• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.621-629
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• 2022

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.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1519-1528
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• 2017

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 $LD_{50}$) 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.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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• pp.1343-1347
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• 2016

Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

• 대한약리학회지
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• 제31권3호
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• pp.271-278
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• 1995

Barbiturates의 분자적 약리학적 작용기전 연구에 기초자료를 제공키 위하여 본연구를 수행하였다. 분자적 약리작용 기전 연구에서는 무엇보다도 선행되어야 하는 것이 barbiturates가 신경세포막에서 어느 부위에 주로 분포되는가를 알아내는 데에 있다. 쥐(Rat)의 뇌로부터 분리한 synaptosomal plasma membrane vesicles (RSPMV)를 분리한 후 이 RSPMV 외측 단층(outer monolayer)의 소수성 부위와 친수성 부위에 barbiturates가 분포되는 경향을 형광 probe 법으로 검색하였다. 세포막 외측 단층의 친수성 부위에 분포되는 형광 probe N-octadecylnaphthyl-2-amino-6-sulfonic acid (ONS)와 소수성 부위에 분포되는 형광 probe12-(9-anthroyloxy)stearic acid (AS)를 각각 봉입한 후 형광소광법으로 barbiturates의 분포를 측정한 결과는 다음과 같다. 1) 대부분의 barbiturates가 RSPMV 외측 단층의 친수성 부위(표면)에 분포되고 소수성 부위 (hydrophobic region)에 극히 소량만이 분포된다는 것을 확인하였다. 2) 마취효과를 크게 일으키는 barbiturates일수록 소수성 부위에 분포되는 양이 증가하였다. barbiturates 종류에 따른 RSPMV 외측 단층 소수성 부위에의 분포 크기는 thiopental sodium > pentobarbital > hexobarbital > amobarbital > phenobarbital의 순위였다.

• 대한약리학회지
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• 제28권2호
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• pp.191-199
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• 1992

한국산 2년생 소의 대뇌피질로부터 synaptosomal plasma membrane vesicles(SPMV)를 분리한 후 이 SPMV로부터 추출한 총지질로서 인공세포막(SPMVTL)을 제제하였다. SPMVTL의 outer monolayer에 trinitrophenyl group을 공유결합시킴으로써 SPMVTL이중층에 분포된 형광 probe 1,6-diphenyl-1,3,5-hexatriene(DPH)중 outer monolayer에 분포된 DPH형광만을 소광케 하였다. 형광분석기를 통하여 SPMVTL의 inner monolayer에 비하여 outer monolayer의 유동성이 크다는 것을 확인하였다. n-Alkanols는 SPMVTL지질 이중층(inner+outer monolayers)의 회전확산운동을 증가시키되 탄소수 두개가 증가될 때마다 그 효력이 약10배 가량 증가된다는 것을 알았다. 그러나 1-decanol의 경우에는 1-nonanol에 비하여 그 효력이 낮아지는 소위 cut-off현상이 나타났다. 뿐만 아니라 n-alkanols는 SPMVTL의 inner monolayer에 비하여 outer monolayer의 회전확산운동을 주로 증가시켰다. 하지만 n-alkanols의 탄소수가 증가됨에 따라 outer monolayer에 대한 선택적인 작용이 감소된다는 것도 확인되었다.

• Applied Microscopy
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• 제28권2호
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• pp.159-170
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• 1998

The glandular secretory system of the capitate gandular trichomes in leaf of Thymus quinquecostatus Celakovsky was examined by transmission electron microscope. The glandular trichome was consisted of three cell layers; an basal cell layer, a stalk cell with single-celled intermediate layer and a discoid secretory layer with thickened cuticle. The secretory cell was dense, rich in mitochondria, rER, plastds, Golgi complex and had many vesicular structure. Typical plastids with reticulate body and plastoglobule were present in glandular trichome. The tytoplasm of secretory cell was filled with osmiophilic secretory materials. The secretory vesicles, originated from Golgi complex, appeared as membrane bounded vesicles and secreted to the outer wall surface. The presences of well developed rER, mitochondria, Golgi complex, and membrane-bounded vesicles fused with plasmalemma in the secreting cells indicate that the granulocrine mechanism of secretion was occurring in T. quinquecostatus. Subcuticular cavity was developed between the cuticular layer and the secretory cell wall, and it formed above the secretory cell upon separation of cuticle-wall.

