Screening for Antifungal Peptides and Their Modes of Action in Aspergillus nidulans
Screening for Antifungal Peptides and Their Modes of Action in Aspergillus nidulans
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DOI：
10.1128/aem.01560-10
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发表时间：
2010-11-01
影响因子：
4.4
通讯作者：
Takeshita, Norio
中科院分区：
文献类型：
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作者：
Mania, Daniel;Hilpert, Kai;Takeshita, Norio
许多短阳离子多肽已被确定为有效的抗菌剂，但其作用机制尚不清楚。在纤维素膜上合成多肽导致了多肽文库的产生，同时高通量分析已经发展起来以测试它们的抗菌活性。本文建立了一种基于微量平板的真菌筛选方法，并用该方法检测了9种抗菌肽对模式真菌Nidulans的抑制作用。使用亚致死性肽浓度的显微镜研究导致了极化生长的缺陷，包括增加了分枝形成和去极化菌丝。我们表征了我们的目标多肽之一Sub5(12个氨基酸)的作用模式，它已经被证明具有作为抗菌剂的药理潜力，并能够与ATP和ATP依赖的酶相互作用。对金黄色葡萄球菌的最低抑菌浓度为2微克/毫升，与报道的细菌最低抑菌浓度范围相同。异硫氰酸荧光素(FITC)标记的Sub5以细胞质膜为靶标，特别是菌丝尖端，在长时间暴露后进入细胞质，与内吞作用无关。有趣的是，Sub5肽处理扰乱了菌丝顶端富含类固醇的膜区，这对菌丝顶端的生长很重要。一种非常相似的多肽FITC-P7也聚集在细胞膜上，但没有抗菌或抗真菌活性，这表明细胞膜是Sub5肽的第一个靶点；然而，抗真菌活性似乎与进入细胞质的能力有关，在那里多肽可能作用于其他靶点。
Many short cationic peptides have been identified as potent antimicrobial agents, but their modes of action are not well understood. Peptide synthesis on cellulose membranes has resulted in the generation of peptide libraries, while high-throughput assays have been developed to test their antibacterial activities. In this paper a microtiter plate-based screening method for fungi has been developed and used to test nine antibacterial peptides against the model fungus Aspergillus nidulans. Microscopical studies using sublethal peptide concentrations caused defects in polarized growth, including increased branch formation and depolarized hyphae. We characterized the mode of action for one of our target peptides, Sub5 (12 amino acids), which has already been shown to possess pharmacological potential as an antibacterial agent and is able to interact with ATP and ATP-dependent enzymes. The MIC for A. nidulans is 2 mu g/ml, which is in the same range as the MICs reported for bacteria. Fluorescein isothiocyanate (FITC)-labeled Sub5 targeted the cytoplasmic membrane, particularly hyphal tips, and entered the cytoplasm after prolonged exposure, independent of endocytosis. Interestingly, Sub5 peptide treatment disturbed sterol-rich membrane domains, important for tip growth, at hyphal tips. A very similar peptide, FITC-P7, also accumulated on the cell membrane but did not have antibacterial or antifungal activity, suggesting that the cytoplasmic membrane is a first target for the Sub5 peptide; however, the antifungal activity seems to be correlated with the ability to enter the cytoplasm, where the peptides might act on other targets.