Department of Veterinary Biosciences, Veterinary Microbiology and Epidemiology, P.O. Box 66, FIN-00014 University of Helsinki, Finland.
Appl Environ Microbiol. 2010 Apr;76(7):2049-57. doi: 10.1128/AEM.01958-09. Epub 2010 Jan 29.
Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.
鼠李糖乳杆菌 GG 是一种成熟的革兰氏阳性益生菌株，其有益健康的特性部分依赖于在胃肠道中的长时间停留，并可能取决于对肠道黏膜的黏附。此前，我们在该益生菌细菌的基因组中鉴定了两个菌毛基因簇（spaCBA 和 spaFED），每个基因簇都包含三个菌毛亚基和一个单一的分选酶的预测基因。我们还证实了 SpaCBA 菌毛存在于细胞表面，并将一个菌毛亚基（SpaC）的肠粘液结合能力归因于它。在这里，我们报告了大肠杆菌中剩余菌毛基因（spaA、spaB、spaD、spaE 和 spaF）的克隆、重组蛋白的生产和纯化，并评估了这些蛋白对人肠粘液的黏附性。我们的研究结果表明，SpaB 和 SpaF 菌毛亚基也表现出对粘液的显著结合，这种结合可以通过竞争方式以剂量相关的方式抑制。此外，SpaB 菌毛亚基与粘膜基质之间的结合似乎通过静电接触起作用，与公认的粘液结合结构域无关。我们从这些结果中得出结论，设想 SpaCBA 菌毛纤维中的两个菌毛亚基（SpaB 和 SpaC）在结合肠粘液中起作用，但对于未表征和推测的 SpaFED 菌毛纤维，只有一个菌毛亚基（SpaF）可能负责与粘液黏附。
