal domain of the minor coat g3p protein and the translocation domain of colicins, with both domains interacting with Tol proteins. In this study, we show that the periplasmic production of either Tol, g3p or colicin domains was able to specifically destabilize the E. coli or Shigella flexneri cell envelope and to induce production of high amounts of vesicles. This technique was further found to work efficiently in Salmonella enterica serovar Typhimurium.     

Sodium-dependent succinate transport in renal outer cortical brush border membrane vesicles

The transport of succinate into outer cortical brush border membrane vesicles (early proximal tubule) was studied. Succinate is taken up into an osmotically active space and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as D-glucose. Succinate uptake is markedly enhanced by sodium and slightly enhanced by lithium but shows no stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of succinate flux indicates a single transport site obeying Michaelis-Menten kinetics (Km = 1 mM and Vmax = 50 nmol X min -1 X mg protein -1 as measured under zero trans conditions at 100 mM NaCl and 28 degrees C with delta psi = 0). Direct evidence is given that succinate transport is coupled to sodium and is rheogenic, involving the net transfer of positive charge. The sodium:succinate coupling stoichiometry is found to be 2:1 by two independent methods.     

Immunization with Vibrio cholerae outer membrane vesicles induces protective immunity in mice

The gram-negative bacterium Vibrio cholerae releases outer membrane vesicles (OMVs) during growth. In this study, we immunized female mice by the intranasal, intragastric, or intraperitoneal route with purified OMVs derived from V. cholerae. Independent of the route of immunization, mice induced specific, high-titer immune responses of similar levels against a variety of antigens present in the OMVs. After the last immunization, the half-maximum total immunoglobulin titer was stable over a 3-month period, indicating that the immune response was long lasting. The induction of specific isotypes, however, was dependent on the immunization route. Immunoglobulin A, for example, was induced to a significant level only by mucosal immunization, with the intranasal route generating the highest titers. We challenged the offspring of immunized female mice with V. cholerae via the oral route in two consecutive periods, approximately 30 and 95 days after the last immunization. Regardless of the route of immunization, the offspring was protected against colonization with V. cholerae in both challenge periods. Our results show that mucosal immunizations via both routes with OMVs derived from V. cholerae induce long-term protective immune responses against this gastrointestinal pathogen. These findings may contribute to the development of “nonliving,” OMV-based vaccines against V. cholerae and other enteric pathogens, using the oral or intranasal route of immunization.     

Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells

Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K.?kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K.?kingae isolate were characterized. Among several major proteins, K.?kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K.?kingae infection. This study is the first report of OMV production by K.?kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.     

Flagellin-deficient outer membrane vesicles as adjuvant induce cross-protection of Salmonella Typhimurium outer membrane proteins against infection by heterologous Salmonella serotypes

Salmonella enteric serovar infections result in high morbidity and mortality worldwide. Cross-protective vaccines are an effective strategy in controlling salmonellosis caused by multiple serotypes. In our previous study, outer membrane vesicles (OMVs) derived from flagellin-deficient Salmonella Typhimurium (S. Typhimurium) were proven effective in mediating cross-protection against infection by multiple Salmonella serotypes; OMVs also exhibit potent adjuvant effects. In this study, we further investigated the adjuvant capacities of flagellin-deficient S. Typhimurium OMVs. Our finding showed that outer membrane proteins (OMPs) in combination with flagellin-deficient S. Typhimurium OMVs could function as adjuvants and invoke stronger humoral, cellular, mucosal, and cross-protective immune responses compared to conventional aluminum (alum). Furthermore, as an adjuvant, OMVs could induce significantly higher cellular immune responses and display enhanced cross-protection for OMPs against wild-type virulent Salmonella Choleraesuis and Salmonella Enteritidis challenge. In summary, OMVs function as a potent adjuvant with the capability of conferring greater cross-protection against infection by multiple Salmonella serotypes, and may be of great value as an effective vaccine adjuvant in enteric diseases.     

The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles

The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.     

Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica.     

Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages

Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ～70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.     

A comparison of the haemagglutinating and enzymic activities ofBacteroides fragiliswhole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacteriumBacteroides fragiliswere examined. Outer membrane vesicles are released from the surface ofB. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide ofB. fragilisconfirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations ofB. fragilisstrains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL; non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence ofB. fragilis.     

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.     

Primary preventive medicine and laboratory medicine

Nutrition plays an important role in the primary preventive medicine. In 1999, we have introduced the Dietary Reference Intakes (DRI) established by the National Academy of Sciences of U.S. into Recommended Dietary Allowances (RDAs) for the Japanese population. Where, reference intervals for the concentration of nutrient in blood are required in setting an Adequate Intakes (AI). For vitamins, also the AI is experimentally determined based on the reference intervals of healthy people, and the assessment of the nutritional status by laboratory investigations is an important part of preventive medicine. In 1997, we founded the Japan Committee for Vitamin Laboratory Standards and standardized whole blood vitamin B1 levels using HPLC method. Reference intervals for vitamin A, vitamin C and vitamin E might be presented in future report. We annually examined 15,000 participants in national nutrition survey. We proposed a use of results of this research for setting AI of vitamins. Genetic investigation should be introduced in future primary preventive medicine, and stress markers such as "biopyrrin" for oxidative stress, uropepsin, and urinary 17KS-S seems to be very important for medical examination.     

sncRNAs packaged by Helicobacter pylori outer membrane vesicles attenuate IL-8 secretion in human cells

Bacterial outer membrane vesicles (OMVs) play a vital role in the mechanism of host―pathogen communication, while emerging evidence suggests that OMVs regulate host immune responses through differentially packaged small noncoding RNAs (sncRNAs) to target host mRNA function. Therefore, we identified differentially packaged sncRNAs in Helicobacter pylori OMVs and showed transfer of OMV sncRNAs to human gastric adenocarcinoma cells in this study. Our data revealed that sncRNAs (sR-2509025 and sR-989262) were enriched in OMVs, and reduced lipopolysaccharide or OMV-induced interleukin 8 (IL-8) secretion by cultured AGS cells. Collectively, these findings are consistent with the hypothesis that sncRNAs in H. pylori OMVs play a novel role in the mechanism of host―pathogen interaction, whereby H. pylori evades the host immune response.     

Helicobacter pylori outer membrane vesicles modulate proliferation and interleukin-8 production by gastric epithelial cells

Helicobacter pylori infection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins by H. pylori. However, H. pylori may not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface of H. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV from cag PAI⁺ toxigenic and cag PAI⁻ nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from the cag PAI⁻ nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated with H. pylori infection.     

Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable Haemophilus influenzae

Outer membrane vesicles (OMVs) are produced by all Gram-negative microorganisms studied to date. The contributions of OMVs to biological processes are diverse and include mediation of bacterial stress responses, selective packaging and secretion of virulence determinants, modulation of the host immune response, and contributions to biofilm formation and stability. First characterized as transformasomes in Haemophilus, these membranous blebs facilitate transfer of DNA among bacteria. Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower respiratory tracts, produces OMVs in vivo, but there is a paucity of information regarding both the composition and role of OMVs during NTHI colonization and pathogenesis. We demonstrated that purified NTHI vesicles are 20 to 200 nm in diameter and contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human pharyngeal epithelial cells, resulting in a time- and temperature-dependent aggregation on the host cell surface, with subsequent internalization. OMVs colocalize with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs stimulate significant release of the immunomodulatory cytokine interleukin-8 (IL-8) as well as the antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain virulence-associated proteins that dynamically interact with and invade host epithelial cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic role for vesiculation in NTHI pathogenesis and clinically relevant disease progression.     

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.