Search

Escherichia coli produces extracellular vesicles called outer membrane vesicles. In this study, we investigated the mechanism underlying the hypervesiculation of deletion mutant ΔrodZ of E. coli. RodZ forms supramolecular complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. Because mreB is an essential gene, an expression-repressed strain (mreBR3) was constructed using CRISPRi, in which the expression of mreB decreased to 20% of that in the wild-type (WT) strain. In shaken-flask culture, the ΔrodZ strain produced >50 times more vesicles than the WT strain. The mreB-repressed strain mreBR3 showed eightfold higher vesicle production than the WT. ΔrodZ and mreBR3 cells were observed using quick-freeze replica electron microscopy. As reported in previous studies, ΔrodZ cells were spherical (WT cells are rod-shaped). Some ΔrodZ cells (around 7% in total) had aberrant surface structures, such as budding vesicles and dented surfaces, or curved patterns on the surface. Holes in the PG layer and an increased cell volume were observed for ΔrodZ and mreBR3 cells compared with the WT. In conditions of osmotic support using sucrose, the OD660 value of the ΔrodZ strain increased significantly, and vesicle production decreased drastically, compared with those in the absence of sucrose. This study first clarified that vesicle production by the E. coli ΔrodZ strain is promoted by surface budding and a burst of cells that became osmotically sensitive because of their incomplete PG structure. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) are non-living spherical nanostructures that derive from the cell envelope of Gram-negative bacteria. OMVs are important in bacterial pathogenesis, cell-to-cell communication, horizontal gene transfer, quorum sensing, and in maintaining bacterial fitness. These structures can be modified to express antigens of interest using glycoengineering and genetic or chemical modification. The resulting OMVs can be used to immunize individuals against the expressed homo- or heterologous antigens. Additionally, cargo can be loaded into OMVs and they could be used as a drug delivery system. OMVs are inherently immunogenic due to proteins and glycans found on Gram negative bacterial outer membranes. This review focuses on OMV manipulation to increase vesiculation and decrease antigenicity, their utility as vaccines, and novel engineering approaches to extend their application.

Citrus Huanglongbing (HLB) is the most devastating citrus disease in the world. Candidatus Liberibacter asiaticus (Las) is the prevalent HLB pathogen, which is yet to be cultivated. A recent study demonstrates that Las does not contain pathogenicity factors that are directly responsible for HLB symptoms. Instead, Las triggers systemic and chronic immune responses, representing a pathogen-triggered immune disease. Importantly, overproduction of reactive oxygen species (ROS) causes systemic cell death of phloem tissues, thus causing HLB symptoms. Because Las resides in the phloem tissues, it is expected that phloem cell might recognize outer membrane proteins, outer membrane vesicle (OMV) proteins and extracellular proteins of Las to contribute to the immune responses. Because Las has not been cultivated, we used Liberibacter crescens (Lcr) as a surrogate to identify proteins in the OM fraction, OMV proteins and extracellular proteins by liquid chromatography with tandem mass spectrometry (LC–MS/MS). We observed OMVs of Lcr under scanning electron microscope, representing the first experimental evidence that Liberibacter can deliver proteins to the extracellular compartment. In addition, we also further analyzed LC–MS/MS data using bioinformatic tools. Our study provides valuable information regarding the biology of Ca. Liberibacter species and identifies many putative proteins that may interact with host proteins in the phloem tissues. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a multifactorial disease with microbial and non-microbial causes. In recent years, Helicobacter pylori infection has been thought to play a critical role in some extra-gastrointestinal manifestations especially liver disorders. Outer membrane vesicles (OMVs) are one of the most important discussed H. pylori virulence factors. In the current study, four different clinical strains of H. pylori were collected and their OMVs were purified using ultra-centrifugation. To investigate their effects on liver cell exosomes, co-incubation with hepatocytes was applied. After a while, hepatocyte-derived exosomes were extracted and incubated with hepatic stellate cells (HSCs) to investigate the HSC activation and fibrosis marker induction. The expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin messenger RNAs (mRNA) was assessed using real-time RT-PCR, and the protein expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin was evaluated by Western blotting. Our results showed that infected hepatocyte-derived exosomes induced the expression of α-SMA, TIMP-1, β-catenin, and vimentin in HSCs and e-cadherin gene and protein expression was downregulated. In the current study, we found that H. pylori -derived OMVs may aid the exosome alternation and modified exosomes may have a possible role in HSC activation and liver fibrosis progression. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a multifactorial disease with microbial and non-microbial causes. In recent years, Helicobacter pylori infection has been thought to play a critical role in some extra-gastrointestinal manifestations especially liver disorders. Outer membrane vesicles (OMVs) are one of the most important discussed H. pylori virulence factors. In the current study, four different clinical strains of H. pylori were collected and their OMVs were purified using ultra-centrifugation. To investigate their effects on liver cell exosomes, co-incubation with hepatocytes was applied. After a while, hepatocyte-derived exosomes were extracted and incubated with hepatic stellate cells (HSCs) to investigate the HSC activation and fibrosis marker induction. The expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin messenger RNAs (mRNA) was assessed using real-time RT-PCR, and the protein expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin was evaluated by Western blotting. Our results showed that infected hepatocyte-derived exosomes induced the expression of α-SMA, TIMP-1, β-catenin, and vimentin in HSCs and e-cadherin gene and protein expression was downregulated. In the current study, we found that H. pylori-derived OMVs may aid the exosome alternation and modified exosomes may have a possible role in HSC activation and liver fibrosis progression.

