Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Appl Environ Microbiol. 2019 May 2;85(10). doi: 10.1128/AEM.02335-18. Print 2019 May 15.
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 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. 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. 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 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 .
由于具有既定的安全性、促进健康的特性和可用的基因组编辑工具，因此有可能将其开发为微生物治疗性递药平台。在这里，我们表明与其他革兰氏阳性菌相比，VPL1014 的突变率较低，我们预计这将有助于稳定遗传修饰菌株。VPL1014 编码两个具有生物活性的原噬菌体，这些原噬菌体在胃肠道转运过程中被诱导。我们假设，在噬菌体介导的裂解后，细胞内积累的重组蛋白可以被释放出来。为了验证这一点，我们设计了 VPL1014 来在细胞内积累我们的模型蛋白瘦素。原噬菌体诱导重组 VPL1014 将瘦素释放到细胞外环境中，这与噬菌体的产生相对应。我们还采用了一种质粒系统，该系统在生长培养基中不需要抗生素来维持质粒。总的来说，这些数据为利用天然原噬菌体来递送治疗分子提供了新途径。在过去的 20 年中，乳酸菌 (LAB) 一直被探索作为潜在的生物治疗载体。为了将治疗剂分泌到细胞外环境中，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途径过载，并影响生物体的适应性，还需要对信号肽进行优化，以最大限度地提高成熟蛋白的释放效率。在这里，我们描述了一种以前未被探索的从益生菌中释放治疗剂的方法。我们证明，在原噬菌体激活后，细胞内积累的重组蛋白被释放出来。由于我们最近证明原噬菌体在胃肠道转运过程中被激活，因此我们提出这种方法将提供一种简单有效的方法来递送治疗剂。
