Wojciechowska Monika, Macyszyn Julia, Miszkiewicz Joanna, Grzela Renata, Trylska Joanna
Centre of New Technologies, University of Warsaw, Warsaw, Poland.
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland.
Front Microbiol. 2021 Dec 13;12:772038. doi: 10.3389/fmicb.2021.772038. eCollection 2021.
Anoplin is a linear 10-amino acid amphipathic peptide (Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu- ) derived from the venom sac of the solitary wasp. It has broad antimicrobial activity, including an antibacterial one. However, the inhibition of bacterial growth requires several dozen micromolar concentrations of this peptide. Anoplin is positively charged and directly interacts with anionic biological membranes forming an α-helix that disrupts the lipid bilayer. To improve the bactericidal properties of anoplin by stabilizing its helical structure, we designed and synthesized its analogs with hydrocarbon staples. The staple was introduced at two locations resulting in different charges and amphipathicity of the analogs. Circular dichroism studies showed that all modified anoplins adopted an α-helical conformation, both in the buffer and in the presence of membrane mimics. As the helicity of the stapled anoplins increased, their stability in trypsin solution improved. Using the propidium iodide uptake assay in and , we confirmed the bacterial membrane disruption by the stapled anoplins. Next, we tested the antimicrobial activity of peptides on a range of Gram-negative and Gram-positive bacteria. Finally, we evaluated peptide hemolytic activity on sheep erythrocytes and cytotoxicity on human embryonic kidney 293 cells. All analogs showed higher antimicrobial activity than unmodified anoplin. Depending on the position of the staple, the peptides were more effective either against Gram-negative or Gram-positive bacteria. Anoplin[5-9], with a lower positive charge and increased hydrophobicity, had higher activity against Gram-positive bacteria but also showed hemolytic and destructive effects on eukaryotic cells. Contrary, anoplin[2-6] with a similar charge and amphipathicity as natural anoplin effectively killed Gram-negative bacteria, also pathogenic drug-resistant strains, without being hemolytic and toxic to eukaryotic cells. Our results showed that anoplin charge, amphipathicity, and location of hydrophobic residues affect the peptide destructive activity on the cell wall, and thus, its antibacterial activity. This means that by manipulating the charge and position of the staple in the sequence, one can manipulate the antimicrobial activity.
阿诺普林是一种由独居黄蜂毒囊中提取的线性十肽（甘氨酸 - 亮氨酸 - 亮氨酸 - 赖氨酸 - 精氨酸 - 异亮氨酸 - 赖氨酸 - 苏氨酸 - 亮氨酸 - 亮氨酸）。它具有广泛的抗菌活性，包括抗菌作用。然而，抑制细菌生长需要几十微摩尔浓度的这种肽。阿诺普林带正电荷，可直接与阴离子生物膜相互作用，形成破坏脂质双层的α螺旋结构。为了通过稳定其螺旋结构来提高阿诺普林的杀菌性能，我们设计并合成了带有烃钉的类似物。在两个位置引入了钉，导致类似物具有不同的电荷和两亲性。圆二色性研究表明，所有修饰的阿诺普林在缓冲液中和存在膜模拟物的情况下都采用α螺旋构象。随着带钉阿诺普林螺旋度的增加，它们在胰蛋白酶溶液中的稳定性提高。使用碘化丙啶摄取试验，我们证实了带钉阿诺普林对细菌膜的破坏作用。接下来，我们测试了这些肽对一系列革兰氏阴性菌和革兰氏阳性菌的抗菌活性。最后，我们评估了肽对绵羊红细胞的溶血活性和对人胚肾293细胞的细胞毒性。所有类似物都显示出比未修饰的阿诺普林更高的抗菌活性。根据钉的位置，这些肽对革兰氏阴性菌或革兰氏阳性菌更有效。带较低正电荷和增加疏水性的阿诺普林[5 - 9]对革兰氏阳性菌具有更高的活性，但对真核细胞也表现出溶血和破坏作用。相反，与天然阿诺普林具有相似电荷和两亲性的阿诺普林[2 - 6]有效地杀死了革兰氏阴性菌，包括致病性耐药菌株，而对真核细胞无溶血和毒性。我们的结果表明，阿诺普林的电荷、两亲性和疏水残基的位置会影响肽对细胞壁的破坏活性，进而影响其抗菌活性。这意味着通过操纵序列中钉的电荷和位置，可以操纵抗菌活性。
