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Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy
天然类黄酮破坏细菌铁稳态以增强粘菌素功效

Zi-xing Zhong 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Shuang Zhou 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Yu-jiao Liang 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Yi-yang Wei 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Yan Li 1 , 2 1 , 2 ^(1,2){ }^{1,2} Teng-fei Long 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Qian He 1 , 2 He 1 , 2 He^(1,2)\mathrm{He}^{1,2}, Meng-yuan Li 1 , 2 Li 1 , 2 Li^(1,2)\mathrm{Li}^{1,2}, Yu-feng Zhou 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Yang Yu 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Liang-xing Fang 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Xiao-ping Liao 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Barry N. Kreiswirth 3 3 ^(3){ }^{3}, Liang Chen 3 3 ^(3){ }^{3}, Hao Ren 1 , 2 ∗ 1 , 2 ∗ ^(1,2**){ }^{1,2 *}, Ya-hong Liu 1 , 2 , 4 ∗ 1 , 2 , 4 ∗ ^(1,2,4**){ }^{1,2,4 *}, Jian Sun 1 , 2 ∗ 1 , 2 ∗ ^(1,2**){ }^{1,2 *} 重试    错误原因

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/ p m r B p m r B pmrBp m r B, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.
面对全球抗生素耐药性的惊人上升，近几十年来只有少数新型抗生素被开发出来，需要在治疗策略上进行创新，以填补抗生素发现的空白。在这里，我们建立了一个模拟宿主环境的筛选平台来选择抗生素佐剂，并发现三种儿茶酚类黄酮-7，8-二羟基黄酮，杨梅素和木犀草素-显着增强粘菌素的功效。进一步的机制分析表明，这些黄酮类化合物能够通过将三价铁转化为亚铁形式来破坏细菌铁稳态。过量的胞内亚铁离子通过干扰双组分系统 pmrA/ p m r B p m r B pmrBp m r B 调节细菌的膜电荷，从而促进黏菌素结合和随后的膜损伤。在体内感染模型中进一步证实了这些黄酮类化合物的增强作用。 总的来说，目前的研究提供了三种黄酮类化合物作为粘菌素佐剂，以补充我们对抗细菌感染的武器库，并揭示了细菌铁信号传导作为抗菌治疗的一个有前途的靶点。
Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
版权所有© 2023 作者，保留部分权利;独家许可证持有人美国科学促进会。不得对原始美国政府工程提出索赔。在知识共享署名非商业许可证 4.0（CC BY-NC）下分发。

INTRODUCTION  介绍

Antibiotics have been widely applied for nearly 100 years since Alexander Fleming heralded the antibiotic era (1). Regrettably, the cumulative consumption of antibiotics has led to the emergence and spread of antibiotic resistance at an alarmingly high rate (2, 3). To date, numerous transferable antibiotic resistance genes have been identified, including those conferring resistance to last-resort antibiotics such as carbapenem, colistin, and tigecycline (4-6). The looming crisis of antimicrobial resistance (AMR) threatens current treatment paradigms based on antibiotics, presenting a worrisome scenario where some bacterial infections that were once easily treatable are now deadly. Clinical antibiotic choices are mainly guided by the antimicrobial susceptibility testing (AST) results, primarily based on minimum inhibitory concentration (MIC) values. However, AST of commonly prescribed antibiotics is often tested in vitro in universal medium, which does not always correlate with the clinical treatment efficacy in vivo. In addition, standard AST, sometimes, inadvertently excludes antibiotics with potent efficacy, due to their high MIC values, despite being synergistic with other antibiotics (7). In concert with the rising antibiotic resistance, the traditional source of antibiotic seems to be overmined, as the discovery of novel antibiotics lags far behind the rapid evolution and dispersion of antibiotic resistances (8). Therefore, more innovative strategies must be included to bridge
自亚历山大弗莱明宣布抗生素时代以来，抗生素已被广泛应用近 100 年（1）。令人遗憾的是，抗生素的累积消费导致了抗生素耐药性的出现和传播，其速度之快令人震惊（2，3）。迄今为止，已经鉴定出许多可转移的抗生素耐药基因，包括那些赋予对最后手段抗生素如碳青霉烯、粘菌素和替加环素的耐药性的基因（4-6）。抗生素耐药性（AMR）的危机迫在眉睫，威胁着目前基于抗生素的治疗模式，呈现出令人担忧的情况，一些曾经容易治疗的细菌感染现在是致命的。临床抗生素的选择主要由抗菌药物敏感性试验（AST）结果指导，主要基于最小抑菌浓度（MIC）值。 然而，常用抗生素的 AST 通常在通用培养基中进行体外检测，其并不总是与体内临床治疗效果相关。此外，标准 AST 有时会因其 MIC 值高而无意中排除具有强效功效的抗生素，尽管与其他抗生素具有协同作用（7）。随着抗生素耐药性的不断上升，传统的抗生素来源似乎被过度开采，因为新型抗生素的发现远远落后于抗生素耐药性的快速演变和分散（8）。因此，必须包括更多的创新战略，
the gap between the availability of new therapy and increasing AMR concerns.
