INSA, UPS, INP, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Université de Toulouse, Toulouse, France.
Biophys J. 2011 Dec 7;101(11):2843-53. doi: 10.1016/j.bpj.2011.10.034.
In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin-based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K(off)). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K(on)). Variations in the loading rate and contact time enabled us to determine K(on) (3.3 × 10(2) M(-1)·s(-1)) and K(off) (0.46 s(-1)), and the latter was consistent with values given in the literature for sugar-protein interactions.
在这项工作中，我们专注于量化乳酸乳球菌（Lactococcus lactis）与粘蛋白之间的黏附作用，乳酸乳球菌是乳酸菌（LAB）的模型。我们测量了两种乳球菌（IBB477 和 MG1820 作为对照）与猪胃粘蛋白涂层之间的相互作用，并使用原子力显微镜进行了比较。分析回缩力-距离曲线揭示了非特异性和特异性力的差异贡献。对于 IBB477，特异性黏附事件的比例增加（对照为 5%，而 IBB477 为 20%）。用游离猪胃粘蛋白及其 O-聚糖部分进行的阻断试验表明，寡糖在乳球菌-粘蛋白相互作用中起主要作用（但不是唯一作用）。特异性相互作用的分析基于动力学常数。原子力显微镜尖端的加载速率增加会导致相互作用的生物实体之间的力增加，这与动力学解离常数（K(off)）直接相关。增加尖端与样品之间的接触时间可以增加相互作用的概率，这可以与动力学缔合常数（K(on)）相关。加载速率和接触时间的变化使我们能够确定 K(on)（3.3×10(2) M(-1)·s(-1)）和 K(off)（0.46 s(-1)），后者与糖-蛋白相互作用的文献值一致。
