Division of General Microbiology, Department of Biosciences, University of Helsinki, Helsinki, Finland.
J Bacteriol. 2012 May;194(10):2509-19. doi: 10.1128/JB.06704-11. Epub 2012 Mar 2.
Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.
谷氨酸合酶（GS）和葡萄糖-6-磷酸异构酶（GPI）被鉴定为卷曲乳杆菌 ST1 的新型黏附性月光蛋白。这两种蛋白都在酸性 pH 值下结合在细菌表面，而将细胞悬浮液的 pH 值调至 8 会导致它们释放到缓冲液中，这与先前观察到的卷曲乳杆菌表面结合的烯醇酶和甘油醛-3-磷酸脱氢酶（GAPDH）的模式相同。这种 pH 变化与细胞壁通透性的快速和短暂增加有关，这可以通过碘化丙啶对细胞进行染色来测量。在用抗菌阳离子肽 LL-37 处理 L. crispatus ST1 细胞后，也观察到这四种月光蛋白的释放逐渐增加，LL-37 通过破坏膜完整性杀死细菌，并且观察到它增加了 L. crispatus ST1 的细胞壁通透性。在 pH 4 时，融合蛋白 His(6)-GS、His(6)-GPI、His(6)-烯醇酶和 His(6)-GAPDH 显示出局部结合到 L. crispatus ST1 细胞的细胞分裂隔膜和极，而没有检测到对 Lactobacillus rhamnosus GG 的结合。ST1 株表现出对基底膜制剂 Matrigel 的 pH 依赖性黏附。纯化的 His(6)-GS 和 His(6)-GPI 蛋白结合到 I 型胶原蛋白上，His(6)-GS 还结合到层粘连蛋白上，并且在 pH 5.5 时的结合水平高于 pH 6.5。His(6)-GS 还表达了纤溶酶原受体功能。结果表明，月光蛋白在乳杆菌中的菌株依赖性表面关联，并且这些蛋白在细胞创伤后、碱性应激条件下或在人细胞产生的抗菌肽 LL-37 存在下从 L. crispatus 表面释放。
