Kanthawong Sakawrat, Puknun Aekkalak, Bolscher Jan G M, Nazmi Kamran, van Marle Jan, de Soet Johannes J, Veerman Enno C I, Wongratanacheewin Surasakdi, Taweechaisupapong Suwimol
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.
Biometals. 2014 Oct;27(5):949-56. doi: 10.1007/s10534-014-9760-5. Epub 2014 Jun 25.
LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.
LF嵌合体是一种结合了牛乳铁蛋白两个抗菌结构域（乳铁蛋白肽265 - 284和乳铁传递蛋白17 - 30）的构建体，具有很强的杀菌活性。然而，目前尚无实验证据来评估LF嵌合体针对伯克霍尔德菌分离株的作用机制。在本研究中，我们分析了LF嵌合体对B类病原体类鼻疽伯克霍尔德菌的杀伤活性，并与通常用作高毒力类鼻疽伯克霍尔德菌模型的低毒力泰国伯克霍尔德菌进行了比较。杀伤动力学表明，泰国伯克霍尔德菌E264比类鼻疽伯克霍尔德菌1026b对LF嵌合体更敏感。有趣的是，LF嵌合体的杀菌活性似乎高度依赖于pH值；在pH 6.4及以下时，泰国伯克霍尔德菌的杀伤作用完全消失。异硫氰酸荧光素标记的LF嵌合体在15分钟内迅速在两种细菌的细胞质中积累。此外，冷冻断裂电子显微镜显示，在孵育1小时内，两种细菌的膜形态都受到了极大影响，同时伴随着核苷酸泄漏所监测到的膜通透性改变。这些数据表明，LF嵌合体对两种细菌的作用机制相似，包括破坏质膜以及随后细胞内核苷酸泄漏导致细胞死亡。
