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 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 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 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 × 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. 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 data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges

“…STG were produced by using the MIC of NaOH as described previously [ 17 – 19 ]. Briefly, the MIC of NaOH against S .…”

“…However, there are some disadvantages of the method such as limitation to Gram-negative bacteria only, potential risks because of difficulty to reach 100% lysis rate of BG strain in a short time [ 22 ], and cost expensive and time consuming, multi-step process. Alternatively, BG produced by the latter method has no limitation to both Gram-positive [ 18 ] and Gram-negative bacteria [ 17 , 19 ]. This method needs a short time to generate BG without any potential risks, cheap cost and time-saving, simple process.…”

“…This method needs a short time to generate BG without any potential risks, cheap cost and time-saving, simple process. Despite some surface structures in BG may be modified or lost by NaOH [ 19 ], the NaOH-induced BG provides efficient protection against specific infections [ 17 , 18 ].…”

Protein and DNA Isolation from Aspergillus Niger as well as Ghost Cells Formation

“…Its main concept is using active chemical compounds that could introduce pores in the microbes and degrade the DNA at concentrations which did not change the surface antigens or the 3D structure [13]. This enables evacuating gram-negative and gram-positive bacteria, eukaryotes and viruses [4,[12][13][14][16][17][18][19][20][21][22][23][24][25][26][27].…”

Preparation and Evaluation of Chemically Inactivated Salmonella Enteritidis Vaccine in Chickens

“…Electrophoresis was performed on PCR product of SEG with modification using electrophoresis [24]. Grade agarose (1 g) was prepared in 100 mL tris-borate-ethylenediaminetetraacetic acid (TBE) buffer in a sterile flask.…”