新疗法的可用性与 AMR 问题日益增加之间的差距。
Such innovations, exemplified by combinatorial therapy, drug repurposing, antiviral therapy, rational optimization of leads, and new leads development from untapped source, have profoundly expanded our arsenal to combat the infections caused by resistant bacteria (9-11). It is notably that all these strategies have pros and cons, and their cost, efficacy, and biosafety should be considered before their introduction to clinical practice. In comparison to the discovery of new antimicrobial leads, the combination strategy offers a promising approach to revitalize existing antibiotics with well-researched and clinically validated status (12). One best example is the syncretic combination of β β beta\beta-lactam antibiotics with β β beta\beta-lactamase inhibitors like clavulanate and avibactam, which have been used to target extended spectrum β β beta\beta-lactamase and carbapenemase-producing bacteria (13). Besides the combination of antibiotic with targetspecific inhibitors, a short linear antibacterial peptide S25 (SLAP25 ), with broad-spectrum adjuvant property, was also reported to be able to potentiate the efficacies of tetracycline, ofloxacin, rifampicin, cefepime, and vancomycin (14). Thus, these examples underscore the promising feasibility of combination therapy to overcome the antibiotic resistance.
这些创新，例如组合疗法，药物再利用，抗病毒疗法，合理优化的线索，以及从未开发的来源开发新的线索，已经深刻地扩大了我们的武器库，以对抗由耐药细菌引起的感染（9-11）。值得注意的是，所有这些策略都有优点和缺点，在将其引入临床实践之前，应考虑其成本，有效性和生物安全性。与发现新的抗菌药物相比，组合策略提供了一种有希望的方法，可以重振现有的抗生素，并具有良好的研究和临床验证状态（12）。一个最好的例子是 β β beta\beta -内酰胺抗生素与 β β beta\beta -内酰胺酶抑制剂如克拉维他和阿维巴坦的融合组合，其已用于靶向超广谱 β β beta\beta -内酰胺酶和产碳青霉烯酶的细菌（13）。 除了抗生素与靶向特异性抑制剂的组合之外，还报道了具有广谱佐剂性质的短线性抗菌肽 S25（SLAP 25）能够增强四环素、氧氟沙星、利福平、头孢吡肟和万古霉素的功效（14）。因此，这些例子强调了联合治疗克服抗生素耐药性的有希望的可行性。
Among the antibiotic of last resort, colistin has been deemed as a viable therapeutic option to eradicate multidrug-resistant (MDR) bacteria, especially carbapenem-resistant Enterobacteriales (15, 16). Although colistin demonstrates a rapid bacterial clearance, its in vivo efficacy has always being suboptimal, as up to 70 % 70 % 70%70 \% of patients responded poorly to colistin treatment (17-20). It suggested that the pathogens at host sites may respond differently to colistin at regular doses. However, using colistin in excess is also not possible due to its nephrotoxicity (21). Unfortunately, the situation has been exacerbated by the emergence of chromosome-mediated and mobile element-mediated colistin resistance (e.g., Mcr-1), which confer colistin resistance by modulating membrane charge (4). In the face of such intrinsic or acquired colistin resistance, current
在最后的抗生素中，粘菌素被认为是根除多重耐药（MDR）细菌，特别是碳青霉烯类耐药肠杆菌的可行治疗选择（15，16）。尽管粘菌素显示出快速的细菌清除，但其体内功效一直是次优的，因为多达 70 % 70 % 70%70 \% 例患者对粘菌素治疗反应不佳（17-20）。表明宿主部位的病原体对常规剂量的粘菌素可能有不同的反应。然而，由于其肾毒性，过量使用粘菌素也是不可能的（21）。不幸的是，由于染色体介导的和移动的元件介导的粘菌素抗性（例如，Mcr-1），其通过调节膜电荷赋予粘菌素抗性（4）。面对这种内在的或获得性的粘菌素抗性，目前的
colistin treatment necessitates innovative strategies to enhance colistin efficacy for better clinical outcomes. To this end, a panel of colistin adjuvants has been developed in previous studies. Liu and co-workers (22) identified that melatonin, a neurohormone, resensitize the Gram-negative bacteria to colistin by targeting the bacterial membrane and promoting oxidative damage. In a recent report, De Oliveira et al. (23) repurposed an ionophore for neurodegenerative disease, PBT-2, to break the resistance to polymyxin by disrupting bacterial intracellular metal homeostasis to a larger extent. In addition, silver was found to substitute the essential zinc ion in the intact enzyme of MCR, to curb the mcr-1-mediated colistin resistance in vitro and in vivo (24). These efforts demonstrated that combinations of colistin with rational adjuvants are of great potential to enhance current treatment paradigms based on colistin.
