PDF(Pubmed)
Abstract：
Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3\'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.
摘要：
粘菌素是一种阳离子环状抗微生物肽，用作抵抗多重耐药性革兰氏阴性菌的最后手段。为了了解粘菌素敏感性的相关因素，我们从大肠杆菌基因敲除文库(Keio集合)中筛选粘菌素敏感突变体。PurA的淘汰赛，其产物在从头嘌呤合成途径中催化IMP合成腺苷琥珀酸，导致对粘菌素的敏感性增加。随后将腺苷琥珀酸酯转化为AMP，磷酸化产生ADP，ATP合成的底物。purA敲除突变体中的ATP量低于野生型菌株中的ATP量。ATP合成与质子转移有关,它有助于膜电位。使用膜电位探针，3,3'-二乙基氧杂碳菁碘化物[DiOC2(3)]，我们发现，与野生型菌株相比，purA敲除突变体的膜是超极化的。用质子解偶联剂处理，羰基氰化物间氯苯基腙(CCCP),消除了突变体的超极化和粘菌素敏感性。purA敲除突变体对氨基糖苷类的敏感性增加，卡那霉素,和庆大霉素；它们的摄取需要膜电位。因此,PurA的淘汰赛，一种腺苷琥珀酸合酶，与膜超极化同时减少ATP合成，导致对粘菌素的敏感性增加。
