Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin, Ireland.
PLoS Pathog. 2012 Dec;8(12):e1003092. doi: 10.1371/journal.ppat.1003092. Epub 2012 Dec 27.
Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the "dock, lock and latch" mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB⁺S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor⁻/⁻) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor⁻/⁻ mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor⁻/⁻ mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.
金黄色葡萄球菌无症状地定植于鼻腔前部，但宿主和细菌因素促进定植的机制仍不完全清楚。金黄色葡萄球菌表面蛋白 ClfB 已被证明可介导体外与鳞状上皮细胞的黏附，并促进小鼠和人类的鼻腔定植。在这里，我们证明了鳞状上皮细胞包膜蛋白丝聚蛋白是金黄色葡萄球菌鼻腔定植过程中 ClfB 的主要靶标配体。体外黏附实验表明，表达 ClfB 的细菌很可能通过“对接、锁定和闩锁”机制结合丝聚蛋白。我们使用表面等离子体共振实验表明，ClfB 结合角蛋白 10（K10），即鳞状上皮细胞的一种结构蛋白，以及丝聚蛋白的亲和力相似，均处于低微摩尔范围。丝聚蛋白由三个独立的区域组成，包含富含 GS 的 ω 环。每个环都单独表达，并发现与 ClfB 结合，但区域 2 的结合亲和力最高。为了研究 ClfB 与丝聚蛋白之间的特异性相互作用是否足以促进金黄色葡萄球菌鼻腔定植，我们比较了 ClfB⁺金黄色葡萄球菌定植野生型和丝聚蛋白缺陷（Lor⁻/⁻）小鼠鼻腔的能力。在缺乏丝聚蛋白的情况下，金黄色葡萄球菌鼻腔定植明显受损。此外，ClfB⁻突变株在野生型小鼠中的定植效率明显低于亲本 ClfB⁺株，而在 Lor⁻/⁻小鼠中，无论是亲本株还是 ClfB⁻突变株，其定植水平都相似较低。通过表达 ClfB 的乳球菌能够在 WT 小鼠的鼻腔中保留而不能在 Lor⁻/⁻小鼠的鼻腔中保留，证实了 ClfB 结合丝聚蛋白在体内支持金黄色葡萄球菌鼻腔定植的能力。通过将体外生化分析与动物模型研究相结合，我们确定了鳞状上皮细胞包膜蛋白丝聚蛋白为金黄色葡萄球菌鼻腔定植过程中 ClfB 的靶标配体。
