A Simple and Rapid Method of Probiotic Bacterial Ghost Cell Preparation to Deliver Mycobacterium tuberculosis Antigen

A bacterial ghost cell is an empty cell envelope of bacteria lacking cytoplasmic content. Bacterial ghost cells (BGs) can be used for various applications such as vaccines, adjuvants, and drug delivery systems. Since BGs offer many advantages over classically prepared vaccines, developing novel methods for the preparation of high-quality BGs remains to be an interesting field of study by various research groups. Several novel methodologies have been reported that involve the biological (gene E mediated) and combination of various chemicals such as NaOH, SDS, H₂O₂, CaCO₃, and ethanol, non-detergent method using Tween80, limulus antimicrobial peptide, and high hydrostatic pressure method, the porcine myeloid antimicrobial peptide (PMPA) 36-lysozyme fusion method, NaOH-Penicillin/Streptolysin combination method. In this study, we have reported a novel methodology that combines the action of chemical and physical factors to produce ghost cells from gram-negative bacteria, the probiotic E.coli Nissle 1917. The mild detergent Triton X-100 and NaCl alter the permeability of the cell membrane which is further amplified by heat shock induction. This enables the cell to expel its cytoplasmic components without affecting the external morphology. The efficiency of this method was analyzed based on viability assay, cell leakage assay, live-dead cell assay, and scanning electron microscopic analysis. Moreover, the protein loading capacity was optimized for Mycobacterium tuberculosis antigen namely, ESAT-6.