This work was developed to the effects of biofilm composite nano-drug delivery system (OMVs-MSN-5-FU) on lymph node metastasis from oral squamous cell carcinoma. Mesoporous silica nanoparticles loaded with 5-FU (MSN-5-FU) were prepared first. Subsequently, the outer membrane vesicles (OMV) of Escherichia coli were collected to wrap MSN-5-FU, and then OMVs-MSN-5-FU was prepared. It was then immersed in artificial gastric juice and artificial intestinal juice to explore the drug release rate. Next, the effects of different concentrations of the nano-drug delivery systems on the proliferation activity of oral squamous carcinoma cell line KOSC-2 cl3-43 were analyzed. Tumor-bearing nude mice models were prepared by injecting human tongue squamous cell carcinoma cells Tca8113 into BALB/c-nu nude mice. They were injected with the OMVs-MSN-5-FU nano drug carrier system, and peri-carcinoma tissue and cervical lymph node tissue were harvested to observe morphological changes by Hematoxylin – eosin (HE) staining. The scanning electron microscope (SEM) results showed that all MSN, MSN-5-FU, OMV, and OMV-MSN-5-FU were spherical and uniformly distributed, with particle sizes of about 60nm, 80nm, 90nm, and 140nm, respectively. Among them, OMV had a directional core-shell structure. The cumulative drug release rates of artificial gastric juice in 48 hours were 61.2 ± 2.3% and 26.5 ± 3.1%, respectively. The 48 hours cumulative drug release rates of artificial intestinal juice were 70.5 ± 6.3% and 32.1 ± 3.8%, respectively. The cumulative release of MSN-5-FU was always higher than OMV-MSN-5-FU. The cumulative release of MSN-5-FU was always higher than OMV-MSN-5-FU. After injection of OMVS-MSN-5-FU, the number of cancer cells was significantly reduced and cervical lymph node metastasis was significantly controlled. HE staining results showed that OMVS-MSN-5-FU injection reduced the number of stained cells. Dense lymphocytes were clearly observed in the cortex of neck lymphocytes. The OMVs-MSN-5-FU drug delivery system can slow down the drug release rate, significantly inhibit the proliferation activity of oral squamous cancer cells, and control the metastasis of cancer cells to cervical lymph nodes. [ABSTRACT FROM AUTHOR]

