Department of Biology, College of Science, Texas A&M University, College Station, Texas, USA.
Department of Biology, College of Science, Texas A&M University, College Station, Texas, USA
Appl Environ Microbiol. 2018 Jul 17;84(15). doi: 10.1128/AEM.00830-18. Print 2018 Aug 1.
Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified. Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.
Mutacin 1140 属于表皮素家族的 AI 型类细菌素。该家族对革兰氏阳性菌具有广泛的活性。Mutacin 1140 与脂质 II 的结合导致细胞壁合成的抑制。由于这些化合物的半衰期短，因此 AI 型类细菌素的药代动力学实验通常不适合临床应用。在生理环境中，兰尼定环外未保护的脱水和易受蛋白酶攻击的残基可能在半衰期短中起作用。以前对 Mutacin 1140 的诱变工作仅限于兰尼定形成残基、C 端脱羧残基以及残基 Phe1、Trp4、Dha5 和 Arg13 处的单个氨基酸取代。为了研究 Mutacin 1140 中脱水（Dha5 和 Dhb14）和易受蛋白酶攻击（Lys2 和 Arg13）残基对稳定性和生物活性的重要性，评估了每个残基对产生和抑制活性的影响。纯化了超过 15 种类似物，使我们能够直接比较针对选定革兰氏阳性菌的活性。Mutacin 1140 的翻译后修饰（PTM）机制对其底物的效率受到高度限制。在第 2、5、13 和 14 位发生单点突变后，观察到各种 PTM 中间阶段的类似物作为次要产物。在 Dha5 和 Dhb14 位置用丙氨酸取代的组合消除了 Mutacin 1140 的产生，而在这些位置之一用甘氨酸取代则恢复了产生。鉴定出具有提高的活性、生产力和蛋白水解稳定性的类似物。我们的研究结果表明，Mutacin 1140 的 PTM 效率高度依赖于核心肽序列。各种 PTM 中间阶段的类似物都可以被细菌运输，这表明 PTM 和运输是为天然 Mutacin 1140 核心肽精心调整的。只有在 Dha5 和 Dhb14 脱水残基位置的某些氨基酸取代组合才被容忍。观察到谷氨酰化核心肽类似物表明脱水是谷氨酸依赖性的。有趣的是，位于环 A 和 B 以外的位置的突变，即脂质 II 结合域，会干扰脂质 II 的结合。纯化的 Mutacin 1140 类似物对不同属的细菌具有不同的活性和选择性，这支持了生成针对致病性细菌具有更高特异性的类似物的努力。发现具有提高的抑制活性的致病性细菌、在存在蛋白酶时提高稳定性和提高的产物产率的类似物可能会促进这种独特的抗菌化合物的临床开发。
