Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

鼠李糖乳杆菌GG（Lactobacillus rhamnosus GG, LGG）可分泌两种主要蛋白，本文将其命名为Msp1（LGG_00324 或称p75）与Msp2（LGG_00031 或称p40），已有研究表明二者可促进肠道上皮细胞的存活与生长。有趣的是，尽管这两种蛋白均与细胞壁水解酶（cell wall hydrolases）具有同源性，但与这类酶活性相关的生理功能在LGG中仍有待证实。为探究该菌的相关功能，我们针对上述基因构建了敲除突变体（knock-out mutants），以期建立基因型-表型的对应关联。对msp1敲除突变体的显微镜检查结果显示，菌体形成了过长且过度延展的细胞链，这表明正常的子细胞分离过程受到阻碍。随后通过免疫荧光显微镜（immunofluorescence microscopy）对LGG野生型细胞的观察发现，Msp1蛋白会在指数期细胞的分裂隔膜处富集。酶谱分析（zymogram analysis）证实了Msp1蛋白的细胞壁水解活性。后续通过反相高效液相色谱（RP-HPLC）与质谱（mass spectrometry）对Msp1酶解LGG肽聚糖（peptidoglycan, PG）的产物进行分析，结果表明Msp1蛋白具有D-谷氨酰-L-赖氨酰内切肽酶活性。免疫荧光显微镜观察结果以及无法成功构建msp2敲除突变体的事实，均表明Msp2在LGG的分裂隔膜形成启动过程中发挥着不可或缺的作用。