Cyanobacterial blooms are a global concern due to their adverse effects on water quality and human health. Therefore, we examined the effects of various compounds on Microcystis aeruginosa growth. We found that Microcystis aeruginosa NIES-298 cells were lysed rapidly by linear six-carbon sugar alcohols including mannitol, galactitol, iditol, fucitol, and sorbitol, but not by other sugar alcohols. Microscopic observations revealed that mannitol treatment induced crumpled inner membrane, an increase in periplasmic space, uneven cell surface with outer membrane vesicles, disruption of membrane structures, release of intracellular matter including chlorophylls, and eventual cell lysis in strain NIES-298, which differed from the previously proposed cell death modes. Mannitol metabolism, antioxidant-mediated protection of mannitol-induced cell lysis by, and caspase-3 induction in strain NIES-298 were not observed, suggesting that mannitol may not cause organic matter accumulation, oxidative stress, and programmed cell death in M. aeruginosa. No significant transcriptional expression was induced in strain NIES-298 by mannitol treatment, indicating that cell lysis is not induced through transcriptional responses. Mannitol-induced cell lysis may be specific to strain NIES-298 and target a specific component of strain NIES-298. This study will provide a basis for controlling M. aeruginosa growth specifically by non-toxic substances. [ABSTRACT FROM AUTHOR]

Cyanobacterial blooms are a global concern due to their adverse effects on water quality and human health. Therefore, we examined the effects of various compounds on Microcystis aeruginosa growth. We found that Microcystis aeruginosa NIES-298 cells were lysed rapidly by linear six-carbon sugar alcohols including mannitol, galactitol, iditol, fucitol, and sorbitol, but not by other sugar alcohols. Microscopic observations revealed that mannitol treatment induced crumpled inner membrane, an increase in periplasmic space, uneven cell surface with outer membrane vesicles, disruption of membrane structures, release of intracellular matter including chlorophylls, and eventual cell lysis in strain NIES-298, which differed from the previously proposed cell death modes. Mannitol metabolism, antioxidant-mediated protection of mannitol-induced cell lysis by, and caspase-3 induction in strain NIES-298 were not observed, suggesting that mannitol may not cause organic matter accumulation, oxidative stress, and programmed cell death in M. aeruginosa. No significant transcriptional expression was induced in strain NIES-298 by mannitol treatment, indicating that cell lysis is not induced through transcriptional responses. Mannitol-induced cell lysis may be specific to strain NIES-298 and target a specific component of strain NIES-298. This study will provide a basis for controlling M. aeruginosa growth specifically by non-toxic substances.

This work was developed to the effects of biofilm composite nano-drug delivery system (OMVs-MSN-5-FU) on lymph node metastasis from oral squamous cell carcinoma. Mesoporous silica nanoparticles loaded with 5-FU (MSN-5-FU) were prepared first. Subsequently, the outer membrane vesicles (OMV) of Escherichia coli were collected to wrap MSN-5-FU, and then OMVs-MSN-5-FU was prepared. It was then immersed in artificial gastric juice and artificial intestinal juice to explore the drug release rate. Next, the effects of different concentrations of the nano-drug delivery systems on the proliferation activity of oral squamous carcinoma cell line KOSC-2 cl3-43 were analyzed. Tumor-bearing nude mice models were prepared by injecting human tongue squamous cell carcinoma cells Tca8113 into BALB/c-nu nude mice. They were injected with the OMVs-MSN-5-FU nano drug carrier system, and peri-carcinoma tissue and cervical lymph node tissue were harvested to observe morphological changes by Hematoxylin – eosin (HE) staining. The scanning electron microscope (SEM) results showed that all MSN, MSN-5-FU, OMV, and OMV-MSN-5-FU were spherical and uniformly distributed, with particle sizes of about 60nm, 80nm, 90nm, and 140nm, respectively. Among them, OMV had a directional core-shell structure. The cumulative drug release rates of artificial gastric juice in 48 hours were 61.2 ± 2.3% and 26.5 ± 3.1%, respectively. The 48 hours cumulative drug release rates of artificial intestinal juice were 70.5 ± 6.3% and 32.1 ± 3.8%, respectively. The cumulative release of MSN-5-FU was always higher than OMV-MSN-5-FU. The cumulative release of MSN-5-FU was always higher than OMV-MSN-5-FU. After injection of OMVS-MSN-5-FU, the number of cancer cells was significantly reduced and cervical lymph node metastasis was significantly controlled. HE staining results showed that OMVS-MSN-5-FU injection reduced the number of stained cells. Dense lymphocytes were clearly observed in the cortex of neck lymphocytes. The OMVs-MSN-5-FU drug delivery system can slow down the drug release rate, significantly inhibit the proliferation activity of oral squamous cancer cells, and control the metastasis of cancer cells to cervical lymph nodes.

