The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25:: role of the microcin Val11-Pro16 β-hairpin region in the recognition mechanism
The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25:: role of the microcin Val11-Pro16 β-hairpin region in the recognition mechanism
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DOI：
10.1042/bj20042107
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发表时间：
2005-08-01
影响因子：
4.1
通讯作者：
Boulanger, P
中科院分区：
文献类型：
--
作者：
Destoumieux-Garzón, D;Duquesne, S;Boulanger, P
通过一系列体内和体外实验研究了外膜铁转运蛋白 FhuA 作为抗菌肽 MccJ25 (microcin J25) 潜在受体的作用。通过使用 fhuA(-) 菌株互补的抗菌测定和使用分别编码蛋白质复合物的基因突变的同基因菌株，证明了对 FhuA 和内膜 TonB-ExbB-ExbD 复合物的需求。此外，MccJ25 在体内通过抑制噬菌体粘附来阻断噬菌体 T5 对大肠杆菌的感染，这表明 MccJ25 可以阻止噬菌体与其受体 FhuA 之间的相互作用。这种体内活性在体外得到了证实，因为 MccJ25 抑制了纯化的 FhuA 触发的噬菌体 T5 DNA 喷射。首次通过尺寸排阻色谱法和等温滴定量热法证明了 MccJ25 与 FhuA 的直接相互作用。 MccJ25 以 2:1 的化学计量比和 1.2 μM 的 K-d 与 FhuA 结合。总之，我们的结果表明 FhuA 是 MccJ25 的受体，并且配体-受体相互作用可能在细菌膜其他成分不存在的情况下发生。最后，差示扫描量热法和抗菌测定均表明，MccJ25 结合涉及 FhuA 的外部环。与天然的 MccJ25 不同，嗜热菌蛋白酶裂解的 MccJ25 变体无法与 FhuA 结合，也无法阻止噬菌体 T5 感染大肠杆菌。因此，MccJ25 的 Val(11)-Pro(16) β-发夹区域在被嗜热菌蛋白酶切割后被破坏，是小菌素识别所必需的。
The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K-d Of 1.2 mu M. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val(11)-Pro(16) beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.