Ohno Maki K, Kirikae Teruo, Yoshihara Eisaku, Kirikae Fumiko, Ishida Isao
Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Tokyo, Japan.
Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japan.
PeerJ. 2020 Nov 30;8:e10176. doi: 10.7717/peerj.10176. eCollection 2020.
Antimicrobial peptides have a broad spectrum of antimicrobial activities and are attracting attention as promising next-generation antibiotics against multidrug-resistant (MDR) bacteria. The all-d-enantiomer [(KLAKLAK)] has been reported to have antimicrobial activity against and , and to be resistant to protein degradation in bacteria because it is composed of D-enantiomer compounds. In this study, we demonstrated that modification of [(KLAKLAK)] by the addition of an L-cysteine residue to its N- or C- terminus markedly enhanced its antimicrobial activities against Gram-negative bacteria such as MDR , , and .
The peptides [(KLAKLAK)] (DP), DP to which L-cysteine was added at the N-terminus C-DP, and DP to which L-cysteine was added at the C-terminus DP-C, were synthesized at >95% purity. The minimum inhibitory concentrations of peptides and antibiotics were determined by the broth microdilution method. The synergistic effects of the peptides and the antibiotics against MDR were evaluated using the checkerboard dilution method. In order to assess how these peptides affect the survival of human cells, cell viability was determined using a Cell Counting Kit-8.
C-DP and DP-C enhanced the antimicrobial activities of the peptide against MDR Gram-negative bacteria, including , and . The antimicrobial activity of DP-C was greater than that of C-DP, with these peptides also having antimicrobial activity against drug-susceptible and drug-resistant overexpressing the efflux pump components. C-DP and DP-C also showed antimicrobial activity against colistin-resistant harboring , which encodes a lipid A modifying enzyme. DP-C showed synergistic antimicrobial activity against MDR when combined with colistin. The LD of DP-C against a human cell line HepG2 was six times higher than the MIC of DP-C against MDR The LD of DP-C was not altered by incubation with low-dose colistin.
Attachment of an L-cysteine residue to the N- or C-terminus of [(KLAKLAK)] enhanced its antimicrobial activity against , , and . The combination of C-DP or DP-C and colistin had synergistic effects against MDR . In addition, DP-C and C-DP showed much stronger antimicrobial activity against MDR and than against .
抗菌肽具有广谱抗菌活性，作为对抗多重耐药（MDR）细菌的有前景的下一代抗生素正受到关注。全D型对映体[（KLAKLAK）]已被报道对[具体细菌1]和[具体细菌2]具有抗菌活性，并且由于它由D型对映体化合物组成，对细菌中的蛋白质降解具有抗性。在本研究中，我们证明在[（KLAKLAK）]的N端或C端添加一个L-半胱氨酸残基对其进行修饰，可显著增强其对革兰氏阴性菌如MDR[具体细菌3]、[具体细菌4]和[具体细菌5]的抗菌活性。
合成了纯度>95%的肽[（KLAKLAK）]（DP）、在N端添加L-半胱氨酸的DP即C-DP以及在C端添加L-半胱氨酸的DP即DP-C。通过肉汤微量稀释法测定肽和抗生素的最低抑菌浓度。使用棋盘稀释法评估肽和抗生素对MDR[具体细菌6]的协同作用。为了评估这些肽如何影响人类细胞的存活，使用细胞计数试剂盒-8测定细胞活力。
C-DP和DP-C增强了该肽对MDR革兰氏阴性菌的抗菌活性，包括[具体细菌7]、[具体细菌8]和[具体细菌9]。DP-C的抗菌活性大于C-DP，这些肽对表达外排泵成分的药敏[具体细菌10]和耐药[具体细菌11]也具有抗菌活性。C-DP和DP-C对携带[具体基因]（编码一种脂质A修饰酶）的耐黏菌素[具体细菌12]也显示出抗菌活性。DP-C与黏菌素联合使用时对MDR[具体细菌13]显示出协同抗菌活性。DP-C对人细胞系HepG2的半数致死剂量比其对MDR[具体细菌14]的最低抑菌浓度高6倍。DP-C与低剂量黏菌素孵育后半数致死剂量未改变。
在[（KLAKLAK）]的N端或C端连接一个L-半胱氨酸残基可增强其对[具体细菌15]、[具体细菌16]和[具体细菌17]的抗菌活性。C-DP或DP-C与黏菌素联合使用对MDR[具体细菌18]具有协同作用。此外，DP-C和C-DP对MDR[具体细菌19]和[具体细菌20]的抗菌活性比对[具体细菌21]强得多。
