Sacramento R S, Martins R M, Miranda A, Dobroff A S S, Daffre S, Foronda A S, De Freitas D, Schenkman S
Departamento de Oftalmologia, Universidade Federal de São Paulo, SP, Brazil.
Parasitology. 2009 Jul;136(8):813-21. doi: 10.1017/S0031182009006283. Epub 2009 Jun 2.
In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.
在这项研究中，我们评估了不同类型抗菌肽促进棘阿米巴角膜炎致病原——卡氏棘阿米巴滋养体通透性改变和生长抑制的能力。我们使用了阳离子α-螺旋肽P5和P6，它们分别对应吸血昆虫大锥蝽成孔蛋白的N端，以及蜘蛛亚马逊巨人食鸟蛛血细胞中的β-发夹两亲分子（戈麦辛）。在与微摩尔浓度的这两种肽孵育1小时后，实现了卡氏棘阿米巴的通透性改变。虽然戈麦辛诱导的通透性改变随着孵育时间延长而增加，但P5诱导的通透性改变并未随时间增加，且在对SIRC细胞毒性更大的剂量下发生。然而，P5在低于用于杀死兔角膜细胞的临界剂量时，在促进生长抑制方面相当有效。同样，在通透性测定中同时添加丝氨酸蛋白酶抑制剂时，P5更有效。高效液相色谱随后进行质谱分析证实，与戈麦辛不同，P5被卡氏棘阿米巴培养上清液水解。我们得出结论，使用抗菌肽治疗卡氏棘阿米巴感染需要寻找更具特异性且抗蛋白水解的肽。
