Vibrio anguillarum O1이 생산하는 Outer Membrane Vesicle (OMV)의 분리 및 OMV 내의 단백질 특성

홍경은,김동균,민문경,공인수,Hong, Gyeong-Eun,Kim, Dong-Gyun,Min, Mun-Kyeong,Kong, In-Soo 한국해양바이오학회 2007 한국해양바이오학회지 Vol.2 No.2

Vibrio anguillarum is a gram-negative bacterium that causes vibriosis in approximately 80 different fish species. V. anguillarum produces several exotoxins are correlated with the pathogenesis of vibriosis. This study is focused on the composition of the outer membrane vesicle. Most of gram-negative bacteria produce outer membrane vesicle (OMV) during cell growth. OMV was formed from the outer membrane surface of cell and than released to extracellular environment. OMV consists of outer membrane lipids, outer membrane protein (OMP), LPS, and soluble periplasmic components. Also, they contain toxins, adhesions, and immunomodulatory. Many gram-negative bacteria were studied out forming OMV. In Vibrio sp., formation of OMV by electron microscopy has been reported from V. cholerae and V. parahaemolyticus. In present study, we isolated OMV from V. anguillarum and OMV protein was separated by SDS-PAGE. Magor band was sliced and analyzed by MALDI-TOF. The major protein band of 38kDa was identified as OmpU by MALDI-TOF MS analysis.

Effective Platform for the Production of Recombinant Outer Membrane Vesicles in Gram-Negative Bacteria

( Anthicha Kunjantarachot ),( Teva Phanaksri ) 한국미생물생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.5

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences―from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA―were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1

윤성호,이상엽,최치원,이하영,노현주,전상미,권용민,권개경,김상진,김건화,김승일 한국미생물학회 2017 The journal of microbiology Vol.55 No.1

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMVNovo) are spherical in shape, and the average diameter of OMVNovo is 25–70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMVNovo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMVNovo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

Native and Foreign Proteins Secreted by the Cupriavidus metallidurans Type II System and an Alternative Mechanism

( Houjuan Xu ),( Timothy P. Denny ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.4

The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.

Microbe-Host Communication by Small RNAs in Extracellular Vesicles: Vehicles for Transkingdom RNA Transportation

MDPI 2019 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.20 No.6

<P>Extracellular vesicles (EVs) are evolutionary well-conserved nano-sized membranous vesicles that are secreted by both prokaryotic and eukaryotic cells. Recently, they have gained great attention for their proposed roles in cell-to-cell communication, and as biomarkers for human disease. In particular, small RNAs (sRNAs) contained within EVs have been considered as candidate interspecies-communication molecules, due to their demonstrated capacity to modulate gene expression in multiple cell types and species. While research into this field is in its infancy, elucidating the mechanisms that underlie host–microbe interactions and communications promises to impact many fields of biological research, including human health and medicine. Thus, this review discussed the results of recent studies that have examined the ways in which EVs and sRNAs mediate ‘microbe–host’ and ‘host–microbe’ interspecies communication.</P>

Bioengineered bacterial outer membrane vesicles: what is their potential in cancer therapy?

Gujrati, Vipul B,Jon, Sangyong Future Medicine Ltd 2014 Nanomedicine Vol.9 No.7

Outer Membrane Vesicles Derived from Salmonella Enteritidis Protect against the Virulent Wild-Type Strain Infection in a Mouse Model

( Qiong Liu ),( Jie Yi ),( Kang Liang ),( Xiangmin Zhang ),( Qing Liu ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.8

Foodborne contamination and salmonellosis caused by Salmonella Enteritidis (S. Enteritidis) are a significant threat to human health and poultry enterprises. Outer membrane vesicles (OMVs), which are naturally secreted by gram-negative bacteria, could be a good vaccine option because they have many biologically active substances, including lipopolysaccharides (LPS), outer membrane proteins (OMPs), and phospholipids, as well as periplasmic components. In the present study, we purified OMVs derived from S. Enteritidis and analyzed their characteristics through silver staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In total, 108 proteins were identified in S. Enteritidis OMVs through liquid chromatography tandem mass spectrometry analysis, and OMPs, periplasmic proteins, and extracellular proteins (49.9% of total proteins) were found to be enriched in the OMVs compared with bacterial cells. Furthermore, native OMVs used in immunizations by either the intranasal route or the intraperitoneal route could elicit significant humoral and mucosal immune responses and provide strong protective efficiency against a lethal dose (~100-fold LD₅₀) of the wild-type S. Enteritidis infection. These results indicated that S. Enteritidis OMVs might be an ideal vaccine strategy for preventing S. Enteritidis diseases.

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles

Federica De Lise,Francesca Mensitieri,Giulia Rusciano,Fabrizio Dal Piaz,Giovanni Forte,Flaviana Di Lorenzo,Antonio Molinaro,Armando Zarrelli,Valeria Romanucci,Valeria Cafaro,Antonio Sasso,Amelia Filip 한국미생물학회 2019 The journal of microbiology Vol.57 No.6

Outer membrane vesicles (OMVs) are nanostructures of 20– 200 nm diameter deriving from the surface of several Gramnegative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical “fingerprint”, suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

Helicobacter pylori-derived outer membrane vesicles stimulate interleukin 8 secretion through nuclear factor kappa B activation

( Mun Sun Choi ),( Eun Young Ze ),( Jae Yong Park ),( Tae-seop Shin ),( Jae Gyu Kim ) 대한내과학회 2021 The Korean Journal of Internal Medicine Vol.36 No.4

Background/Aims: Bacteria-derived outer membrane vesicles (OMVs) are commonly associated with various biological activities and functions. Helicobacter pylori-derived OMVs are thought to contribute to pathogenesis. This study aimed to investigate the effects of H. pylori-derived OMVs. Methods: H. pylori strains were isolated from patients with gastritis, gastric ulcer, or gastric cancer using endoscopic biopsy. The U-937, AGS, and MKN-45 cell lines were exposed to H. pylori and H. pylori-derived OMVs. The expression of interleukin 8 (IL-8) messenger RNA (mRNA) was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR, and IL-8 secretion was analyzed using enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB) activation was evaluated by Western blotting. Results: H. pylori and H. pylori-derived OMVs induced the expression of IL-8 mRNA and protein. Importantly, the bacteria induced higher IL-8 mRNA and protein expression than the OMVs. IL-8 expression was induced to different levels in response to H. pylori-derived OMVs from hosts with different gastric diseases. Western blotting revealed the increased phosphorylation and reduced degradation of inhibitor of NF-κB alpha in cells exposed to OMVs. Conclusions: H. pylori-derived OMVs may aid the development of various gastric diseases by inducing IL-8 production and NF-κB activation.

Bacterial Outer Membrane Vesicles as Drug Delivery Vehicles for Cancer Therapy

Sangyong JON,Vipul BRIJMOHA,Sang-Hyun KIM,Sunghyun KIM,Sangeun LEE 한국생물공학회 2012 한국생물공학회 학술대회 Vol.2012 No.4