The development of more effective, accessible, and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector, and whole inactivated virus vaccines is essential to curtailing the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six-proline-stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to outer membrane vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells, and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum-neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination, hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs 7 days after challenge in OMV-mC-Spike-vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction.

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum -derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum -Siglec-7 interaction. [ABSTRACT FROM AUTHOR]

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE , related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of Δ mlaE Δ nlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants Δ nlpI and Δ mlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the Δ mlaE Δ nlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the Δ mlaE Δ nlpI cells was more than 100 times higher than that of WT and Δ nlpI , and about 50 times higher than that of Δ mlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that Δ mlaE Δ nlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in Δ mlaE Δ nlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs. [ABSTRACT FROM AUTHOR]

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

Colibacillosis is an economically important infectious disease in poultry, caused by avian pathogenic Escherichia coli (APEC). Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne diseases in human circulated through poultry-derived products, including meat and chicken eggs. Vaccine control is the mainstream approach for combating these infections, but it is difficult to create a vaccine for the broad-spectrum protection of poultry due to multiple serotypes of these pathogens. Our previous studies have shown that outer membrane vesicles (OMVs) derived from S. enterica serovar Typhimurium mutants with a remodeled outer membrane could induce cross-protection against heteroserotypic Salmonella infection. Therefore, in this study, we further evaluated the potential of broad-spectrum vaccines based on major outer membrane protein (OMP)-deficient OMVs, including ΔompA, ΔompC, and ΔompD, and determined the protection effectiveness of these candidate vaccines in murine and chicken infection models. The results showed that ΔompA led to an increase in the production of OMVs. Notably, ΔompAΔompCΔompD OMVs showed significantly better cross-protection against S. enterica serovar Choleraesuis, S. Enteritidis, APEC O78, and Shigella flexneri 2a than did other omp-deficient OMVs, with the exception of ΔompA OMVs. Subsequently, we verified the results in the chicken model, in which ΔompAΔompCΔompD OMVs elicited significant cross-protection against S. Enteritidis and APEC O78 infections. These findings further confirmed the feasibility of improving the immunogenicity of OMVs by remodeling the outer membrane and provide a new perspective for the development of broad-spectrum vaccines based on OMVs.

Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics.