粘菌素治疗需要创新的策略来增强粘菌素的功效以获得更好的临床结果。为此，在先前的研究中已经开发了一组粘菌素佐剂。Liu 及其同事（22）发现，褪黑激素（一种神经激素）通过靶向细菌膜并促进氧化损伤，使革兰氏阴性菌对粘菌素重新敏感。在最近的一份报告中，De Oliveira 等人（23）重新利用了一种用于神经退行性疾病的离子载体 PBT-2，通过在更大程度上破坏细菌细胞内金属稳态来打破对多粘菌素的抗性。此外，发现银可替代完整 MCR 酶中的必需锌离子，以抑制 mcr-1 介导的体外和体内粘菌素耐药性（24）。这些努力表明，粘菌素与合理佐剂的组合具有极大的潜力，以增强目前基于粘菌素的治疗范例。
Here, we established a screening platform based on host-mimicking condition, in which three natural catechol-type flavonoids that synergized with colistin were selected. The three flavonoids substantially potentiated colistin efficacy against both colistin-sensitive and colistin-resistant isolates. Our mechanistic analysis demonstrated that these flavonoids disrupt bacterial iron homeostasis, dysregulating the iron signaling to promote colistin binding on bacterial membrane and subsequent accumulation of reactive oxygen species (ROS). Collectively, the current study unveiled the great potential of these catechol-type flavonoids as colistin adjuvants and highlighted iron signaling as an ideal target for colistin treatment.
本文建立了一个基于模拟宿主条件的筛选平台，筛选出三种与黏菌素具有协同作用的天然儿茶酚类黄酮化合物。这三种黄酮类化合物显著增强了粘菌素对粘菌素敏感和粘菌素耐药菌株的功效。我们的机制分析表明，这些黄酮类化合物破坏细菌的铁稳态，失调的铁信号，以促进粘菌素结合在细菌膜上，随后积累的活性氧（ROS）。总的来说，目前的研究揭示了这些儿茶酚类黄酮作为粘菌素佐剂的巨大潜力，并强调了铁信号作为粘菌素治疗的理想靶点。

RESULTS  结果

Primary screening identified catechol-type flavonoids as adjuvants to colistin
初步筛选确定儿茶酚类黄酮作为粘菌素的佐剂

The suboptimal clinical response of colistin in vivo has revealed that the resistances against polymyxin antibiotic can also be conferred to Gram-negative bacteria by intrinsic mechanism under defined host conditions (25). Therefore, we sought to establish a screening method that mimics the host condition to more precisely select adjuvants that can potentiate colistin efficacy in vivo. To this end, we adopted the low-phosphate, low-magnesium media (LPM) medium (26), which was constructed to resemble the condition in macrophage (Fig. 1A). The colistin MIC of Salmonella typhimurium strain ATCC14028s (S. Tm str. 14028s, colistin-sensitive) in LPM was 16 -fold higher than that from the Mueller Hinton (MH) medium, likely due to the acidification and magnesium unavailability in macrophage-mimicking conditions (table S1). Subsequently, a total of 37 phytochemicals were screened according to a previous method using the LPM medium (27). The growth profiles [optical density at 600 nm ( OD 600 nm ) 600 nm OD 600 nm 600nm(OD_(600nm))600 \mathrm{~nm}\left(\mathrm{OD}_{600 \mathrm{~nm}}\right) ] of bacteria in the presence of colistin, phytochemicals, or their combinations relative to that of no-drug control were presented as W X , W Y W X , W Y W_(X),W_(Y)W_{X}, W_{Y}, and W X Y W X Y W_(XY)W_{X Y}, respectively. The ε ~ ε ~ tilde(epsi)\tilde{\varepsilon} value for defining the interaction between colistin and phytochemicals was introduced as ( W X Y − W X W Y ) / ( W ~ X Y − W X W Y ) W X Y − W