Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.
Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.
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DOI：
10.1007/s00253-015-6867-8
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发表时间：
2015-10
影响因子：
5
通讯作者：
Nugen SR
中科院分区：
文献类型：
--
作者：
Alcaine SD;Tilton L;Serrano MA;Wang M;Vachet RW;Nugen SR
噬菌体是用于检测细菌病原体的快速、容易靶向和容易产生的分子探针。分子生物学技术使研究人员在噬菌体生物工程方面取得了重大进展，进一步提高了检测的速度和灵敏度。尽管噬菌体具有宿主特异性，但在细菌病原体的多重检测中并没有被有效地利用。我们提出了一种基于主噬菌体证明的方案来实现多路检测。我们的方案包括对噬菌体进行生物工程，使其携带一种特定蛋白水解酶的基因，该基因在感染目标细胞时表达。在裂解时，蛋白水解酶被释放以裂解报告肽，并检测到信号。在这里，我们展示了成功的：i)修饰T7噬菌体以携带TEV蛋白水解酶；ii)在大肠杆菌中表达TEV蛋白水解酶。在我们改进的T7感染后，平均每个噬菌体产生2000个单位的蛋白水解酶；以及iii)使用荧光肽和设计的用于MALDI-TOF MS分析的目标肽，通过TEV酶活性的初步浓缩，在3小时内检测到大肠杆菌。这种原理证明可以翻译成其他噬菌体-蛋白酶-多肽组合，以实现多重细菌检测，并易于在多个平台上采用，如MALDI-TOF MS或实验室中常见的荧光阅读器。
Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host-specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful: i) modification of T7 bacteriophage to carry TEV protease; ii) expression of TEV protease by E. coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and iii) proof-of-principle detection of E. coli in 3 hours after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for MALDI-TOF MS analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection, and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.