Exploiting Prophage-Mediated Lysis for Biotherapeutic Release by Lactobacillus reuteri
Exploiting Prophage-Mediated Lysis for Biotherapeutic Release by Lactobacillus reuteri
复制标题
罗伊氏乳杆菌利用噬菌体介导的裂解作用释放双歧杆菌
DOI：
10.1128/aem.02335-18
复制
发表时间：
2019-05-01
影响因子：
4.4
通讯作者：
van Pijkeren, Jan-Peter
中科院分区：
文献类型：
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作者：
Alexander, Laura M.;Oh, Jee-Hwan;van Pijkeren, Jan-Peter
罗伊氏乳杆菌具有开发作为微生物治疗递送平台的潜力，因为它具有既定的安全性、促进健康的特性和可用的基因组编辑工具。在这里，我们发现与其他革兰氏阳性菌相比，罗伊氏乳杆菌VPL1014表现出较低的突变率，我们期望这将有助于转基因菌株的稳定性。VPL1014编码两种具有生物活性的前噬菌体，它们在胃肠道转运过程中被诱导。我们假设细胞内积累的重组蛋白可以在噬菌体介导的裂解后释放。为了验证这一点，我们设计了VPL1014，让它在细胞内积累瘦素，也就是我们的模型蛋白。在体外噬菌体诱导下，重组VPL1014将瘦素释放到细胞外环境中，这与噬菌体的产生相对应。我们还采用了一种不需要在生长培养基中使用抗生素的质粒系统来维持质粒。总的来说，这些数据为利用天然噬菌体递送治疗分子提供了新的途径。乳酸菌（LAB）作为潜在的生物治疗载体已被探索了近20年。为了在细胞外环境中分泌治疗药物，通常依赖于细菌分泌途径，即Sec途径。分泌蛋白的过表达会使分泌通路过载，影响生物体的适应度，同时还需要对信号肽进行优化，使成熟蛋白的释放效率最大化。在这里，我们描述了一种以前未开发的方法，从益生菌罗伊氏乳杆菌释放治疗。我们证明了细胞内积累的重组蛋白在噬菌体激活后被释放。由于我们最近证明了噬菌体在胃肠道转运过程中被激活，我们提出这种方法将提供一种直接有效的方法来在体内输送治疗药物。
Lactobacillus reuteri has the potential to be developed as a microbial therapeutic delivery platform because of an established safety profile, health-promoting properties, and available genome editing tools. Here, we show that L. reuteri VPL1014 exhibits a low mutation rate compared to other Gram-positive bacteria, which we expect will contribute to the stability of genetically modified strains. VPL1014 encodes two biologically active prophages, which are induced during gastrointestinal transit. We hypothesized that intracellularly accumulated recombinant protein can be released following bacteriophage-mediated lysis. To test this, we engineered VPL1014 to accumulate leptin, our model protein, inside the cell. In vitro prophage induction of recombinant VPL1014 released leptin into the extracellular milieu, which corresponded to bacteriophage production. We also employed a plasmid system that does not require antibiotic in the growth medium for plasmid maintenance. Collectively, these data provide new avenues to exploit native prophages to deliver therapeutic molecules.IMPORTANCE Lactic acid bacteria (LAB) have been explored as potential biotherapeutic vehicles for the past 20 years. To secrete a therapeutic in the extracellular milieu, one typically relies on the bacterial secretion pathway, i.e., the Sec pathway. Overexpression of a secreted protein can overload the secretory pathway and impact the organism's fitness, and optimization of the signal peptide is also required to maximize the efficiency of the release of mature protein. Here, we describe a previously unexplored approach to release therapeutics from the probiotic Lactobacillus reuteri. We demonstrate that an intracellularly accumulated recombinant protein is released following prophage activation. Since we recently demonstrated that prophages are activated during gastrointestinal transit, we propose that this method will provide a straightforward and efficient approach to deliver therapeutics in vivo.