X W Y / W ~ X Y − W X W Y (W_(XY)-W_(X)W_(Y))//( tilde(W)_(XY)-W_(X)W_(Y))\left(W_{X Y}-W_{X} W_{Y}\right) /\left(\tilde{W}_{X Y}-W_{X} W_{Y}\right), where W ~ X Y W ~ X Y tilde(W)_(XY)\tilde{W}_{X Y} was equal to min [ W X , W Y W X , W Y W_(X),W_(Y)W_{X}, W_{Y} ] for W X Y > W X W Y W X Y > W X W Y W_(XY) > W_(X)W_(Y)W_{X Y}>W_{X} W_{Y} and 0 otherwise. The ε ~ ε ~ tilde(epsi)\tilde{\varepsilon} value of -0.5 was set as a cutoff to select the potential compounds that synergize with colistin. The primary screening identified three hits ( 8.1 % 8.1 % 8.1%8.1 \% ) as lead compounds that could potentiate colistin in LPM (Fig. 1B and table S2).
粘菌素在体内的次优临床反应表明，在确定的宿主条件下，革兰氏阴性菌也可通过内在机制对多粘菌素抗生素产生耐药性（25）。因此，我们试图建立一种模拟宿主条件的筛选方法，以更精确地选择可以增强黏菌素体内功效的佐剂。为此，我们采用了低磷酸盐、低镁培养基（LPM）培养基（26），其构建类似于巨噬细胞中的条件（图 1A）。鼠伤寒沙门氏菌 ATCC 14028s（S. Tm str.14028s，粘菌素敏感）的浓度是 Mueller 欣顿（MH）培养基的 16 倍，这可能是由于在巨噬细胞模拟条件下酸化和镁不可用（表 S1）。随后，根据先前的方法使用 LPM 培养基筛选了总共 37 种植物化学物质（27）。 在粘菌素、植物化学物质或其组合存在下的细菌相对于无药物对照的生长曲线[ 600 nm ( OD 600 nm ) 600 nm OD 600 nm 600nm(OD_(600nm))600 \mathrm{~nm}\left(\mathrm{OD}_{600 \mathrm{~nm}}\right) 处的光密度]分别表示为 W X , W Y W X , W Y W_(X),W_(Y)W_{X}, W_{Y} 和 W X Y W X Y W_(XY)W_{X Y} 。用于定义粘菌素和植物化学物质之间相互作用的 ε ~ ε ~ tilde(epsi)\tilde{\varepsilon} 值被引入为 ( W X Y − W X W Y ) / ( W ~ X Y − W X W Y ) W X Y − W X W Y / W ~ X Y − W X W Y (W_(XY)-W_(X)W_(Y))//( tilde(W)_(XY)-W_(X)W_(Y))\left(W_{X Y}-W_{X} W_{Y}\right) /\left(\tilde{W}_{X Y}-W_{X} W_{Y}\right) ，其中对于 W X Y > W X W Y W X Y > W X W Y W_(XY) > W_(X)W_(Y)W_{X Y}>W_{X} W_{Y} ， W ~ X Y W ~ X Y tilde(W)_(XY)\tilde{W}_{X Y} 等于 min [ W X , W Y W X , W Y W_(X),W_(Y)W_{X}, W_{Y} ]，否则等于 0。将-0.5 的 ε ~ ε ~ tilde(epsi)\tilde{\varepsilon} 值设定为截止值以选择与粘菌素协同作用的潜在化合物。初步筛选鉴定了三种命中物（ 8.1 % 8.1 % 8.1%8.1 \% ）作为可以增强 LPM 中的粘菌素的先导化合物（图 1B 和表 S2）。
Intriguingly, the three hits were all flavonoids and shared the same modification of catechol (1,2-dihydroxy benzene) moiety. Hence, we hypothesized that the catechol moiety might be vital
有趣的是，这三个命中都是黄酮类化合物，并共享相同的邻苯二酚（1，2-二羟基苯）部分的修饰。因此，我们假设儿茶酚部分可能是至关重要的
for the activity of these compounds. To address this hypothesis, a structure-activity relationship (SAR) analysis was conducted by comparing the activities of catechol-type flavonoids to their derivatives without the catechol-type flavonoids (Fig. 1C). As shown in Fig. 1D, the catechol-type flavonoids, including 7,8-dihydroxyflavone (7,8-DHF), myricetin (MYR), and luteolin (LUT), profoundly restored the colistin sensitivity against bacteria, yet no synergy was observed on the resorcinol-type flavonoids (5,7-DHF) and hydroxyflavone ( 7 − HF 7 − HF 7-HF7-\mathrm{HF} and 8-HF). Further details were given in fig. S1. Together, these findings suggest that the three catechol-type flavonoids identified in our primary screening are promising adjuvants to restore colistin activity in host-mimicking condition.
