Table_1_Induction of the Stringent Response Underlies the Antimicrobial Action of Aliphatic Isothiocyanates.DOCX

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain—enterohemorrhagic Escherichia coli (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress. The important source of potentially beneficial compounds are secondary plant metabolites, isothiocyanates (ITC), and phytoncides from the Brassicaceae family. We reported previously that sulforaphane and phenethyl isothiocyanate, already known for their chemopreventive and anticancer features, exhibit significant antibacterial effects against various pathogenic bacteria. The mechanism of their action is based on the induction of the stringent response and accumulation of its alarmones, the guanosine penta- and tetraphosphate. In this process, the amino acid starvation path is employed via the RelA protein, however, the precise mechanism of amino acid limitation in the presence of ITCs is yet unknown. In this work, we asked whether ITCs could act synergistically with each other to increase the antibacterial effect. A set of aliphatic ITCs, such as iberin, iberverin, alyssin, erucin, sulforaphen, erysolin, and cheirolin was tested in combination with sulforaphane against E. coli. Our experiments show that all tested ITCs exhibit strong antimicrobial effect individually, and this effect involves the stringent response caused by induction of the amino acid starvation. Interestingly, excess of specific amino acids reversed the antimicrobial effects of ITCs, where the common amino acid for all tested compounds was glycine. The synergistic action observed for iberin, iberverin, and alyssin also led to accumulation of (p)ppGpp, and the minimal inhibitory concentration necessary for the antibacterial effect was four- to eightfold lower than for individual ITCs. Moreover, the unique mode of ITC action is responsible for inhibition of prophage induction and toxin production, in addition to growth inhibition of EHEC strains. Thus, the antimicrobial effect of plant secondary metabolites by the stringent response induction could be employed in potential therapeutic strategies.

细菌对现有抗生素（antibiotics）的耐药性已对公共卫生构成严重威胁。多重耐药致病菌株的传播，促使科研人员基于新开发的化学化合物与天然存在的活性制剂，探寻全新的治疗策略。此外，针对携带肠毒素的菌株——肠出血性大肠埃希菌（enterohemorrhagic Escherichia coli, EHEC）引发的感染实施抗生素治疗，因抗生素介导的应激可能诱导噬菌体编码的毒素产生，故而被视为具有危险性且存在争议。
潜在有益化合物的重要来源，当属十字花科（Brassicaceae）植物的次生代谢产物、异硫氰酸盐（isothiocyanates, ITC）以及植物杀菌素。我们此前的研究证实，萝卜硫素（sulforaphane）与苯乙基异硫氰酸盐（phenethyl isothiocyanate）——二者以化学预防与抗癌特性闻名——对多种致病菌具备显著的抗菌活性。其作用机制基于诱导严谨反应（stringent response）并积累其报警素：五磷酸鸟苷与四磷酸鸟苷。该过程经由RelA蛋白触发氨基酸饥饿通路，但异硫氰酸盐存在时氨基酸限制的精确机制仍未阐明。
本研究旨在探究异硫氰酸盐之间是否可产生协同作用，以增强抗菌效果。我们将一系列脂肪族异硫氰酸盐（aliphatic ITCs），包括iberin、iberverin、alyssin、erucin、sulforaphen、erysolin与cheirolin，与萝卜硫素（sulforaphane）联合，针对大肠埃希菌开展抗菌实验。实验结果显示，所有受试异硫氰酸盐单独使用时均具有强劲的抗菌活性，且该活性依赖于氨基酸饥饿诱导的严谨反应。有趣的是，过量添加特定氨基酸可逆转异硫氰酸盐的抗菌效果，其中所有受试化合物共同针对的氨基酸为甘氨酸。iberin、iberverin与alyssin联用所展现的协同作用，同样可导致（p）ppGpp的积累，且其发挥抗菌效果所需的最低抑菌浓度，较单独使用异硫氰酸盐降低了4至8倍。此外，除抑制肠出血性大肠埃希菌的生长外，异硫氰酸盐的独特作用模式还可抑制前噬菌体诱导与毒素产生。因此，通过诱导严谨反应发挥抗菌活性的植物次生代谢产物，有望应用于潜在的治疗策略中。