Yamauchi K, Tomita M, Giehl T J, Ellison R T
Medical Service, Department of Veterans Affairs Medical Center, Denver, Colorado.
Infect Immun. 1993 Feb;61(2):719-28. doi: 10.1128/iai.61.2.719-728.1993.
Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.
尽管乳铁蛋白的抗菌活性已得到充分描述，但其作用机制却鲜为人知。最近的研究表明，除了结合铁之外，人乳铁蛋白还会破坏革兰氏阴性菌的外膜。在本研究中，我们测定了牛乳铁蛋白和一种胃蛋白酶衍生的牛乳铁蛋白肽（乳铁蛋白素）片段是否具有相似的活性。我们发现，20微摩尔的牛乳铁蛋白和20微摩尔的乳铁蛋白素均可从三种细菌菌株，即大肠杆菌CL99 1-2、鼠伤寒沙门氏菌SL696和蒙得维的亚沙门氏菌SL5222中释放出内在标记的[3H]脂多糖（[3H]LPS）。在大多数情况下，肽片段释放的LPS比完整的牛乳铁蛋白更多。在乳铁蛋白或乳铁蛋白素存在的情况下，溶菌酶对大肠杆菌CL99 1-2的杀伤作用增强。与人类乳铁蛋白一样，牛乳铁蛋白和乳铁蛋白素都能够结合游离的内在标记的[3H]LPS分子。除了这些作用之外，虽然牛乳铁蛋白最多具有抑菌作用，但乳铁蛋白素对革兰氏阴性菌表现出持续的杀菌活性。这种杀菌作用受到阳离子Ca2+、Mg2+和Fe3+的调节，但与培养基的渗透压无关。对暴露于乳铁蛋白素的细菌细胞进行透射电子显微镜观察显示，立即出现了电子致密的“膜泡”。这些实验提供了证据，证明牛乳铁蛋白和乳铁蛋白素会破坏革兰氏阴性菌的外膜。此外，肽片段乳铁蛋白素具有直接的杀菌活性。由于乳铁蛋白在体内会暴露于蛋白水解因子，这些因子可能会切割乳铁蛋白素片段，因此该肽的作用在机制和生理方面都具有相关性。
