Uptake, Stability, and Activity of Antisense Anti-acpP PNA-Peptide Conjugates in Escherichia coli and the Role of SbmA.
Uptake, Stability, and Activity of Antisense Anti-acpP PNA-Peptide Conjugates in Escherichia coli and the Role of SbmA.
复制标题
反义抗 acpP PNA-肽缀合物在大肠杆菌中的摄取、稳定性和活性以及 SbmA 的作用。
DOI：
10.1021/acschembio.0c00822
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发表时间：
2021
影响因子：
4
通讯作者：
P. Nielsen
中科院分区：
文献类型：
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作者：
Niloofar Yavari;Lise Goltermann;P. Nielsen
与细菌穿透肽(BPP)(如(Kff)3K)偶联的PNA寡聚体，靶向关键细菌基因，如acpP，可在一位数的微摩尔浓度下抑制细菌生长。已发现革兰氏阴性菌外膜的内毒素是细胞摄取(Kff)3K-eg1-PNA的屏障，而SBMA转运蛋白参与内膜的转运。我们现在进一步阐明(Kff)3K-eg1-PNA的摄取机制，因为(Kff)3K-eg1-PNA的多肽部分是不稳定的，在细菌培养介质(T1/2<；5分钟)和细菌内被多肽酶降解。对存在于周质间隙和细胞质中的多肽-PNA结合物的分析表明，主要存在PNA，仅含有FFK三肽，而不含多肽，浓度为添加到培养基中的10倍。此外，当两种主要降解产物直接加入细菌培养物中时，没有显示出抗菌作用，并且随着肽长度的增加，抗菌作用减弱，从而证明完整的肽确实是摄取的关键，但不是细胞内反义活性的关键。最令人惊讶的是，研究发现，尽管相应的一系列蛋白水解性稳定的D-型(Kff)3k-eg1-PNAs的活性随肽长度的增加而类似地降低，但对于较短的多肽，其活性依赖于SBMA的存在(全长多肽则不是这样)。因此，我们的结果表明，BPP既是穿过内膜也是穿过内膜所必需的，全长(Kff)3K可能自发地穿过内膜。因此，(Kff)3K-eg1-PNA对SBMA的依赖归因于细菌介质和周质空间中的多肽降解。结果表明，在BPP(和抗菌肽)的设计和活性/摄取分析中，应考虑稳定性和新陈代谢(由细菌蛋白水解酶/多肽酶)。
PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t1/2 < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).