Colibacillosis is an economically important infectious disease in poultry, caused by avian pathogenic Escherichia coli (APEC). Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne diseases in human circulated through poultry-derived products, including meat and chicken eggs. Vaccine control is the mainstream approach for combating these infections, but it is difficult to create a vaccine for the broad-spectrum protection of poultry due to multiple serotypes of these pathogens. Our previous studies have shown that outer membrane vesicles (OMVs) derived from S. enterica serovar Typhimurium mutants with a remodeled outer membrane could induce cross-protection against heteroserotypic Salmonella infection. Therefore, in this study, we further evaluated the potential of broad-spectrum vaccines based on major outer membrane protein (OMP)-deficient OMVs, including Δ ompA , Δ ompC , and Δ ompD , and determined the protection effectiveness of these candidate vaccines in murine and chicken infection models. The results showed that Δ ompA led to an increase in the production of OMVs. Notably, Δ ompA Δ ompC Δ ompD OMVs showed significantly better cross-protection against S. enterica serovar Choleraesuis, S. Enteritidis, APEC O78, and Shigella flexneri 2a than did other omp -deficient OMVs, with the exception of Δ ompA OMVs. Subsequently, we verified the results in the chicken model, in which Δ ompA Δ ompC Δ ompD OMVs elicited significant cross-protection against S. Enteritidis and APEC O78 infections. These findings further confirmed the feasibility of improving the immunogenicity of OMVs by remodeling the outer membrane and provide a new perspective for the development of broad-spectrum vaccines based on OMVs. [ABSTRACT FROM AUTHOR]

Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics. [ABSTRACT FROM AUTHOR]

Dynamic Light Scattering (DLS), Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) are physical techniques widely employed to characterize the morphology and the structure of vesicles such as liposomes or human extracellular vesicles (exosomes). Bacterial extracellular vesicles are similar in size to human exosomes, although their function and membrane properties have not been elucidated in such detail as in the case of exosomes. Here, we applied the above cited techniques, in synergy with the thermotropic characterization of the vesicles lipid membrane using a turbidimetric technique to the study of vesicles produced by Gram-negative bacteria (Outer Membrane Vesicles, OMVs) grown at different temperatures. This study demonstrated that our combined approach is useful to discriminate vesicles of different origin or coming from bacteria cultured under different experimental conditions. We envisage that in a near future the techniques employed in our work will be further implemented to discriminate complex mixtures of bacterial vesicles, thus showing great promises for biomedical or diagnostic applications.

Myxobacteria are ubiquitous in soil environments. They display a complex life cycle: vegetatively growing cells coordinate their motility to form multicellular swarms, which upon starvation aggregate into large fruiting bodies where cells differentiate into spores. In addition to growing as saprophytes, Myxobacteria are predators that actively kill bacteria of other species to consume their biomass. In this review, we summarize research on the predation behavior of the model myxobacterium Myxococcus xanthus, which can access nutrients from a broad spectrum of microorganisms. M. xanthus displays an epibiotic predation strategy, i.e., it induces prey lysis from the outside and feeds on the released biomass. This predatory behavior encompasses various processes: Gliding motility and induced cell reversals allow M. xanthus to encounter prey and to remain within the area to sweep up its biomass, which causes the characteristic “rippling” of preying populations. Antibiotics and secreted bacteriolytic enzymes appear to be important predation factors, which are possibly targeted to prey cells with the aid of outer membrane vesicles. However, certain bacteria protect themselves from M. xanthus predation by forming mechanical barriers, such as biofilms and mucoid colonies, or by secreting antibiotics. Further understanding the molecular mechanisms that mediate myxobacterial predation will offer fascinating insight into the reciprocal relationships of bacteria in complex communities, and might spur application-oriented research on the development of novel antibacterial strategies.

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.

Dynamic Light Scattering (DLS), Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) are physical techniques widely employed to characterize the morphology and the structure of vesicles such as liposomes or human extracellular vesicles (exosomes). Bacterial extracellular vesicles are similar in size to human exosomes, although their function and membrane properties have not been elucidated in such detail as in the case of exosomes. Here, we applied the above cited techniques, in synergy with the thermotropic characterization of the vesicles lipid membrane using a turbidimetric technique to the study of vesicles produced by Gram-negative bacteria (Outer Membrane Vesicles, OMVs) grown at different temperatures. This study demonstrated that our combined approach is useful to discriminate vesicles of different origin or coming from bacteria cultured under different experimental conditions. We envisage that in a near future the techniques employed in our work will be further implemented to discriminate complex mixtures of bacterial vesicles, thus showing great promises for biomedical or diagnostic applications. [ABSTRACT FROM AUTHOR]

