Generation of Edwardsiella tarda ghosts by bacteriophage PhiX174 lysis gene E

Kwon, Se Ryun; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Dong Soo; Kim, Ki Hong

doi:10.1016/j.aquaculture.2005.02.052

Cited by 45 publications

(38 citation statements)

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“…To examine whether the expression of mmAQP genes is induced by inflammatory stimuli and immune challenge, lipopolysaccharide (LPS; Escherichia coli 0111:B4) or polyinosinic: polycytidylic acid [poly(I:C)] injection and bacterial challenge (E. tarda; Gram negative) (Kwon et al 2005) were performed in vivo. First, LPS (Sigma-Aldrich, St Louis, MO, USA; 5 μg/g body weight [BW]) or poly(I:C) (Sigma-Aldrich, 25 μg/g BW) was injected intraperitoneally into fish individuals (10.5 ± 2.1 g; n = 8 for each group).…”

Section: Immune Challengesmentioning

confidence: 99%

Characterization and expression profiles of aquaporins (AQPs) 1a and 3a in mud loach Misgurnus mizolepis after experimental challenges

2017

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Two distinct cDNAs encoding aquaporins (mmAQPs 1a and 3a) were isolated and characterized from mud loach Misgurnus mizolepis. The identified mud loach AQP cDNAs encode for polypeptides of 260 and 302 amino acids. Topology predictions confirmed six putative membrane-spanning domains connected by five loops and the Nand C-terminal domains being cytoplasmic. The mud loach AQPs 1a and 3a showed broad distribution in multiple tissues including immune-responsive tissues as well as osmoregulatory tissues. Hence, the diversity of AQP distribution and expression possibly indicated its differential functions in the regulation of fluid movement in response to environmental stimuli. The transcription of mmAQP genes was differentially modulated by immune challenges. In particular, the mmAQP3a expression level in the liver was more responsive to immune challenges than that of mmAQP1a. Taken together, fish stimulation or infection resulted in significant modulation of mud loach AQP genes, suggesting potential functional roles of these proteins in piscine pathophysiological process.

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Section: Immune Challengesmentioning

confidence: 99%

Characterization and expression profiles of aquaporins (AQPs) 1a and 3a in mud loach Misgurnus mizolepis after experimental challenges

2017

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“…Previously, acetic acid, hydrochloric acid and lactic acid inhibited Helicobacter pylori growth in a pH-dependent manner [23], while butyric acid inhibited the bacterial growth in a pH-independent manner [24]. In this study, the MICs of different chemicals changed culture medium pH from pH 7.0 to 9.99 (NaOH), 4.28 (acetic acid), 3.11 (citric acid), 3.95 (hydrochloric acid), 3.56 (maleic acid) and 5.83 (sulfuric acid). The data indicate that different chemicals above does not inhibit the V. parahaemolyticus growth in a pH-dependent manner but they inhibit the bacterial growth in a concentration-dependent manner.…”

Section: Effects Of Chemicals On Bacterial Cell Envelopesmentioning

confidence: 78%

“…In the lysis E gene-mediated method, it took almost 8 h to produce non-living BGs with complete lysis [8] and the lysis efficiency was commonly 99.9% [3,25,26]. Thus, production of BGs by the chemical mediated lysis is more simple and rapid process than that by the genetically engineered E gene-mediated lysis method.…”

Section: Effects Of Chemicals On Bacterial Cell Envelopesmentioning

confidence: 99%

“…To consider various potential candidates, bacterial ghosts (BGs) have been proposed as a multivalent vaccine candidate [2]. A typical method to produce BGs is by controlled expression of the cloned lysis gene E of bacteriophage PhiX174 [3], which forms trans-membrane lysis tunnel structures on the bacterial cell surfaces [4][5][6][7]. The resultant BGs are non-living whole cell envelopes lacking cytoplasmic contents but retaining basic bacterial cell surface structures such as lipopolysaccharides (LPS), lipids and peptidoglycan [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced <em>Vibrio parahaemolyticus </em>Ghosts (VPGs)

Park¹,

Oh²,

Vinod³

et al. 2016

Preprint

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Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, < 50.0, 25.0, 6.25, 1.56 and 0.781 mg/ml. Except boric acid, the lysis efficiency was reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs showed completely DNA-free that was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 10 6 CFU/mL showed cell viability of 97.9%, however the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1β and iNOS), anti-inflammatory cytokine (IL-10) and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these results indicated that the NaOH-induced VPGs show the potency of safe, economical and effective inactivated bacterial vaccine candidate.

