title: Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs).
abstract: Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis geneEof bacteriophage PhiX174. In this study,Vibrio parahaemolyticusghosts (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 for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which 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 amoebocytelysate 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 × 10⁶ CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. LikeEscherichia coliLPS, 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 data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. parkhj0524@pcu.ac.kr.
authors.forename: Hyun Jung
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. 5star@pcu.ac.kr.
authors.forename: Sung
authors.initials: S
authors.lastname: Oh
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. biovinz@gmail.com.
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Vinod
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. dongny12@pcu.ac.kr.
authors.forename: Seongmi
authors.initials: S
authors.lastname: Ji
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. creator1018@pcu.ac.kr.
authors.forename: Han Byul
authors.initials: HB
authors.lastname: Noh
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. jungmo9@gmail.com.
authors.forename: Jung Mo
authors.initials: JM
authors.lastname: Koo
authors.affiliation: Cheongshim International Academy, Gapyeong-gun, Gyeonggi-do 12461, Korea. potatoclsrn@naver.com.
authors.forename: Su Hyeong
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. kimsc@pcu.ac.kr.
authors.forename: Sei Chang
authors.initials: SC
authors.lastname: Kim
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. kslee@pcu.ac.kr.
authors.forename: Ki-Sung
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, Korea. choicw@pcu.ac.kr.
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Choi
mesh_terms: D019342:Acetic Acid / Q000494:pharmacology; D000818:Animals; D001888:Boric Acids / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects; D019343:Citric Acid / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism*; D015870:Gene Expression; D006851:Hydrochloric Acid / Q000494:pharmacology; D016753:Interleukin-10 / Q000235:genetics / Q000276:immunology; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D008298:Maleates / Q000494:pharmacology; D051379:Mice; D008826:Microbial Sensitivity Tests; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000276:immunology; D012972:Sodium Hydroxide / Q000494:pharmacology*; D013464:Sulfuric Acids / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology; D014736:Vibrio parahaemolyticus / Q000737:chemistry / Q000187:drug effects / Q000276:immunology
chemical_list: D001888:Boric Acids; D004269:DNA, Bacterial; C556062:IL10 protein, mouse; C587672:IL1B protein, mouse; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008298:Maleates; D013464:Sulfuric Acids; D014409:Tumor Necrosis Factor-alpha; C577686:interleukin-6, mouse; D016753:Interleukin-10; D019343:Citric Acid; D012972:Sodium Hydroxide; C030272:maleic acid; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C033158:sulfuric acid; D019342:Acetic Acid; D006851:Hydrochloric Acid; C032688:boric acid
keywords: Vibrio parahaemolyticus; bacterial ghosts (BGs); chemically-induced lysis; cytokine; cytotoxicity; endotoxic activity; lipopolysaccharides (LPS); macrophages; minimum inhibition concentration (MIC); sodium hydroxide (NaOH)