Construction and characterization of cortex-stripped Clostridium perfringens spores

The complex multilayered structure of bacterial spores poses a challenge to investigating the mechanisms governing inner membrane responses. However, when the extracellular wall, spore coat, and cortex are effectively removed, revealing the vesicles of the spore inner membrane, it would provide a more direct insight into the changes in the inner membrane. To achieve complete exposure of the intima, the effectiveness of four methods for removing the cell wall, spore coat, and cortex was evaluated. The treatment with alkaline protein denaturant (SDS-DTT) and lysozyme maintained membrane integrity, with 1.2 log ±0.23 CFU/mL plate counts, while other methods caused membrane damage. The morphological analysis revealed a reduction in the spore's long and short axis from 1.77 ± 0.17 μm to 1.42 ± 0.15 μm, and from 1.12 ± 0.04 μm to 0.83 ± 0.08 μm, respectively, while the inner membrane remained intact. Germination efficiency increased from 0.274 to 0.338, and DPA release increased from 1.46 to 1.59, with both properties closely resembling the original spores (>95 % similarity). However, heat resistance decreased by 20 %. The combined treatment preserved the biological activity of cortex-free spores, showing similar changes to those in the original spores, confirming that the cortex mainly enhances heat resistance. Removing the cortex allowed more direct observation of inner membrane dynamics and offers a valuable tool for studying spore biology. This approach provides a foundation for future research into spore regulation and inner membrane functions.