Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Rd. CB#3290, Chapel Hill, NC, 27599, USA.
Department of Chemistry, High Point University, High Point, NC, USA.
BMC Microbiol. 2021 Apr 12;21(1):110. doi: 10.1186/s12866-021-02176-4.
Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays.
Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe and chelate Fe at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors.
This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.
植物防御素是一类广泛分布的抗菌肽家族，主要研究其在农业相关抗真菌活性方面的应用。最近的研究探索了防御素对革兰氏阴性菌的作用，揭示了多种作用机制，包括膜裂解和核糖体抑制。在此，含有苋菜防御素 2（Atr-DEF2）γ-核心基序的截断合成类似物显示出革兰氏阴性抗菌活性，并通过蛋白质组学、外膜通透性研究和铁还原/螯合测定来研究其作用机制。
Atr-DEF2(G39-C54)对两种革兰氏阴性人类细菌病原体大肠杆菌和肺炎克雷伯菌表现出活性。定量蛋白质组学揭示了亚致死浓度的截断防御素处理后大肠杆菌蛋白质组的变化，包括细菌外膜（OM）和铁获取/处理相关蛋白。改变革兰氏阴性细菌外膜的电荷是其对膜裂解抗菌肽（AMP）的常见反应，以减少静电相互作用，通过 N-苯基萘-1-胺摄取试验证实了 Atr-DEF2(G39-C54)的这种作用机制。此外，体外试验证实了 Atr-DEF2(G39-C54)在细胞培养相关浓度下还原 Fe 和螯合 Fe 的能力，从而限制了必需酶辅因子的可用性。
本研究强调了植物防御素 γ-核心基序合成类似物在表征新型防御素活性方面的应用。Atr-DEF2(G39-C54)处理大肠杆菌后的蛋白质组变化支持了膜裂解是 γ-核心基序介导的抗菌活性的重要组成部分的假设，但也强调了其他特性，如金属螯合，可能有助于发挥多效作用机制。
