Laboratory of Biochemistry and Biotechnology, Institute for Marine Sciences, Federal University of Ceará, Av. Da Abolição, 3207, Meireles, Fortaleza-CE, 60165081, Brazil.
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará (UECE), Fortaleza-CE, Brazil.
Curr Pharm Biotechnol. 2022;23(2):172-179. doi: 10.2174/1389201022666210322123903.
Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (KRFKKFFKK and KRLKKIFKK; consensus: KRhKKhFKK, h = hydrophobic amino acid) as an integral part of its structure.
The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamine- B‒conjugated Ctn[1-9], designated as RhoB-Ctn[1-9].
The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis.
RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials.
These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.
Crotalicidin (Ctn) 是一种 34 个氨基酸组成的线性赖氨酸丰富的蛇毒抗菌肽，是从南美响尾蛇 Crotalus durissus terrificus 中获得的 Vipericidin 相关抗菌肽。Ctn 包含串联重复的九个氨基酸残基（KRFKKFFKK 和 KRLKKIFKK；共识：KRhKKhFKK，h = 疏水性氨基酸），作为其结构的一部分。
本研究旨在评估加密 Vipericidin 九肽 KRFKKFFKK，命名为 Ctn[1-9]，及其结构类似物，罗丹明 B 缀合 Ctn[1-9]，命名为 RhoB-Ctn[1-9] 的抗菌活性。
采用肉汤微量稀释法测定最低抑菌浓度（MIC）法测定代表病原菌和酵母对抗菌剂的敏感性。采用溶血试验估计细胞毒性。通过荧光显微镜观察 RhoB-Ctn[1-9]在微生物细胞中的积累。采用棋盘分析评价 RhoB-Ctn[1-9]与抗菌药物的协同作用。
RhoB 缀合的 Ctn[1-9]对感染性革兰氏阴性菌（如大肠杆菌、铜绿假单胞菌）和致病性念珠菌具有选择性抗菌活性，对人红细胞的溶血作用较小，而未缀合的 Ctn[1-9]则没有观察到。RhoB-Ctn[1-9]可穿透细胞膜并在微生物细胞内积累。RhoB-Ctn[1-9]与抗生素或抗真菌药物联合使用时具有协同作用，并降低肽和抗生素的 MIC。
这些发现表明，Crotalicidin 相关短肽具有作为生物功能多样化的结构基序的潜力。此外，它们为研究化学修饰的 Vipericidin 重复序列如何调节细胞命运的分子机制奠定了基础。