这些化合物的活性。为了解决这一假设，通过比较儿茶酚型黄酮类化合物与其不含儿茶酚型黄酮类化合物的衍生物的活性来进行结构-活性关系（SAR）分析（图 1C）。如图 1D 所示，儿茶酚型类黄酮，包括 7，8-二羟基黄酮（7，8-DHF）、杨梅素（MYR）和毛地黄黄酮（LUT），极大地恢复了粘菌素对细菌的敏感性，但在间苯二酚型类黄酮（5，7-DHF）和羟基黄酮（ 7 − HF 7 − HF 7-HF7-\mathrm{HF} 和 8-HF）上没有观察到协同作用。图 S1 中给出了进一步的细节。总之，这些发现表明，在我们的初步筛选中鉴定的三种儿茶酚型类黄酮是有希望的佐剂，以恢复黏菌素在模拟宿主条件下的活性。

Candidate flavonoids restore colistin activity against chromosome and plasmid-mediated colistin-resistant bacteria and minimize the emergence of resistance
候选类黄酮恢复粘菌素对染色体和质粒介导的粘菌素抗性细菌的活性，并最大限度地减少抗性的出现

With findings that three candidate flavonoids were able to potentiate colistin efficacy, we next determined whether these combinations are also effective against the colistin-resistant bacteria bearing m c r m c r mcrm c r genes or chromosomal m g r B m g r B mgrBm g r B mutation. The tests were performed on four representative MDR isolates of Gram-negative bacteria: mcr-1-positive 17ES (S. typhimurium), 2012FS (Escherichia coli), CMG (Klebsiella pneumoniae), and an mgrBdisrupted ZJ18-19 (K. pneumoniae). As illustrated in isobolograms, a typical synergistic effect between the candidate flavonoids and the colistin was observed with fractional inhibitory concentration index (FICI) ranging from 0.125 ± 0 0.125 ± 0 0.125+-00.125 \pm 0 to 0.458 ± 0.072 0.458 ± 0.072 0.458+-0.0720.458 \pm 0.072 (Fig. 2A and fig. S2). To reinforce the notion of synergism between the candidate flavonoids and colistin, a direct synergistic bactericidal assay was conducted on the aforementioned colistin-resistant strains. In the monotherapy assay, the application of colistin, 7 , 8 − DHF , MYR 7 , 8 − DHF , MYR 7,8-DHF,MYR7,8-\mathrm{DHF}, \mathrm{MYR}, and LUT could hardly kill the bacteria over time. In contrast, the combination of colistin plus any of the three candidate flavonoids rapidly eradicated the colistin-resistant strains, and the bacterial loads were reduced by 10 2 10 2 10^(2)10^{2} - to 10 5 10 5 10^(5)10^{5}-fold after treatments (Fig. 2B and fig. S3).
