Abstract
Lactobacillus rhamnosus p75 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 of Bacillus subtilis spores 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 from B. subtilis was 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 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was 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.
Highlights
Lactobacillus rhamnosus GG (LGG) is one of the most widely studied probiotic strains and has been reported to be effective in treating and preventing ulcerative colitis, diarrhea, and atopic dermatitis in numerous clinical studies (Zocco et al 2006; Szajewska et al 2011; Doron et al 2005). p75, known as major secreted protein 1, is one of the soluble proteins derived from LGG and is a key molecule that shows probiotic features. p75 regulates the proliferation and survival of intestinal epithelial cells through stimulation of Akt activation, inhibition of probiotics have numerous health benefits including treating and preventing intestinal disease, they have limitations
Peptidoglycan hydrolase activity of CotG‐p75 A ninhydrin test was performed to verify that the CotG-p75 displayed on the spore surface maintained its intrinsic peptidoglycan hydrolase (PGH) activity
PG was treated with different concentrations (1.2, 1.8, 2.4, 3.0, and 3.6 × 105 colony forming units (CFU)/ mL) of CotG-p75 and the degree of PG degradation was measured by the ninhydrin test
Summary
Lactobacillus rhamnosus GG (LGG) is one of the most widely studied probiotic strains and has been reported to be effective in treating and preventing ulcerative colitis, diarrhea, and atopic dermatitis in numerous clinical studies (Zocco et al 2006; Szajewska et al 2011; Doron et al 2005). p75, known as major secreted protein 1, is one of the soluble proteins derived from LGG and is a key molecule that shows probiotic features. p75 regulates the proliferation and survival of intestinal epithelial cells through stimulation of Akt activation, inhibition of probiotics have numerous health benefits including treating and preventing intestinal disease, they have limitations. Lactobacillus rhamnosus GG (LGG) is one of the most widely studied probiotic strains and has been reported to be effective in treating and preventing ulcerative colitis, diarrhea, and atopic dermatitis in numerous clinical studies (Zocco et al 2006; Szajewska et al 2011; Doron et al 2005). Since probiotics are living microorganisms, they tend to have low viability during food processing and passage through the gastrointestinal tract (MattilaSandholm et al 2002). To overcome these limitations, there have been many efforts to develop new probiotics through bioengineering. Probiotic strains and E. coli were engineered to produce antimicrobial peptides or compounds to enhance its antimicrobial actions (Goh et al 2012; Chen et al 2010)
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