• Translational and Clinical Pharmacology
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• 제26권3호
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• pp.103-110
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• 2018

The human microbiome is known to play an essential role in influencing host health. Extracellular vesicles (EVs) have also been reported to act on a variety of signaling pathways, distally transport cellular components such as proteins, lipids, and nucleic acid, and have immunomodulatory effects. Here we shall review the current understanding of the intersectionality of the human microbiome and EVs in the emerging field of microbiota-derived EVs and their pharmacological potential. Microbes secrete several classes of EVs: outer membrane vesicles (OMVs), membrane vesicles (MVs), and apoptotic bodies. EV biogenesis is unique to each cell and regulated by sophisticated signaling pathways. EVs are primarily composed of lipids, proteins, nucleic acids, and recent evidence suggests they may also carry metabolites. These components interact with host cells and control various cellular processes by transferring their constituents. The pharmacological potential of microbiome-derived EVs as vaccine candidates, biomarkers, and a smart drug delivery system is a promising area of future research. Therefore, it is necessary to elucidate in detail the mechanisms of microbiome-derived EV action in host health in a multi-disciplinary manner.

• Journal of Photoscience
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• 제8권3_4호
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• pp.87-92
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• 2001

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to investigate the effects of parathyroid hormone (PTH) on the bulk bilayer fluidity of the plasma membrane vesicles isolated from cultured osteoblasts (OB-PMV). In a dose-dependent manner, rat PTH-(1-34) [rPTH-(1-34)] increased the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py and decreased the anisotropy (r) of DPH in OB-PMV. This indicates that PTH increased both the lateral and rotational diffusion of the probes in OB-PMY. Selective quenching of DPH fluorescence by trinitrophenyl groups was utilized to examine the transbilayer fluidity asymmetry of OB-PMV. The anisotropy, limiting anisotropy, and order parameter of DPH in the inner monolayer were 0.024, 0.032, and 0.062 greater than calculated for the outer monolayer of OB-PMY. Selective quenching of DPH fluorescence by trinitrophenyl groups was also utilized to examine the transbilayer effects of PTH on the fluidity of OB-PMV. rPTH-(1-34) had a greater fluidizing effect on the outer monolayer as compared to the inner monolayer of OB-PMV. Thus, it has been proven that PTH exhibits a selective rather than nonselective fluidizing effect within transbilayer domains of OB-PMV.

• 대한약리학회지
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• 제28권1호
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• pp.103-114
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• 1992

한국산 2년생 소의 신선한 대뇌피질로 부터 synaptosomal plasma membrane vesicles (SPMV)를 분리한 후 이 SPMV로 부터 추출한 총지질 (SPMVTL) 및 총인지질 (SPMVPL)로서 인공세포막을 제제하였다. SPMVTL 및 SPMVPL의 outer monolayer에 trinitrophenyl groups로 공유결합시킴으로써 형광 probe 1,6-diphenyl-1,3,5-hexatriene (DPH)의 outer monolayer 형광을 선택적으로 소광케한 후 SPMVTL 및 SPMVPL의 inner와 outer monolayer 사이의 비대칭적 유동성 존재 유무를 확인하였던 바 inner monolayer에 비하여 outer monolayer가 유동성이 크다는 것을 확인하였으며 SPMVTL에 비하여 SPMVPL의 유동성이 높았다. 뿐만 아니라 barbiturates는 SPMVTL의 유동성에 대하여는 유의성이 있을 만큼의 증감 효과를 나타내지 않았고 SPMVPL의 유동성은 감소시키되 주로 outer monolayer의 유동성을 감소시킨다는 것을 확인하였으며 barbiturates 종류에 따른 SPMVPL의 총유동성 감소의 크기는 pentobarbital > hexobarbital > amobarbital > phenobarbital의 순이었다.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.791-807
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• 2017

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.