Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota.
Biotechnol Bioeng. 2019 Oct;116(10):2439-2450. doi: 10.1002/bit.27092. Epub 2019 Jul 21.
Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin ( ) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1-16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p < .05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2-10-fold (p < .05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1-16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.
富含脯氨酸的抗菌肽 (PrAMPs) 通过非溶细胞机制杀死细菌，该机制包括它们穿透外膜、利用蛋白介导的方式穿过内膜运输，并靶向核糖体以抑制蛋白质合成。我们之前报道过，用一对阳离子残基替换 oncocin ( ) 可提高抗菌活性。在这项研究中，我们应用该设计方案对另外三种 PrAMPs（apidaecin-1b、pyrrhocoricin 和 bactenecin 7(1-16)）进行了研究，发现 apidaecin-1b 的取代（R4K 和 I8K/R）将活性提高了两倍（p <.05），对非致病性大肠杆菌有效。此外，pyrrhocoricin 的取代（L7K/R 和 R14K）将活性提高了 2-10 倍（p <.05），对一些大肠杆菌和鼠伤寒沙门氏菌菌株有效。我们还对内膜蛋白（SbmA 或 YgdD）敲除菌株进行了活性测试。结果与之前的研究一致，SbmA 是 apidaecin-1b 和 pyrrhocoricin 衍生物的主要转运蛋白。然而，bactenecin 7(1-16) 独立于这些转运蛋白发挥作用。此外，一些 apidaecin-1b 衍生物在没有 SbmA 的情况下相对于野生型表现出增强的活性，这与增强跨内膜转运的突变一致。基于高效液相色谱的细胞结合和内化动力学测定表明，所选的阳离子突变可以提高最小培养基中的细胞结合，但这种增强的结合对于提高活性并非必需，这表明内膜转运的重要性。这些 PrAMPs 阳离子突变体的功能研究增进了对效力和机制的理解，并推进了工程化改良抗菌剂的能力，这从鉴定出对致病性大肠杆菌具有 10 倍增强效力的 pyrrhocoricin 突变体（L7R 和 R14K）中得到了证明。
