Veerman Enno C I, Nazmi Kamran, Van't Hof Wim, Bolscher Jan G M, Den Hertog Alice L, Nieuw Amerongen Arie V
Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands.
Biochem J. 2004 Jul 15;381(Pt 2):447-52. doi: 10.1042/BJ20040208.
The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.
抗菌肽的作用机制仍是一个有争议的问题。活性氧(ROS)的形成被认为是包括组蛋白5和乳铁蛋白衍生肽在内的多种抗菌肽杀菌机制中的关键步骤。在本研究中,我们使用二氢乙锭作为ROS的指示剂,研究了组蛋白5和一种更具两亲性的合成衍生物dhvar4对白色念珠菌中ROS生成的影响。使用这两种肽时,均观察到荧光显著增强。然而,细胞可渗透的ROS清除剂TEMPO(2,2,6,6-四甲基哌啶-N-氧基)对杀伤或荧光增强没有抑制作用。此外,据报道在体外可诱导ROS的抗霉素和叠氮化物不能增强二氢乙锭荧光,而非特异性防腐剂洗必泰增强二氢乙锭荧光的程度与肽相同。荧光显微镜显示荧光增强是细胞线粒体内未结合的预先形成的乙锭释放的结果。得出的结论是,ROS在组蛋白5介导的白色念珠菌杀伤中不起作用。