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.

中文翻译：

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p75（鼠李糖乳杆菌GG衍生蛋白）在枯草芽孢杆菌孢子上的表面展示及其对单核细胞增生李斯特菌的抗菌活性。

具有肽聚糖水解酶（PGH）活性的鼠李糖乳杆菌p75蛋白是对人肠上皮细胞具有抗凋亡和细胞保护活性的关键分子之一。在这项研究中，以开发新的益生菌为目标，使用孢子外壳蛋白CotG作为锚定基序，在枯草芽孢杆菌孢子表面展示了p75蛋白。还研究了孢子展示的p75（CotG-p75）蛋白的PGH活性，稳定性和抗菌活性。CotG-p75对枯草芽孢杆菌提取的肽聚糖的PGH活性通过茚三酮测试证实。与对照组相比，在各种恶劣条件下，CotG-p75的PGH活性在pH 3-7范围内非常稳定，并在50°C时保持70％。另外，通过时间杀灭试验评价了CotG-p75对单核细胞增生性李斯特菌的抗菌活性。在磷酸盐缓冲液中孵育6小时后，CotG-p75将单核细胞增生李斯特氏菌的活细胞数量减少了2.0 log。扫描电子显微镜分析显示单核细胞增生李斯特氏菌的细胞壁被CotG-p75处理部分破坏。我们的初步结果表明，CotG-p75可以作为进一步研究开发新的基因工程益生菌的良好候选者。