Myxobacteria are ubiquitous in soil environments. They display a complex life cycle: vegetatively growing cells coordinate their motility to form multicellular swarms, which upon starvation aggregate into large fruiting bodies where cells differentiate into spores. In addition to growing as saprophytes, Myxobacteria are predators that actively kill bacteria of other species to consume their biomass. In this review, we summarize research on the predation behavior of the model myxobacterium Myxococcus xanthus, which can access nutrients from a broad spectrum of microorganisms. M. xanthus displays an epibiotic predation strategy, i.e., it induces prey lysis from the outside and feeds on the released biomass. This predatory behavior encompasses various processes: Gliding motility and induced cell reversals allow M. xanthus to encounter prey and to remain within the area to sweep up its biomass, which causes the characteristic "rippling" of preying populations. Antibiotics and secreted bacteriolytic enzymes appear to be important predation factors, which are possibly targeted to prey cells with the aid of outer membrane vesicles. However, certain bacteria protect themselves from M. xanthus predation by forming mechanical barriers, such as biofilms and mucoid colonies, or by secreting antibiotics. Further understanding the molecular mechanisms that mediate myxobacterial predation will offer fascinating insight into the reciprocal relationships of bacteria in complex communities, and might spur application-oriented research on the development of novel antibacterial strategies. [ABSTRACT FROM AUTHOR]

The interactions between the gut microbiota and its host are of central importance to the health of the host. Outer membrane vesicles (OMVs) are produced ubiquitously by Gram-negative bacteria including the gut commensal Bacteroides thetaiotaomicron. These vesicles can interact with the host in various ways but until now their complement of small molecules has not been investigated in this context. Using an untargeted high-coverage metabolomic approach we have measured the small molecule content of these vesicles in contrasting in vitro conditions to establish what role these metabolites could perform when packed into these vesicles. B. thetaiotaomicron packs OMVs with a highly conserved core set of small molecules which are strikingly enriched with mouse-digestible metabolites and with metabolites previously shown to be associated with colonization of the murine GIT. By use of an expanded genome-scale metabolic model of B. thetaiotaomicron and a potential host (the mouse) we have established many possible metabolic pathways between the two organisms that were previously unknown, and have found several putative novel metabolic functions for mouse that are supported by gene annotations, but that do not currently appear in existing mouse metabolic networks. The lipidome of these OMVs bears no relation to the mouse lipidome, so the purpose of this particular composition of lipids remains unclear. We conclude from this analysis that through intimate symbiotic evolution OMVs produced by B. thetaiotaomicron are likely to have been adopted as a conduit for small molecules bound for the mammalian host in vivo.

The interactions between the gut microbiota and its host are of central importance to the health of the host. Outer membrane vesicles (OMVs) are produced ubiquitously by Gram-negative bacteria including the gut commensal Bacteroides thetaiotaomicron. These vesicles can interact with the host in various ways but until now their complement of small molecules has not been investigated in this context. Using an untargeted high-coverage metabolomic approach we have measured the small molecule content of these vesicles in contrasting in vitro conditions to establish what role these metabolites could perform when packed into these vesicles. B. thetaiotaomicron packs OMVs with a highly conserved core set of small molecules which are strikingly enriched with mouse-digestible metabolites and with metabolites previously shown to be associated with colonization of the murine GIT. By use of an expanded genome-scale metabolic model of B. thetaiotaomicron and a potential host (the mouse) we have established many possible metabolic pathways between the two organisms that were previously unknown, and have found several putative novel metabolic functions for mouse that are supported by gene annotations, but that do not currently appear in existing mouse metabolic networks. The lipidome of these OMVs bears no relation to the mouse lipidome, so the purpose of this particular composition of lipids remains unclear. We conclude from this analysis that through intimate symbiotic evolution OMVs produced by B. thetaiotaomicron are likely to have been adopted as a conduit for small molecules bound for the mammalian host in vivo. [ABSTRACT FROM AUTHOR]