革兰氏阴性菌多药外排泵MacAB-TolC的功能与结构

病原菌日益增长的多药耐药性已成为人类健康和生命的主要威胁之一。多药外排泵介导的药物主动外排是细菌产生耐药性的主要原因之一,给感染性疾病的防治带来了极大挑战。深入研究多药外排泵,了解其作用机制,揭示药物结合位点,可以为临床抗感染治疗提供新思路。在革兰氏阴性菌中,一些多药外排泵形成跨越细菌胞外被膜的三联复合物。MacAB-TolC是普遍存在于革兰氏阴性菌中的ABC家族三联外排泵,在近些年逐渐被关注。本文综述了关于MacAB-TolC外排泵的功能与结构研究以及相应抑制剂研发方面的进展。MacAB-TolC外排泵的底物种类多样,包含抗生素、毒力因子以及代谢产物。本文据此将MacAB-TolC外排泵的功能归纳为3类：耐药功能、病理功能和生理功能,并对相应功能分别进行了阐述。在结构研究方面,本文总结了MacAB-TolC外排泵单个组分的晶体结构和组装完全的MacAB-TolC三联外排泵的冷冻电镜单颗粒结构,并对结构数据所揭示的MacAB-TolC外排泵发挥功能的机制进行了论述。最后,本文介绍了MacAB-TolC外排泵抑制剂的最新研究进展,指出解析MacAB-TolC的原位结构,以及MacB结合底...     

大肠杆菌的AcrAB-TolC多药外排泵及其调控研究进展

多药外排泵造成了细菌的多种药物的耐药现象, 这对感染性疾病的防治提出了挑战。对于多药外排泵的研究不仅使人们认识细菌耐药性机制, 而且为细菌耐药性的防治提供思路。大肠杆菌AcrAB-TolC外排泵系统的结构和调控机制研究取得了一些新进展, 这为病原菌的相关研究提供了参考, 本文对其进行了综述。     

重组细菌多药外排泵AcrAB-TolC的转运功能和动态组装

【背景】多药外排泵多以膜蛋白复合体形式存在,是导致细菌耐药性的重要原因。外排泵的转运功能和组装过程对于细菌耐药性和药物研发具有重要意义。【目的】以多药外排泵耐药结节细胞分化家族(resistance-nodulation-division family, RND)的重要成员AcrAB-TolC复合体为对象,研究其转运活性和体外组装特性。【方法】基于大肠杆菌AcrAB-TolC复合体基因序列,分别构建含有acrA、acrB、tolC基因的重组质粒,表达和纯化复合体各亚基,利用荧光光谱、等温滴定量热法(isothermal titration calorimetry,ITC)等技术分析复合体及亚基的转运功能、亚基与底物的相互作用,以及亚基间的相互作用和动态装配。【结果】实现了AcrAB-TolC复合体各组分的表达和纯化(纯度>98%),证实表达有各组分的活细胞提高了对于溴化乙锭(ethidium bromide,EB)的转运活性,并发现群体感应效应信号分子N-hexanoyl-L-homoserine lactone (C6-HSL)能够抑制AcrB、TolC对于EB的转运活性。ITC结果进一步证实了C6-HSL与AcrB、TolC的相互作用。ITC结果还显示AcrA分别与AcrB、TolC之间存在明显的相互作用,而AcrB与TolC之间无明显的相互作用。在体外装配实验中观测到AcrAB-TolC亚基的单分子荧光强度随时间增加,证实了复合体亚基在膜上的动态组装过程。【结论】实现了AcrAB-TolC外排泵及亚基的表达和纯化,证实了AcrAB-TolC对底物的转运活性及与底物的相互作用,观察到AcrAB-TolC的动态组装过程。以上结果为研究多药外排泵导致的细菌耐药性及抗菌策略具有重要意义。     

细菌AcrAB-TolC型外排泵中AcrA蛋白的进化分析

研究不同耐药细菌AcrAB-Tolc型外排泵中关键蛋白AcrA的序列,针对其保守及非保守氨基酸残基进行该类蛋白的进化分析,构建蛋白进化树。收集来源于不同细菌的已知序列的AcrA蛋白,去除冗余并进行序列比对之后,根据其序列比对结果的相似性、氨基酸残基的保守性研究其进化特征。结果表明,不同细菌的AcrA蛋白部分氨基酸残基具有高度的保守性,这与其实现生物学功能有关,非保守区域是主要的进化区域。可为不同菌株的进化提供参考,同时为以AcrAB-Tolc型外排泵为靶标的新药研究提供相关数据。     

结核分枝杆菌外排泵研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种传染病。随着多药耐药和广泛耐药结核分枝杆菌的出现,结核病的治疗变得更为艰难。近年来研究发现,结核分枝杆菌存在外排泵是其耐药的原因之一,现已发现结核分枝杆菌的主要易化子超家族(major facilitator superfamily,MFS)、三磷酸腺苷(adenosine-triphosphate,ATP)结合盒超家族(ATP-Binding Cassette,ABC)、耐受小节分裂区家族(resistance-nodulation-division,RND)和小耐多药性家族(small multidrug resistance,SMR)外排泵。但是人们对结核分枝杆菌外排泵介导的耐药现象认识不足,仍缺乏从新药发现角度研发外排泵抑制剂的研究。本文拟对结核分枝杆菌的ABC、MFS、RND和SMR外排泵的结构和功能,以及结核分枝杆菌外排泵抑制剂的研究进展进行综述。     

