Abstract
BackgroundBacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. However, the efficient production of BGs from bacteria in a short period of time is challenging.ObjectiveThe purpose of this study was to investigate the possibility of producing BGs in the Gram-positive Bacillus subtilis using various chemicals, and the potential application of BGs as a novel immunomodulatory agent.ResultsIn this study, Bacillus subtilis ghosts (BSGs) were generated, for the first time to the best of our knowledge, using the minimum inhibitory concentration (MIC) of hydrochloric acid (HCl; 6.25 mg/mL), sulfuric acid (H2SO4; 3.125 mg/mL), and nitric acid (HNO3; 6.25 mg/mL). Among the BSGs generated using these chemicals, HCl-induced BSGs were completely DNA-free as confirmed by real-time polymerase chain reaction. Scanning electron microscopy showed the formation of transmembrane lysis tunnel structures in HCl-induced BSGs. Murine macrophages exposed to the HCl-induced BSGs at a concentration of 1 × 105 CFU/mL showed a cell viability of 97.8%. Additionally, HCl-induced BSGs upregulated the expression of pro-inflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor alpha, and IL-6. Furthermore, we found differences in the protein expression profiles between intact live bacteria and BSGs using two-dimensional electrophoresis coupled with peptide mass fingerprinting/matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis.ConclusionThese data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants.
Highlights
Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced via controlled expression of the cloned lysis gene E of bacteriophage phi174 (Hajam et al 2015) that forms transmembrane lysis tunnel structures on bacterial cell surfaces (Eko et al 2000)
We investigated the cytotoxic effect of hydrochloric acid (HCl)-induced Bacillus subtilis ghosts (BSGs) on murine macrophage RAW 264.7 cells and their immunomodulatory activities based on the mRNA expression of pro-inflammatory cytokines
We found that B. subtilis did not form any colonies on LB agar plates on treatment with the chemicals at their corresponding minimum inhibitory concentration (MIC) (Fig. 1B), indicating that the MIC of the BGs was determined based on cell death within 60 min of exposure to each chemical
Summary
Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced via controlled expression of the cloned lysis gene E of bacteriophage phi174 (Hajam et al 2015) that forms transmembrane lysis tunnel structures on bacterial cell surfaces (Eko et al 2000). The use of whole cells of killed bacteria as a potential vaccine may result in the introduction of antibiotic resistance genes or pathogenic islands into host microbes (Frosch and Meyer 1992). These properties make BGs an attractive tool for vaccine development and antigen delivery systems in both humans and animals (Hajam et al 2017). Bacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. Conclusion These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants
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