随着三种候选类黄酮能够增强粘菌素功效的发现，我们接下来确定这些组合是否也对携带 m c r m c r mcrm c r 基因或染色体 m g r B m g r B mgrBm g r B 突变的粘菌素抗性细菌有效。对革兰氏阴性菌的 4 种代表性 MDR 分离株进行了试验：mcr-1 阳性 17 ES（S.鼠伤寒沙门氏菌）、2012 FS（大肠杆菌）、CMG（肺炎克雷伯氏菌）和 mgrB 破坏的 ZJ 18 -19（克雷伯氏菌）。肺炎）。如等效线图所示，观察到候选类黄酮和粘菌素之间的典型协同效应，部分抑制浓度指数（FICI）范围为 0.125 ± 0 0.125 ± 0 0.125+-00.125 \pm 0 至 0.458 ± 0.072 0.458 ± 0.072 0.458+-0.0720.458 \pm 0.072 （图 2A 和图 S2）。为了加强候选类黄酮和粘菌素之间协同作用的概念，对上述粘菌素抗性菌株进行直接协同杀菌测定。在单一疗法测定中，随着时间的推移，粘菌素、 7 , 8 − DHF , MYR 7 , 8 − DHF , MYR 7,8-DHF,MYR7,8-\mathrm{DHF}, \mathrm{MYR} 和 LUT 的应用几乎不能杀死细菌。 相比之下，粘菌素加上三种候选类黄酮中的任何一种的组合快速根除了粘菌素抗性菌株，并且在处理后细菌载量减少了 10 2 10 2 10^(2)10^{2} -至 10 5 10 5 10^(5)10^{5} -倍（图 2B 和图 S3）。
To gain the insights on resistance development, both colistinsensitive and colistin-resistant strains ( S S SS. Tm str. 14028s, 17ES, and ZJ18-19) were serially passaged in the medium supplemented with colistin with or without candidate flavonoids. The development of resistance to colistin was observed to be reduced by the application of candidate flavonoids in three tested strains, whereas the colistin alone rapidly resulted in an increment of MIC up to eightfold (Fig. 2C). These results collectively suggested that the synergistic combinations of colistin with 7,8-DHF, MYR, and LUT were an efficient approach to eliminate the bacteria and minimize the potential emergence of resistance.
为了获得关于抗性发展的见解，将粘菌素不敏感和粘菌素抗性菌株（ S S SS . 14028 s、17 ES 和 ZJ 18 -19）在补充有粘杆菌素（含有或不含候选类黄酮）的培养基中连续传代。在三种测试菌株中观察到通过应用候选类黄酮降低了对粘菌素的抗性的发展，而单独的粘菌素迅速导致 MIC 增加高达八倍（图 2C）。这些结果共同表明，粘菌素与 7，8-DHF、MYR 和 LUT 的协同组合是消除细菌并最小化潜在耐药性出现的有效方法。

Candidate flavonoids target bacterial iron homeostasis to synergize with colistin
候选类黄酮靶向细菌铁稳态以与黏菌素协同作用

In view of the promising synergism between 7,8-DHF, MYR, and LUT with colistin, we sought to determine the underlying mechanisms. We first performed the RNA sequencing (RNA-seq) on the bacteria treated with single colistin or colistin in combination with 7,8-DHF, as a representative candidate of flavonoid. Transcriptomic data revealed that a total of 647 genes were differentially regulated after the treatment of the flavonoid-colistin combination compared with cells treated by colistin alone (fig. S4). The Gene Ontology (GO) enrichment analysis demonstrated that the differentially
鉴于 7，8-DHF、MYR 和 LUT 与粘菌素之间有希望的协同作用，我们试图确定潜在的机制。我们首先对用单一粘菌素或粘菌素与 7，8-DHF 组合处理的细菌进行 RNA 测序（RNA-seq），作为类黄酮的代表性候选物。转录组学数据显示，与单独用粘菌素处理的细胞相比，在类黄酮-粘菌素组合处理后，总共 647 个基因受到差异调节（图 S4）。基因本体（GO）富集分析表明，差异
A
B
C
O=C1CC(c2ccccc2)Oc2cc(O)ccc21OOOH
7-hydroxyflavone (7-HF)  7-羟基黄酮（7-HF）
C=C1c2c(O)cc(O)cc2Oc2cc(O)c(O)c(O)c2c1OHOHOOHOHOH
Myricetin(MYR)  马币（MYR）
O=c1cc(-c2ccccc2)oc2c(O)cccc12OOOH
8-hydroxyflavone (8-HF)  8-羟基黄酮（8-HF）
O=c1cc(-c2ccc(O)c(O)c2)oc2cc(O)cc(O)c12OOHOHOOHOH
Luteolin (LUT)  木犀草素（LUT）
O=c1cc(-c2ccccc2)oc2cc(O)cc(O)c12OOOHOH
5,7-dihydroxyflavone (5,7-DHF)
5，7-二羟基黄酮
D
Fig. 1. Primary screening identified three catechol-type flavonoids as colistin adjuvants. (A) Schematic illustration of the screening procedure. (B) Three flavonoids were identified as potent colistin adjuvants from the phytochemical collection. © Structure-activity relationship (SAR) analysis demonstrated the presence of catechol moiety on flavonoids as a prerequisite for observed synergy. (D) Fractional inhibitory concentration indices (FICls) showing the combination between colistin and cat-echol-type/non-catechol-type flavonoids.