结核杆菌的药物外排泵

外排泵基因过度表达促使细胞内的药物转移到细胞外,胞内药物浓度下降,致使药物临床疗效降低或无效。外排泵上有多个与识别底物结构无关的阴离子凹穴(底物结合位点),通过静电引力非特异性结合各种含阳离子的药物,在能量协助下将药物排到细胞外,进而导致细菌抗药性的产生。研究表明,结核杆菌基因组中存在外排泵基因,但仅分离到几个有功效的外排泵。本文就相关研究进展进行综述。     

外排泵介导肺炎克雷伯菌耐药的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是重要的条件致病菌,近年来肺炎克雷伯菌感染在医院内感染中所占的比率持续上升,耐药率也不断攀升,这给临床治疗带来极大的困难。肺炎克雷伯菌发生耐药的重要机制之一就是其细胞膜上存在的外排泵系统,它们将渗入细菌体内的药物不断泵出,导致菌体内的药物浓度过低,不足以发挥抗菌作用。本文主要针对外排泵介导肺炎克雷伯菌的耐药现状,外排泵的分子结构和基因调节,外排泵抑制剂以及传统中药在耐药菌治疗方面的应用等做系统性梳理,以期为临床治疗耐药肺炎克雷伯菌提供一些新思路。     

新型可移动RND家族外排泵TMexCD-TOprJ的研究进展

抗生素被认为是现代医学的基石之一,但包括抗生素在内抗菌药物的滥用也加速了可抵抗多种抗菌药物“超级细菌”的出现。耐药基因是导致细菌产生耐药性的关键因素,可通过质粒、转座子(transposon, Tn)、插入序列(insertion sequence, IS)等可移动元件(mobile genetic elements, MGEs)进行水平转移,严重威胁公共卫生安全。近年来,面对碳青霉烯类药物和多黏菌素耐药性的暴发,替加环素被视为人类面临多重耐药细菌感染的最后一道防线。近期发现了一种主要存在于质粒上的新型可移动外排泵基因簇tmexCD-toprJ,可编码耐药结节细胞分化家族(resistance-nodulation-cell division, RND)外排泵,排出菌体内包括替加环素在内的多种抗生素,大幅提升了细菌的耐药性。tmexCD-toprJ基因簇可以随质粒等可移动元件进行水平转移,已经传播至人、动物和环境中,给公共卫生健康造成了严重威胁。然而,目前人们对于其具体结构和功能作用机制等研究仍不透彻。本文系统总结tmexCD-toprJ耐药基因的分布特征、传播机制及外排泵结构等研究现状,并基于同一健康(One Health)理念提出了阻遏其扩散的措施,为减缓tmexCD-toprJ传播提供科学依据。     

单增李斯特菌耐药外排泵研究进展

单增李斯特菌是一种重要的人兽共患食源性胞内致病菌,广泛存在于自然环境中且易污染动物性食品,人及动物感染后可引起严重的李斯特菌病,死亡率高达30%。单增李斯特菌通常对多种药物敏感,然而,因不合理使用抗菌药或消毒剂形成的选择压力导致李斯特菌多重耐药情况的报道日渐增多。外排泵蛋白是细菌中一类重要的蛋白,可参与机体多种生物学过程,包括影响细菌对抗生素敏感性、促进有毒化合物泵出、影响细菌毒力等。本文综述了近年来关于单增李斯特菌耐药外排泵的功能及调控机制的研究进展,为深入理解李斯特菌耐药等环境适应机制及有效控制该病原污染传播和筛选抗感染药物新靶点提供理论基础。     

双平板对照筛选跟踪分离细菌外排泵抑制剂

建立了细菌外排泵抑制剂的筛选与活性跟踪方法.准备2个平板,一个为普通营养琼脂平板,另一个为舍小蘖碱的普通营养琼脂平板,通过比较两个平板含药纸片周围抑菌圈的直径大小判断筛选结果,方法可靠稳定.筛选发现某霉菌提取物对细菌外排泵有抑制活性,经活性跟踪分离,得到单体化合物,经NMR鉴定为4′,5,7-三羟基异黄酮.方法简便易行,成本低,适宜于对大批样本进行快速筛选并在分离时进行活性跟踪.     

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution

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Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system

ABSTRACT

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL^?1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol^?1) styrene, respectively.     

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas β-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.     

Molecular cloning and characterization of the HmrM multidrug efflux pump from Haemophilus influenzae Rd

We cloned a gene responsible for norfloxacin resistance from the chromosomal DNA of Haemophilus influenzae Rd, and designated the gene as hmrM. HmrM showed sequence similarity with NorM of Vibrio parahaemolyticus and YdhE of Escherichia coli and others that belong to the MATE family multidrug efflux pumps. The recombinant plasmid carrying the hmrM gene conferred elevated resistance not only to norfloxacin but also to acriflavine, 4 ', 6-diamidino-2-phenylindole, doxorubicin, ethidium bromide, tetraphenylphosphonium chloride, Hoechst 33342, daunomycin, berberine, and sodium deoxycholate in Escherichia coli KAM32, a drug-hypersensitive strain. We observed an Na+-dependent efflux of ethidium and an ethidium-induced efflux of Na+ in E. coli KAM32 cells harboring the plasmid carrying the hmrM gene. These results indicate that HmrM is an Na+/drug antiporter-type multidrug efflux pump. A difference in substrate preference was observed between HmrM, NorM, and YdhE.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.