Silvestro L, Gupta K, Weiser J N, Axelsen P H
Department of Pharmacology, Infectious Diseases Section, and Johnson Foundation for Molecular Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6084, USA.
Biochemistry. 1997 Sep 23;36(38):11452-60. doi: 10.1021/bi9630826.
Cecropin A is a naturally occurring, linear, cationic, 37-residue antimicrobial peptide. The precise mechanism by which it kills bacteria is not known, but its site of action is believed to be the cell membrane. To investigate the nature of its membrane activity, we examined the ability of cecropin A to alter membrane permeability in synthetic lipid vesicles and in Gram-negative bacteria. Cecropin A exerted distinctly different types of membrane activity depending on its concentration. In synthetic lipid vesicles, cecropin A dissipated transmembrane electrochemical ion gradients at relatively low concentrations, but much higher concentrations were required to release an encapsulated fluorescent probe. Cecropin A dissipated ion gradients whether or not the vesicle membranes contained anionic lipid, although the presence of anionic lipid dramatically increased peptide binding, and modestly increased the release of an encapsulated probe. Cholesterol did not prevent the dissipation of ion gradients by low concentrations of peptide, but it did inhibit release of the encapsulated probe by high concentrations of peptide. At the highest concentrations examined, cecropin A remained monomeric in solution, and did not aggregate, lyse, or otherwise alter vesicle size. In Gram-negative bacteria, cecropin A was potently bactericidal at concentrations which dissipated ion gradients in lipid vesicles, but much higher concentrations were required to cause the release of cytoplasmic contents. These findings point to the conclusion that cecropin A kills bacteria by dissipating transmembrane electrochemical ion gradients. They weigh against theories comparing the antimicrobial activity of cecropin A to the release of encapsulated probes from lipid vesicles, and against roles for cholesterol or anionic lipid headgroups in the selectivity of peptide action against bacteria.
天蚕素A是一种天然存在的、线性的、阳离子型的、由37个氨基酸残基组成的抗菌肽。其杀死细菌的确切机制尚不清楚，但据信其作用位点是细胞膜。为了研究其膜活性的本质，我们检测了天蚕素A改变合成脂质体和革兰氏阴性菌中膜通透性的能力。天蚕素A根据其浓度表现出明显不同类型的膜活性。在合成脂质体中，天蚕素A在相对较低的浓度下就能消散跨膜电化学离子梯度，但需要更高的浓度才能释放包封的荧光探针。无论脂质体膜是否含有阴离子脂质，天蚕素A都能消散离子梯度，尽管阴离子脂质的存在显著增加了肽的结合，并适度增加了包封探针的释放。胆固醇并不能阻止低浓度肽消散离子梯度，但它确实抑制了高浓度肽释放包封探针。在所检测的最高浓度下，天蚕素A在溶液中保持单体状态，不会聚集、裂解或以其他方式改变脂质体大小。在革兰氏阴性菌中，天蚕素A在能消散脂质体中离子梯度的浓度下具有强效杀菌作用，但需要更高的浓度才能导致细胞质内容物的释放。这些发现得出的结论是，天蚕素A通过消散跨膜电化学离子梯度来杀死细菌。它们与将天蚕素A的抗菌活性与从脂质体中释放包封探针进行比较的理论相悖，也与胆固醇或阴离子脂质头部基团在肽对细菌作用选择性方面的作用相悖。
