Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.
Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.
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DOI：
10.1371/journal.pone.0031588
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lebeer S
中科院分区：
文献类型：
--
作者：
Claes IJ;Schoofs G;Regulski K;Courtin P;Chapot-Chartier MP;Rolain T;Hols P;von Ossowski I;Reunanen J;de Vos WM;Palva A;Vanderleyden J;De Keersmaecker SC;Lebeer S
鼠李糖乳杆菌GG （Lactobacillus rhamnosus GG， LGG）产生两种主要的分泌蛋白，分别为Msp1 （LGG_00324或p75）和Msp2 (LGG_00031或p40)，据报道这两种蛋白可促进肠上皮细胞的存活和生长。有趣的是，尽管这些蛋白都与细胞壁水解酶具有同源性，但与这种酶活性相关的生理功能在LGG中仍有待证实。为了研究细菌的功能，我们构建了相应基因的敲除突变体，旨在建立基因型与表型的关系。显微镜检查显示msp1突变体存在相当长的和过度延伸的细胞链，这表明正常的子细胞分离受到阻碍。随后用免疫荧光显微镜观察LGG野生型细胞，发现Msp1蛋白聚集在指数期细胞的隔膜上。酶谱分析证实了Msp1蛋白的细胞壁水解活性。Msp1对LGG肽聚糖（PG）酶切产物的RP-HPLC和质谱分析表明，Msp1蛋白具有d -谷氨酰基-l -赖氨酸内肽酶活性。免疫荧光显微镜和构建敲除突变体的失败表明，Msp2在启动LGG中隔形成中起着不可或缺的作用。
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.
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DOI：
10.1073/pnas.0908876106
发表时间：
2009-10-06
影响因子：
11.1
作者：
Kankainen, Matti;Paulin, Lars;de Vos, Willem M.
通讯作者：
de Vos, Willem M.
影响因子：
1.2
作者：
Baeuerl, Christine;Perez-Martinez, Gaspar;Monedero, Vicente
通讯作者：
Monedero, Vicente
影响因子：
3.2
作者：
Lebeer, Sarah;De Keersmaecker, Sigrid C. J.;Vanderleyden, Jos
通讯作者：
Vanderleyden, Jos
影响因子：
4.4
作者：
Prado Acosta, Mariano;Mercedes Palomino, Maria;Ruzal, Sandra M.
通讯作者：
Ruzal, Sandra M.
影响因子：
4.8
作者：
Yan, F;Polk, DB
通讯作者：
Polk, DB