Cellular Uptake of Apidaecin 1b and Related Analogs in Gram-negative Bacteria Reveals Novel Antibacterial Mechanism for Proline-rich Antimicrobial Peptides
Cellular Uptake of Apidaecin 1b and Related Analogs in Gram-negative Bacteria Reveals Novel Antibacterial Mechanism for Proline-rich Antimicrobial Peptides
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DOI：
10.2174/09298665113206660104
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发表时间：
2014-04-01
影响因子：
1.6
通讯作者：
Hoffmann, Ralf
中科院分区：
文献类型：
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作者：
Berthold, Nicole;Hoffmann, Ralf
富含脯氨酸的抗微生物肽（PrAMP）自由地穿过外膜进入革兰氏阴性菌的周质中，然后它们通过通透酶/转运蛋白介导的摄取主动易位到细胞质中，在细胞质中它们被报道抑制伴侣DnaK。在这里，我们研究了PrAMP apidaecin 1b，这是在蜜蜂中产生的细菌感染，并优化apidaecin类似物的细菌摄取。用5（6）-羧基荧光素标记肽，通过荧光显微镜观察它们在大肠杆菌和肺炎克雷伯氏菌中的内化，并通过流式细胞术在4小时的孵育期内的4个不同时间点进行定量。Apidaecin 1b仅以可检测的量进入40%至50%的细胞，而设计肽Api 88、Api 134和Api 155在30分钟内以比天然肽高约四倍的量进入超过95%的细菌。有趣的是，命名为（1-17）Api 88的缩短版本与18个残基长的Api 88一样有效地结合DnaK，并且以与Api 88相似的动力学进入细菌，但不能抑制细菌生长。与目前提出的PrAMP机制的类似冲突也得到了一些Ala取代的类似物和反向apidaecin序列。尽管具有C-末端酰胺的肽比同源C-末端酸更有效地进入细胞，但这种改善的细胞渗透并不改善抗菌活性。这些研究表明，PrAMP利用其他作用模式杀死敏感生物体。
Proline-rich antimicrobial peptides (PrAMPs) freely penetrate through the outer membrane into the periplasm of Gram-negative bacteria, before they are actively translocated by a permease/transporter-mediated uptake into the cytoplasm where they are reported to inhibit chaperone DnaK. Here we have studied the PrAMP apidaecin 1b, which is produced in honey bees in response to bacterial infections, and optimized apidaecin analogs for their bacterial uptake. The peptides were labeled with 5(6)-carboxyfluorescein and their internalization in Escherichia coli and Klebsiella pneumoniae was visualized by fluorescence microscopy and quantified by flow cytometry for four different time points over an incubation period of 4 h. Apidaecin 1b entered only 40% to 50% of the cells at detectable quantities, whereas designer peptides Api88, Api134 and Api155 entered more than 95% of the bacteria within 30 min at around fourfold higher quantities than the native peptide. Interestingly, a shortened version designated as (1-17)Api88, bound DnaK as efficiently as the 18-residue long Api88 and entered the bacteria at similar kinetics as Api88, but was unable to inhibit the bacterial growth. Similar conflicts with currently proposed mechanisms of PrAMPs were also obtained for some Ala-substituted analogs and reverse apidaecin sequences. Although peptides with C-terminal amides enter the cells much more efficiently than homologous C-terminal acids, this improved cell penetration does not improve the antibacterial activities. These studies suggest that PrAMPs utilize additional modes of action to kill sensitive organisms.