Programa de Pós-Graduação em Saúde e Desenvolvimento na Região Centro Oeste, Universidade Federal do Mato Grosso do Sul, Brazil; Laboratório de Purificação de Proteínas e suas Funções Biológicas, Universidade Federal de Mato Grosso do Sul, Cidade Universitária S/N, Caixa Postal 549, Campo Grande, MS 79070-900, Brazil.
Laboratório de Purificação de Proteínas e suas Funções Biológicas, Universidade Federal de Mato Grosso do Sul, Cidade Universitária S/N, Caixa Postal 549, Campo Grande, MS 79070-900, Brazil.
Biomed Pharmacother. 2021 Dec;144:112198. doi: 10.1016/j.biopha.2021.112198. Epub 2021 Oct 15.
Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37-100 °C) and pH (2-10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5-10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.
山黧豆属（Fabaceae 科）（副科 Papillionoideae）植物分布于巴西的热带和亚热带地区。本研究从山黧豆属植物种子中分离纯化出一种新的 Kunitz 型抑制剂，命名为 EpTI。EpTI 由三个分子质量在 17-20 kDa 之间且氨基端序列完全一致的同工型组成。EpTI 的理化性质与 Kunitz 抑制剂相似，包括解离常数（13.1 nM）、热稳定性（37-100°C）和 pH 稳定性（2-10），并且存在二硫键稳定其活性部位。此外，我们研究了 EpTI 对革兰氏阳性菌和阴性菌的抗菌、抗黏附和抗生物膜作用。结果表明，该抑制剂对阴沟肠杆菌、普通变形杆菌、肺炎克雷伯菌、金黄色葡萄球菌和溶血性葡萄球菌的最低抑菌浓度（MIC，5-10 μM）和最低杀菌浓度（MBC）均为 10 μM。EpTI 与环丙沙星联合使用具有明显的协同作用，使抗生素浓度降低 150%。结晶紫摄取实验表明 EpTI 可增加金黄色葡萄球菌和肺炎克雷伯菌的摄取量，分别增加约 30%和 50%，提示 EpTI 的作用靶点可能是细菌的质膜。用 EpTI 处理 1x 和 10x MIC 可显著减少生物膜的形成并促进成熟生物膜的破坏。在 MIC/2 时，EpTI 可在 2 h 内降低细菌对聚苯乙烯表面的黏附。最后，EpTI 在动物模型大蜡螟中显示出低毒性。鉴于 EpTI 的抗菌和抗生物膜特性，其序列可能被用于设计新型药物原型。
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