Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called D-CONGA, which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational D-CONGA variants, we tested multiple sequence-structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, D-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, D-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of K. pneumoniae, innate resistance to D-CONGA was surprisingly common despite a lack of inducible resistance in P. aeruginosa reported previously. Yet, these same isolates were susceptible to D-CONGA-Q7. D-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, D-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.

中文翻译：

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优化与宿主细胞相容的抗菌肽，有效对抗生物膜和耐药细菌的临床分离株

在这里，我们描述了宿主兼容的抗菌肽 (AMP) 谱系的持续合成分子进化，该谱系用于治疗感染了耐药性生物膜形成细菌的伤口。测试的肽是一种称为 D-CONGA 的进化 AMP 的变体，它在体外和体内都具有出色的抗菌活性. 在最新一代的有理 D-CONGA 变体中，我们测试了前几代未测试过的多个序列-结构-功能假设。许多肽变体对革兰氏阳性或革兰氏阴性病原体的抗菌活性较低，尤其是疏水性、二级结构电位或带电和疏水残基空间分布发生改变的变体。因此，D-CONGA 通常针对抗菌活性进行了很好的调整。然而，我们发现了一个变体 D-CONGA-Q7，其序列中间插入了一个极性谷氨酰胺，它对浮游细菌和生物膜形成细菌具有更高的活性，并且对人类成纤维细胞的细胞毒性更低。针对肺炎克雷伯氏菌的临床分离株, 尽管先前报道的铜绿假单胞菌缺乏诱导抗性，但对 D-CONGA 的先天抗性出奇地普遍。然而，这些相同的分离株对 D-CONGA-Q7 敏感。D-CONGA-Q7 比其前身更不容易受到革兰氏阴性细菌的 AMP 耐药性的影响。与合成分子进化的精神一致，D-CONGA-Q7 实现了关键的功能增益，并具有明显更好的活性特征。