Surface display of p75, a Lactobacillus rhamnosusGG derived protein, onBacillus subtilisspores and its antibacterial activity againstListeria monocytogenes

Lactobacillus rhamnosusp75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface ofBacillus subtilisspores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted fromB. subtiliswas confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3-7 and maintained at 70% at 50 degrees C. In addition, the antibacterial activity of CotG-p75 againstListeria monocytogeneswas evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells ofL. monocytogenesby up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall ofL. monocytogeneswas partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics.