Cardoso Marlon H, Cândido Elizabete S, Chan Lai Y, Der Torossian Torres Marcelo, Oshiro Karen G N, Rezende Samilla B, Porto William F, Lu Timothy K, de la Fuente-Nunez Cesar, Craik David J, Franco Octávio L
Programa de Pós-Graduação em Patologia Molecular, Faculdade de Medicina , Universidade de Brasília , Campus Darcy Ribeiro, Asa Norte , Brasília , DF 70910900 , Brazil.
Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia , Universidade Católica de Brasília , SGAN 916 Módulo B, Asa Norte , Brasília , DF 70790160 , Brazil.
ACS Infect Dis. 2018 Dec 14;4(12):1727-1736. doi: 10.1021/acsinfecdis.8b00219. Epub 2018 Nov 1.
Computer-aided screening of antimicrobial peptides (AMPs) is a promising approach for discovering novel therapies against multidrug-resistant bacterial infections. Here, we functionally and structurally characterized an Escherichia coli-derived AMP (EcDBS1R5) previously designed through pattern identification [α-helical set (KK[ILV][AILV])], followed by sequence optimization. EcDBS1R5 inhibited the growth of Gram-negative and Gram-positive, susceptible and resistant bacterial strains at low doses (2-32 μM), with no cytotoxicity observed against non-cancerous and cancerous cell lines in the concentration range analyzed (<100 μM). Furthermore, EcDBS1R5 (16 μM) acted on Pseudomonas aeruginosa pre-formed biofilms by compromising the viability of biofilm-constituting cells. The in vivo antibacterial potential of EcDBS1R5 was confirmed as the peptide reduced bacterial counts by two-logs 2 days post-infection using a skin scarification mouse model. Structurally, circular dichroism analysis revealed that EcDBS1R5 is unstructured in hydrophilic environments, but has strong helicity in 2,2,2-trifluoroethanol (TFE)/water mixtures (v/v) and sodium dodecyl sulfate (SDS) micelles. The TFE-induced nuclear magnetic resonance structure of EcDBS1R5 was determined and showed an amphipathic helical segment with flexible termini. Moreover, we observed that the amide protons for residues Met2-Ala8, Arg10, Ala13-Ala16, and Trp19 in EcDBS1R5 are protected from the solvent, as their temperature coefficients values are more positive than -4.6 ppb·K. In summary, this study reports a novel dual-antibacterial/antibiofilm α-helical peptide with therapeutic potential in vitro and in vivo against clinically relevant bacterial strains.
计算机辅助筛选抗菌肽（AMPs）是发现针对多重耐药细菌感染的新型疗法的一种有前景的方法。在此，我们对一种先前通过模式识别[α-螺旋组（KK[ILV][AILV]）]设计、随后进行序列优化的大肠杆菌衍生抗菌肽（EcDBS1R5）进行了功能和结构表征。EcDBS1R5在低剂量（2 - 32 μM）时可抑制革兰氏阴性和革兰氏阳性、敏感和耐药细菌菌株的生长，在所分析的浓度范围（<100 μM）内，未观察到对非癌细胞系和癌细胞系的细胞毒性。此外，EcDBS1R5（16 μM）通过损害构成生物膜的细胞的活力作用于铜绿假单胞菌预先形成的生物膜。使用皮肤划痕小鼠模型证实了EcDBS1R5的体内抗菌潜力，该肽在感染后2天使细菌数量减少了两个对数。在结构上，圆二色性分析表明EcDBS1R5在亲水环境中无结构，但在2,2,2-三氟乙醇（TFE）/水混合物（v/v）和十二烷基硫酸钠（SDS）胶束中有很强的螺旋性。确定了TFE诱导的EcDBS1R5的核磁共振结构，显示出一个具有柔性末端的两亲性螺旋片段。此外，我们观察到EcDBS1R5中Met2 - Ala8、Arg10、Ala13 - Ala16和Trp19残基的酰胺质子受溶剂保护，因为它们的温度系数值比-4.6 ppb·K更正。总之，本研究报道了一种新型的具有双重抗菌/抗生物膜作用的α-螺旋肽，在体外和体内对临床相关细菌菌株具有治疗潜力。
