Abstract:
Bacteria secrete various iron-chelators (siderophores), which scavenge Fe3+ from the environment, bind it with high affinity, and retrieve it inside the cell. After the Fe3+ uptake, bacteria extract the soluble iron(II) from the siderophore. Ferric siderophores are transported inside the cell via the TonB-dependent receptor system. Importantly, siderophore uptake paths have been also used by sideromycins, natural antibiotics. Our goal is to hijack the transport system for hydroxamate-type siderophores to deliver peptide nucleic acid oligomers into Escherichia coli cells. As siderophore mimics we designed and synthesized linear and cyclic Nδ-acetyl-Nδ-hydroxy-l-ornithine based peptides. Using circular dichroism spectroscopy, we found that iron(III) is coordinated by the linear trimer with hydroxamate groups but not by the cyclic peptide. The internal flexibility of the linear siderophore oxygen atoms and their interactions with Fe3+ were confirmed by all-atom molecular dynamics simulations. Using flow cytometry we found that the designed hydroxamate trimer transports PNA oligomers inside the E. coli cells. Growth recovery assays on various E. coli mutants suggest the pathway of this transport through the FhuE outer-membrane receptor, which is responsible for the uptake of the natural iron chelator, ferric-coprogen. This pathway also involves the FhuD periplasmic binding protein. Docking of the siderophores to the FhuE and FhuD receptor structures showed that binding of the hydroxamate trimer is energetically favorable corroborating the experimentally suggested uptake path. Therefore, this siderophore mimic, as well as its conjugate with PNA, is most probably internalized through the hydroxamate pathway.
摘要:
细菌分泌各种铁螯合剂(铁载体),从环境中清除Fe3+,以高亲和力结合,并在牢房里取回它。Fe3+吸收后,细菌从铁载体中提取可溶性铁(II)。铁铁载体通过TonB依赖性受体系统在细胞内运输。重要的是,铁载体吸收途径也被铁霉素使用,天然抗生素我们的目标是劫持异羟肟酸盐型铁载体的运输系统,以将肽核酸寡聚物递送到大肠杆菌细胞中。作为铁载体模拟物,我们设计并合成了基于线性和环状Nδ-乙酰基-Nδ-羟基-1-鸟氨酸的肽。使用圆二色性光谱,我们发现铁(III)由具有异羟肟酸基团的线性三聚体配位,而不是由环肽配位。通过全原子分子动力学模拟证实了线性铁载体氧原子的内部柔性及其与Fe3的相互作用。使用流式细胞术,我们发现设计的异羟肟酸盐三聚体在大肠杆菌细胞内转运PNA寡聚物。对各种大肠杆菌突变体的生长恢复试验表明了这种通过FhuE外膜受体转运的途径,负责吸收天然铁螯合剂,三价三价铁。该途径还涉及FhuD周质结合蛋白。铁载体与FhuE和FhuD受体结构的对接表明,异羟肟酸盐三聚体的结合在能量上是有利的,证实了实验建议的摄取途径。因此,这个铁载体模拟物,以及它与PNA的缀合物,最有可能通过异羟肟酸盐途径内化。