scholarly journals Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

2018 ◽  
Vol 84 (8) ◽  
pp. e02567-17 ◽  
Author(s):  
H. Bart van den Berg van Saparoea ◽  
Diane Houben ◽  
Marien I. de Jonge ◽  
Wouter S. P. Jong ◽  
Joen Luirink
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Therapeutic Agents ◽  
Outer Membrane Vesicles ◽  
High Density ◽  
Content Type ◽  
Protein Ligation ◽  
Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

scholarly journals Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Andreas Bauwens ◽  
Lisa Kunsmann ◽  
Helge Karch ◽  
Alexander Mellmann ◽  
Martina Bielaszewska
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shiga Toxin ◽  
Membrane Vesicles ◽  
Polymyxin B ◽  
Outer Membrane Vesicles ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

scholarly journals Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

2014 ◽  
Vol 80 (18) ◽  
pp. 5854-5865 ◽  
Author(s):  
Maria H. Daleke-Schermerhorn ◽  
Tristan Felix ◽  
Zora Soprova ◽  
Corinne M. ten Hagen-Jongman ◽  
David Vikström ◽  
...  
Keyword(s):  
Immune Responses ◽  
Outer Membrane ◽  
Vaccine Development ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Heterologous Proteins ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Heterologous Antigens

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

scholarly journals Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

2015 ◽  
Vol 81 (17) ◽  
pp. 5900-5906 ◽  
Author(s):  
Yoshihiro Ojima ◽  
Minh Hong Nguyen ◽  
Reiki Yajima ◽  
Masahito Taya
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Laser Scanning Microscopy ◽  
Content Type ◽  
E Coli ◽  
Enhanced Production ◽  
K 12

ABSTRACTMicrobial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation ofEscherichia colicells by overexpression of thebcsBgene was investigated. The flocculation induced by overexpression ofbcsBwas consistent among the variousE. colistrains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeledE. colicells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) inE. coliflocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore,bcsB-inducedE. coliflocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbAand ΔdsbBstrains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production ofE. colicells.

scholarly journals Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

2021 ◽  
Author(s):  
Dominika Houserova ◽  
Yulong Huang ◽  
Kasukurthi Kasukurthi ◽  
Brianna Watters ◽  
Fiza Khan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Dependent Manner ◽  
Antiviral Defense ◽  
Bacteriophage P22 ◽  
Bacteriophage Infection ◽  
Serovar Typhimurium ◽  
Dose Dependent

Abstract Salmonella Outer Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Interestingly, we identify 31 recurrent tRFs abundantly expressed by Salmonella enterica serovar Typhimurium and find these tRFs are highly complementary to known Salmonella enterica-infecting bacteriophage (17 averaging 97.4% complementarity over 22.9 nt) and specifically enriched in S. Typhimurium OMVs. Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% complementary over its entire 30 nt length to 29 distinct Salmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22. Furthermore, we find P22 pre-incubation with OMVs isolated from naïve S. Typhimurium, rescues the ability of S. Typhimurium depleted of tRNA-Thr-CGT-1-1, 44-73 tRF to defend against P22. Collectively, these experiments confirm tRFs secreted in S. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of known Salmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized ancient antiviral defense.

scholarly journals Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

2011 ◽  
Vol 18 (11) ◽  
pp. 1803-1808 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
George P. Munson ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protective Efficacy ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Virulence Proteins ◽  
Protective Antigens ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines.

scholarly journals Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

2014 ◽  
Vol 82 (10) ◽  
pp. 4001-4010 ◽  
Author(s):  
Jaewoo Bai ◽  
Seul I Kim ◽  
Sangryeol Ryu ◽  
Hyunjin Yoon
Keyword(s):  
Outer Membrane ◽  
Virulence Factors ◽  
Salmonella Enterica ◽  
Minimal Medium ◽  
Environmental Changes ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Associated Proteins

ABSTRACTSalmonella entericaserovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enableSalmonellato survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influencedSalmonellasurvival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into howSalmonellautilizes OMVs to adapt to environmental changes.

scholarly journals Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

2021 ◽  
Author(s):  
Dominika Houserova ◽  
Yulong Huang ◽  
Mohan V. Kasukurthi ◽  
Brianna C. Watters ◽  
Fiza F. Khan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Dependent Manner ◽  
Rna Seq ◽  
Bacteriophage P22 ◽  
Bacteriophage Infection ◽  
Serovar Typhimurium ◽  
Dose Dependent

Salmonella Outer Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Furthermore, despite there being several published reports now independently describing (1) the marked prevalence of tRFs within secreted vesicle transcriptomes and (2) roles for specific tRFs in facilitating/inhibiting viral replication, there have been no examinations of the effects of vesicle-secreted tRFs on viral infection reported to date. Notably, while specific tRFs have been reported in a number of bacteria, the tRFs expressed by salmonellae have not been previously characterized. As such, we recently screened small RNA-seq datasets for the presence of recurrent, specifically excised tRFs and identified 31 recurrent, relatively abundant tRFs expressed by Salmonella enterica serovar Typhimurium (SL1344). Furthermore, we find S. Typhimurium OMVs contain significant levels of tRFs highly complementary to known Salmonella enterica-infecting bacteriophage with 17 of 31 tRFs bearing marked complementarity to at least one known Salmonella enterica-infecting phage (averaging 97.4% complementarity over 22.9 nt). Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% sequence complementary over its entire 30 nt length to 29 distinct, annotated Salmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22 infectivity. Furthermore, we find P22 phage pre-incubation with OMVs isolated from naive, control SL1344 S. Typhimurium, successfully rescues the ability of S. Typhimurium transformed with a specific tRNA-Thr-CGT-1-1, 44-73 tRF inhibitor to defend against P22. Collectively, these experiments confirm tRFs secreted in S. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of known Salmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized innate antiviral defense.

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