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

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 (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.

Conclusion: These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants.

Supplementary information: The online version contains supplementary material available at 10.1007/s13273-022-00221-5.