• Journal of Microbiology and Biotechnology
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• v.32 no.3
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• pp.278-286
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• 2022

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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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.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.407-409
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• 2000

Outer envelope membrane proteins of chloroplasts encoded by the nuclear genome are transported without the N-terminal transit peptide. Here, we investigated the targeting mechanism of AtOEP7, an Arabidopsis homolog of small outer envelope membrane proteins in vivo. AtOEP7 was expressed transiently in protoplasts or stably in transgenic plants as fusion proteins with GFP. In both cases AtOEP7:GFP was targeted to the outer envelope membrane when assayed under a fluorescent microscope or by Western blot analysis. Except the transmembrane domain, deletions of the N- or C-terminal regions of AtOEP7 did not affect targeting although a region closed to the C-terminal side of the transmembrane domain affected the targeting efficiency. Targeting experiments with various hybrid transmembrane mutants revealed that the amino acid sequence of the transmembrane domain determines the targeting specificity The targeting mechanism was further studied using a fusion protein, AtOEP7：NLS：GFP, that had a nuclear localization signal. AtOEP7：NLS：GFP was efficiently targeted to the chloroplast envelope despite the presence of the nuclear localization signal. Taken together, these results suggest that the transmembrane domain of AtOEP7 functions as the sole determinant of targeting specificity and that AtOEP7 may be associated with a cytosolic component during translocation to the chloroplast envelope membrane.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• Food Science of Animal Resources
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• v.33 no.2
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• pp.281-286
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• 2013

Salmonella Gallinarum (SG) is known as an important pathogen that causes fowl typhoid in chickens. To investigate SG outer-membrane proteins (OMPs) as a vaccine candidate, we used proteomic mapping and database analysis techniques with extracted OMPs. Also, extracted OMPs were evaluated in several aspects to their safety, immune response in their host and protective effects. Our research has established a proteomic map and database of immunogenic SG-OMPs used as inactive vaccine against salmonellosis in chickens. A total of 22 spots were detected by 2-dimensional gel electrophoresis and immunogenic protein analysis. Eight spots were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprinting (PMF) and categorized into four different types of proteins. Among these proteins, OmpA is considered to be an immunogenic protein and involved in the hosts' immune system. To estimate the minimum safety dose in chickens, 35 brown layers were immunized with various concentrations of OMPs, respectively. Consequently, all chickens immunized with more than a $50{\mu}g$ dose were protected against challenges. Moreover, intramuscular administration of OMPs to chickens was more effective compared to subcutaneous administration. These results suggest that the adjuvanted SG-OMP vaccine not only induces both the humoral and cellular immune response in the host but also highly protects the hosts' exposed to virulent SG with $50{\mu}g$ OMPs extracted by our method.

• Journal of fish pathology
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• v.12 no.2
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• pp.89-99
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• 1999

New sixteen heat shock proteins (Hsps) and ten ethanol shock proteins were appeared on the analysis with SDS-PAGE when cultivation temperature for the Vibrio vulnifrcus ATCC 27562 strain was shifted-up to $42^{\circ}C$ from $30^{\circ}C$ for 20 mins and treated with of 6% ethanol for 10 mins, respectively. Even the induction of thermotolerance in V. vulnificus was coincided with the induction of Hsps if the pre-shock was adjusted to thermal temperature. Outer membrane proteins (OMPs) that were purified from the membrane of cells after heat shock showed more immunodominant pattern to the immunized rabbit anti-V. vulnificus O serum in enzyme-linked immunosorbent assay (ELISA). On the western immunoblot analysis it was confirmed that both 62 kDa IMP and 69 kDa OMP in the Hsps and 48 kDa IMP a major OMP in the ethanol shock proteins were reacted with rabbit anti-V. vulnificus O sera. Agglutination titer of the heat shocked V. vulnificus with rabbit anti-V. vulnificus O serum was higher than that of the untreated bacteria.

• Journal of Photoscience
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• v.9 no.2
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• pp.47-50
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• 2002

Raft is a distinctive membrane domain enriched in a certain class of lipids, cholesterol, and proteins observed on the plasma membrane. Growing evidence has revealed that such membrane domains play key roles in signal transduction, fertilization, development, transmitter release, and so on. Recently, we have isolated raft-like detergent-resistant membrane (DRM) fraction from bovine photoreceptor rod outer segments. Transducin and its effecter, cGMP-phosphodiesterase, elicited stimulus-dependent translocation between detergent-soluble membrane and DRM. This suggested potential importance of such distinct membrane domains in vertebrate phototransduction. Here, we will discuss physiological meaning of the translocation of major components of cGMP cascade to raft-like membrane in phototransduction. We would like to propose a hypothesis that raft-like membrane domains on the disk membrane are the place where cGMP cascade system could be quenched.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1034-1040
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• 2009

Porin proteins of Gram-negative bacteria are outer membrane proteins that act as receptors for bacteriophages and are involved in a variety of functions like solute transport, pathogenesis, and immunity. Salmonella enterica serovar Typhi (S. Typhi), a Gram-negative bacterium, is the causative agent of typhoid fever. Porins of S. Typhi have been shown to have a potential role in diagnostics and vaccination. In the present study, the major outer membrane proteins OmpF and OmpC from S. Typhi were cloned in pQE30UA vector and expressed in E. coli. The immunogenic nature of the recombinant porin proteins were evaluated by ELISA by raising hyperimmune sera in Swiss Albino mice with three different adjuvants (i.e., Freund's adjuvant and two human-compatible adjuvants like montanide and aluminium hydroxide gel) and proved to be immunogenic. The recombinant OmpF and OmpC generated in this work may be used for further studies for vaccination and diagnostics.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.645-658
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• 2021

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.