Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica
Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica
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DOI：
10.1073/pnas.93.1.412
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发表时间：
1996-01-09
影响因子：
11.1
通讯作者：
Elliott, S
中科院分区：
文献类型：
--
作者：
Brogden, KA;DeLucca, AJ;Elliott, S
当表面活性剂和细菌混合物与正常绵羊血清一起孵育时，绵羊肺表面活性剂对溶血巴斯德氏菌具有杀菌作用。为了分离该组分，将表面活性剂（lmg/ml）以100，000 x g（av）离心，并通过HPLC分级分离上清液。用乙腈（10-100%）/0.1%三氟乙酸洗脱组分并测试杀菌活性。对三种杀菌组分的氨基酸和序列分析表明，组分2含有H-GDDDDDD-OH，组分3含有H-DDDDDDD-OH，组分5含有H-GADDDDD-OH。0.14 M NaCl/10 μ M ZnCl 2中的肽（锌盐溶液）诱导的溶血性芽孢杆菌和其它细菌的杀伤与防御素和β-防御素相当[最小杀菌浓度（MBC）（50）范围，0.01-0.06 mM]，但在0.14 M NaCl/10 mM磷酸钠缓冲液，pH 7.2/0.5 mM CaCl 2/0.15 mM MgCl 2中不诱导（MBC（50）范围，2.8-11.5 mM）。杀菌活性位于天冬氨酸六肽均聚物的核心区域，天冬氨酸二肽-七肽均聚物的MBC（50）值与肽中天冬氨酸残基的数量成反比。在锌盐溶液中与H-DDDDDD-OH孵育的溶血性P.表面活性剂相关的阴离子肽尺寸较小，电荷相反，并且在锌盐溶液中具有杀菌性。它们是含有天冬氨酸的另一类肽抗生素的成员，当存在于肺分泌物中时，天冬氨酸可以帮助清除细菌，作为先天性肺防御系统的一部分。
Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum, To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x g(av), and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity, Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 5 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 mu M ZnCl2 (zinc saline solution) induced killing of P, haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)(50) range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC(50) range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC(50) values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide, P, haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min, Ultrastructurally, cells contained flocculated intracellular constituents, In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution, They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.