Fig. 1.初步筛选确定了三种儿茶酚类黄酮作为黏菌素佐剂。(A)筛选程序示意图。(B)从植物化学品收集中鉴定出三种黄酮类化合物是有效的粘菌素佐剂。结构-活性关系（SAR）分析证明类黄酮上儿茶酚部分的存在是观察到协同作用的先决条件。(D)显示粘菌素与儿茶酚型/非儿茶酚型黄酮类化合物之间的组合的部分抑制浓度指数（FICl）。
expressed genes (DEGs) were highly associated with several pathways in biological processes, cellular components, and molecular functions. Of note, as highlighted in Fig. 3A, the pathways involved in iron homeostasis and uptake were significantly enriched. Iron uptake occurs mainly through two primary routes in bacteria (28). On one hand, the bacteria are able to import either ferric or ferrous cation through iron-chelating siderophore, and, on the other hand, the free ferrous ions can diffuse into periplasm of bacteria via porins, where they are subsequently transported into the cytoplasm by a panel of cytoplasmic proteins. Considering that iron is vital for bacterial physiology, it is possible that the candidate flavonoids resensitize the bacteria to colistin by disrupting their iron homeostasis. In this regard, we profiled the expression of genes associated with iron uptake and found that nearly all selected genes were repressed in response to 7 , 8 − DHF 7 , 8 − DHF 7,8-DHF7,8-\mathrm{DHF} (Fig. 3B and fig. S4). To test whether the flavonoid-mediated iron disruption was responsible for the observed synergy, we applied the colistin in combination with candidate flavonoids on the strain defective in iron transport ( ton B B BB-deficient and f e o B f e o B feoBf e o B-deficient) as well as their isogenic parental strain. The results indicated that the deficiency in ton B B BB, the major transmembrane importer of ferric cation, abolished the synergism of MYR and LUT. As to the 7,8-DHF, knockout of tonB and f e o B f e o B feoBf e o B also significantly impair its synergism with colistin, but to a lesser extent (Fig. 3C).
表达基因（DEG）与生物学过程、细胞组分和分子功能中的几个途径高度相关。值得注意的是，如图 3A 中突出显示的，参与铁稳态和摄取的途径显著富集。细菌主要通过两种主要途径吸收铁（28）。一方面，细菌能够通过铁螯合铁载体输入三价铁或亚铁阳离子，另一方面，游离的亚铁离子可以通过孔蛋白扩散到细菌的周质中，在那里它们随后通过一组细胞质蛋白转运到细胞质中。考虑到铁对细菌生理学至关重要，候选类黄酮可能通过破坏细菌的铁稳态来使细菌对粘菌素重新敏感。在这方面，我们分析了与铁摄取相关的基因的表达，发现几乎所有选择的基因响应于 7 , 8 − DHF 7 , 8 − DHF 7,8-DHF7,8-\mathrm{DHF} 而被抑制（图 3B 和图 S4）。 为了测试类黄酮介导的铁破坏是否是所观察到的协同作用的原因，我们将粘菌素与候选类黄酮组合应用于铁转运缺陷型菌株（ton B B BB -缺陷型和 f e o B f e o B feoBf e o B -缺陷型）以及它们的同基因亲本菌株。结果表明，铁离子的主要跨膜输入体 3 号吨的缺乏，破坏了最高产量和最低产量的协同作用。至于 7，8-DHF，tonB 和 f e o B f e o B feoBf e o B 的敲除也显著损害其与粘菌素的协同作用，但程度较小（图 3C）。
Then, we investigated the mechanistic insights into iron dysregulation mediated by flavonoids. The intracellular iron contents of bacteria were measured, and sharp decreases in total iron were observed after treatment with 7,8-DHF, MYR, and LUT (Fig. 3D). Note that the decline in ferric iron accounted for the majority of intracellular iron loss comparing with ferrous iron, and the iron was more in the ferrous form