Viral infection modulation and neutralization by camelid nanobodies
Viral infection modulation and neutralization by camelid nanobodies
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DOI：
10.1073/pnas.1301336110
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发表时间：
2013-04-09
影响因子：
11.1
通讯作者：
Cambillau, Christian
中科院分区：
文献类型：
--
作者：
Desmyter, Aline;Farenc, Carine;Cambillau, Christian
乳球菌噬菌体属于Siphoviridae的一个大家族，感染乳酸乳球菌，一种用于商业乳制品发酵的革兰氏阳性细菌。这些噬菌体被认为能够识别并特异性结合覆盖整个细菌的细胞膜多糖。位于尾部尖端的噬菌体TP901-1基板含有18种三聚体受体结合蛋白（rbp），可促进与特定乳球菌菌株的粘附。与其他乳球菌噬菌体相比，TP901-1噬菌体的粘附不需要主要的构象改变或Ca2+。在这里，我们制备并表征了针对纯化的噬菌体TP901-1的基板和Tal蛋白的骆驼纳米体，作为分析噬菌体TP901-1感染的分子决定因素的工具。使用一组互补技术，表面等离子体共振，EM和x射线晶体学混合方法，我们鉴定了基板的三个组成部分的结合物，分析了它们对目标的亲和力，确定了它们的表位以及它们对TP901-1噬菌体感染性的功能影响。我们测定了三种纳米体与RBP复合物的x射线结构。其中两种与RBP的糖结合位点结合，即使经过15次传代也能完全中和TP901-1噬菌体的感染性。这些结果为纳米体在乳制品发酵中规避乳球菌噬菌体病毒感染的实际应用提供了明确的证据。
Lactococcal phages belong to a large family of Siphoviridae and infect Lactococcus lactis, a Gram-positive bacterium used in commercial dairy fermentations. These phages are believed to recognize and bind specifically to pellicle polysaccharides covering the entire bacterium. The phage TP901-1 baseplate, located at the tip of the tail, harbors 18 trimeric receptor binding proteins (RBPs) promoting adhesion to a specific lactococcal strain. Phage TP901-1 adhesion does not require major conformational changes or Ca2+, which contrasts other lactococcal phages. Here, we produced and characterized llama nanobodies raised against the purified baseplate and the Tal protein of phage TP901-1 as tools to dissect the molecular determinants of phage TP901-1 infection. Using a set of complementary techniques, surface plasmon resonance, EM, and X-ray crystallography in a hybrid approach, we identified binders to the three components of the baseplate, analyzed their affinity for their targets, and determined their epitopes as well as their functional impact on TP901-1 phage infectivity. We determined the X-ray structures of three nanobodies in complex with the RBP. Two of them bind to the saccharide binding site of the RBP and are able to fully neutralize TP901-1 phage infectivity, even after 15 passages,. These results provide clear evidence for a practical use of nanobodies in circumventing lactococcal phages viral infection in dairy fermentation.