Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells
Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells
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DOI：
10.1128/aem.02402-06
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Loessner, Martin J.
中科院分区：
文献类型：
--
作者：
Kretzer, Jan W.;Lehmann, Rainer;Loessner, Martin J.
用于特定富集微生物细胞（例如病原体单核细胞增生李斯特氏菌）的固定和磁力分离取决于合适的亲和分子的可用性。我们在此报告了一种通过用噬菌体编码的肽聚糖水解酶（内溶素）的细胞壁结合域（CBD）替换抗体来固定和分离细菌细胞的新概念。这些多肽模块非常特异性地识别并以高亲和力结合革兰氏阳性细胞壁上的配体。使用涂有重组李斯特菌噬菌体内溶素衍生的 CBD 分子的顺磁珠，可以在 20 至 40 分钟内固定并从稀释的悬浮液中固定超过 90% 的活单核细胞增生李斯特菌细胞。不同种类和李斯特菌血清型的回收率相似，并且不受其他微生物存在的影响。对基于 CBD 的磁分离 (CBD-MS) 程序进行了评估，以捕获和检测人工和自然污染的食品样品中的单核细胞增生李斯特菌。 CBD 分离方法被证明优于既定的标准程序；它需要的时间更短（48 小时与 96 小时相比）并且是更灵敏的方法。此外，通过使用分别特异性识别蜡状芽孢杆菌和产气荚膜梭菌细胞的特定噬菌体编码的 CBD，证明了 CBD-MS 方法的普遍性。总而言之，CBD 多肽代表了结合和捕获细菌细胞的新颖和创新工具，在微生物学和诊断学方面具有许多可能的应用。
Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wallbinding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.