than in the ferric form (Fig. 3E). This is interesting because iron normally occurs in its biologically relevant ferric form because the ferrous ion is unstable under aerobic conditions (29). The excessive ferrous iron binds with Fur (ferric uptake regulator) as a cofactor, which thereafter represses iron acquisition via gene regulation (30). Thus, we assumed that the application of 7,8-DHF, MYR, and LUT decreased pool of accessible ferric iron by converting iron to ferrous form and subsequent blocking iron uptake via Fur regulon. To test this, the free ferric iron was incubated with 7,8-DHF, MYR, and LUT, and the majority of them were transformed into ferrous form, probably due to the reducibility of the flavonoids (Fig. 3F). As a comparison, the flavonoids without synergy (non-catechol-type) showed negligible ferrous iron conversion. In addition, the isothermal titration calorimetry (ITC) tests were performed to determine the interaction between the ferric iron with three candidate flavonoids. The result demonstrated that equilibrium dissociation constant ( K d K d K_(d)K_{\mathrm{d}} ) values between ferric iron and candidate flavonoids are 1.231 × 10 − 6 M 1.231 × 10 − 6 M 1.231 xx10^(-6)M1.231 \times 10^{-6} \mathrm{M}
然后，我们研究了黄酮类化合物介导的铁失调的机制。测定了细菌的细胞内铁含量，在用 7，8-DHF、MYR 和 LUT 处理后，观察到总铁急剧下降（图 3D）。请注意，与二价铁相比，三价铁的减少占细胞内铁损失的大部分，且二价铁形式的铁多于三价铁形式的铁（图 3E）。这一点很有趣，因为铁通常以其生物相关的三价铁形式存在，因为亚铁离子在有氧条件下不稳定（29）。过量的亚铁与作为辅因子的 Fur（铁摄取调节剂）结合，其随后通过基因调节抑制铁的获得（30）。因此，我们假设应用 7，8-DHF、MYR 和 LUT 通过将铁转化为亚铁形式并随后通过 Fur 调节子阻断铁摄取来减少可接近的三价铁池。 为了测试这一点，将游离三价铁与 7，8-DHF、MYR 和 LUT 一起孵育，并且它们中的大多数转化为亚铁形式，这可能是由于类黄酮的还原作用（图 3F）。作为比较，没有协同作用的黄酮类化合物（非儿茶酚型）表现出可忽略的亚铁转化。此外，等温滴定量热法（ITC）测试，以确定三价铁与三个候选黄酮之间的相互作用。结果表明，三价铁与候选黄酮类化合物之间的平衡解离常数（ K d K d K_(d)K_{\mathrm{d}} ）值为 1.231 × 10 − 6 M 1.231 × 10 − 6 M 1.231 xx10^(-6)M1.231 \times 10^{-6} \mathrm{M}
  1. ¹Guangdong Laboratory for Lingnan Modern Agriculture, National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China. 2 2 ^(2){ }^{2} Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics, Development and Safety Evaluation, South China Agricultural University, Guangzhou 510642, PR China. 3 3 ^(3){ }^{3} Hackensack-Meridian Health Center for Discovery and Innovation, Nutley, NJ, USA. 4 4 ^(4){ }^{4} Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, PR China.
    †岭南现代农业广东实验室，华南农业大学兽医学院动物源菌耐药性国家风险评估实验室，广州 510642，中国。 2 2 ^(2){ }^{2} 华南农业大学广东省兽药开发与安全性评价重点实验室，广州 510642。 3 3 ^(3){ }^{3} Hackensack-Meridian Health Center for Discovery and Innovation，Nutley，NJ，USA。 4 4 ^(4){ }^{4} 江苏省重大动物传染病与人畜共患病防控协同创新中心，扬州大学，扬州 225009。
    *Corresponding author. Email: hao.ren@scau.edu.cn (H.R.); gale@scau.edu.cn (Y.-h. L.); jiansun@scau.edu.cn (J.S.)
    * 通讯作者。电子邮件：hao.ren @ scau.edu.cn（人力资源）; gale@scau.edu.cn（Y.-），网址为：H. L.）; jiansun@scau.edu.cn（J.S.）