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Background 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. However, the efficient production of BGs from bacteria in a short period of time is challenging. Objective The 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. Results In 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 (H 2 SO 4 ; 3.125 mg/mL), and nitric acid (HNO 3 ; 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 × 10 5  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. Conclusion These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants.

Hydrochloric acid-treated Bacillus subtilis ghosts induce IL-1 beta, IL-6, and TNF-alpha in murine macrophage