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

Hyun Park,Seongmi Ji,Nagarajan Vinod,Sei Kim,Sung Oh,Chang Choi,Jung Koo,Su Lee,Ki-Sung Lee,Han Noh

doi:10.3390/ijms17111904

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.

Highlights

Vaccines are very efficient in protecting human and animal hosts from bacterial pathogens
No colony was formed on LB agar plates spread with V. parahaemolyticus bacteria treated with respective chemicals at their minimum inhibition concentration (MIC) (Figure S2)
The data indicated that the different chemicals above did not inhibit the V. parahaemolyticus growth in a pH-dependent manner but they inhibited the bacterial growth in a concentration-dependent manner

Summary

Introduction

Vaccines are very efficient in protecting human and animal hosts from bacterial pathogens Their roles against infections are limited because currently available vaccines are usually serotype- or species-specific [1]. 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]. These functional and antigenic determinants possessed the intrinsic adjuvant properties and induced both humoral and cell-mediated immune responses against virulent challenge in various animal models [8,11]. Vibrio cholrae BGs induced antibodies showing vibriocidal activity and these antibodies provided protection from homologous and heterologous challenges in experimental animals [4,5]

Methods

Results

Conclusion