Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E-coli
Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E-coli
复制标题
DOI：
10.1021/cb300434e
复制
发表时间：
2013-02-01
影响因子：
4
通讯作者：
Nielsen, Peter E.
中科院分区：
文献类型：
--
作者：
Ghosal, Anubrata;Vitali, Ally;Nielsen, Peter E.
靶向必需基因（acpP或ftsZ）并与递送肽L（（KFF）（3）K）缀合的反义PNA寡聚物显示野生型E的完全生长抑制。大肠杆菌MG 1655的MIC为亚微摩尔。在这项研究中，我们表明，对这种PNA-肽缀合物产生的耐药突变体在sbmA，编码内膜肽转运蛋白的基因的区域中有破坏。通过与sbmA互补，在抗性突变体中重新建立了野生型对PNA缀合物的敏感性。此外，在E. coliAS 19对未修饰的PNA敏感，导致对PNA产生抗性。最后，与相应的非生物H-D（（KFF）（3）K）肽缀合的PNA在sbmA缺失菌株中保留了抗菌活性，而与蛋白酶敏感接头的相同缀合物则没有。这些结果清楚地鉴定了SbmA作为细菌内膜上的裸PNA的载体，从而推断该肽正在外膜上转运PNA缀合物。使用缺乏SbmA的菌株来筛选SbmA非依赖性的新型肽-PNA载体。鉴定了四种这样的PNA-肽缀合物，H-D（（KFF）（3）K）、H-（RFR）（4）-Ahx-β Ala、H-（R-Ahx-R）（4）-Ahx-β Ala和H-（R-Ahx）（6）-β Ala，其利用替代的摄取机制但保留其抗微生物效力。此外，SbmA是第一个识别PNA的蛋白质。
Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-D((KFF)(3)K), H-(RFR)(4)-Ahx-beta Ala, H-(R-Ahx-R)(4)-Ahx-beta Ala, and H-(R-Ahx)(6)-beta Ala, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA.