Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System
Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System
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从头聚集抗菌肽和基于细菌缀合的递送系统的设计
DOI：
10.1021/acs.biochem.8b00888
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Chatterjee, Anushree
中科院分区：
文献类型：
--
作者：
Collins, Logan T.;Otoupal, Peter B.;Campos, Jocelyn K.;Courtney, Colleen M.;Chatterjee, Anushree
抗生素耐药性需要开发新的感染治疗方法。蛋白质聚集体最近已被用作抗微生物剂以破坏细菌体内平衡。过去关于蛋白质聚集体的工作集中在细菌基因组中易于聚集的序列的基因组挖掘，而不是聚集的抗菌肽的合理设计。在这里，我们使用合成生物学的方法来设计一个人工基因编码从头聚集抗菌肽。这种人工基因，opaL（过度表达的亲脂蛋白聚集剂），通过表达极端疏水的肽破坏细菌的体内平衡。当这种疏水序列被酸性残基破坏时，随之而来的聚集和抗微生物效果降低。此外，我们开发了一种益生菌递送系统，使用广泛宿主范围的接合质粒RK2将基因从供体转移到受体细菌。我们利用RK2通过添加RK2转移起点来移动携带OpaL的穿梭质粒。我们表明，opaL是无毒的捐助者，允许维护和转移，因为它的表达是在一个启动子的控制下，与有害的特异性T7 RNA聚合酶。在供体和宿主大肠杆菌交配后，我们观察到T7聚合酶表达受体的选择性生长抑制。该技术可用于通过选择病原体特异性启动子来控制T7 RNA聚合酶表达来靶向所需病原体，并为聚集性抗菌肽的设计和递送提供了基础。
Antibacterial resistance necessitates the development of novel treatment methods for infections. Protein aggregates have recently been applied as antimicrobials to disrupt bacterial homeostasis. Past work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding a de novo aggregating antimicrobial peptide. This artificial gene,opaL(overexpressed protein aggregator lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decrease. Further, we developed a probiotic delivery system using the broad-host range conjugative plasmid RK2 to transfer the gene from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carryingopaLby adding the RK2 origin of transfer. We show thatopaLis nontoxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipientEscherichia coli, we observe selective growth repression in T7 polymerase-expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control T7 RNA polymerase expression and provides a basis for the design and delivery of aggregating antimicrobial peptides.