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“…Recently, we have generated Edwardsiella tarda ghosts (ETG) by gene E mediated lysis (Kwon et al 2005), and have demonstrated significantly higher protection against infection of E. tarda in tilapia and olive flounder immunized with ETG than in fish immunized with formalin-killed E. tarda (Kwon et al 2006(Kwon et al , 2007. In these studies, although the bacteria were inactivated by induction of E gene expression, large-sized genomic DNA or plasmid DNA was detected from the produced ETG, suggesting the presence of nonlysed or partially lysed inactivated cells within the ghost preparation.…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

Generation of safety enhancedEdwardsiella tardaghost vaccine

Lee

Kwon²,

Zenke³

et al. 2008

Dis. Aquat. Org.

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A dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of Edwardsiella tarda ghosts (ETG). The SNA (devoid of secretion signal sequence and the nuclease B amino terminus sequence), fused with the 26 amino acid N-terminal sequence of the λ phage Cro gene, showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of the SNA gene using site-directed mutagenesis. ETG were generated via coexpression of the SNA gene and lysis gene E under the control of each λP R promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria, and reduces production costs through cheap means of induction that use only temperature shifts. KEY WORDS: Edwardsiella tarda · Ghost bacteria · Staphylococcal nuclease A · Dual vector · Safety enhancement Resale or republication not permitted without written consent of the publisherDis Aquat Org 81: [249][250][251][252][253][254] 2008 Katinger et al. 1999, Panthel et al. 2002, Marchart et al. 2003.Recently, we have generated Edwardsiella tarda ghosts (ETG) by gene E mediated lysis (Kwon et al. 2005), and have demonstrated significantly higher protection against infection of E. tarda in tilapia and olive flounder immunized with ETG than in fish immunized with formalin-killed E. tarda (Kwon et al. 2006(Kwon et al. , 2007. In these studies, although the bacteria were inactivated by induction of E gene expression, large-sized genomic DNA or plasmid DNA was detected from the produced ETG, suggesting the presence of nonlysed or partially lysed inactivated cells within the ghost preparation. The presence of genomic DNA with intact pathogenic islands and/or antibiotic resistance genes in the ETG preparation would be problematic to use as a practical vaccine for cultured fish. In this study, we constructed a dual vector expressing both the ghost inducing PhiX174 E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene to minimize the presence of antibiotic resistance genes and genomic DNA with pathogenic islands in the ETG vaccine. We also examined the potential utility of the vector in producing safety-improved ETG. MATERIALS AND METHODSBacterial strains. Edwardsiella tarda FSW910410, isolated in 1991 from moribund olive flounder in a natural outbreak of edwardsiellosis on a commercial farm in Korea (Bang et al. 1992), was used. This strain is used in a commercial edwardsiellosis vaccine in Korea, and was kindly provided by the National Fisheries Research & Development Institute, Korea. For cloning of nuclease gene, Staphylococcus aureus KCCM 11335 was purchased from the Korean Culture Center of Microorganisms.Construc...

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Generation of Edwardsiella tarda ghosts by bacteriophage PhiX174 lysis gene E

Cited by 45 publications

References 19 publications

Characterization and expression profiles of aquaporins (AQPs) 1a and 3a in mud loach Misgurnus mizolepis after experimental challenges

Characterization and expression profiles of aquaporins (AQPs) 1a and 3a in mud loach Misgurnus mizolepis after experimental challenges

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced <em>Vibrio parahaemolyticus </em>Ghosts (VPGs)

Generation of safety enhancedEdwardsiella tardaghost vaccine

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