Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide
Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide
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DOI：
10.1128/msphere.00146-20
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发表时间：
2020-03-01
期刊：
影响因子：
4.8
通讯作者：
Bitoun, Jacob P.
中科院分区：
文献类型：
--
作者：
Kiefer, Mallory C.;Motyka, Natalya, I;Bitoun, Jacob P.
产肠毒素大肠杆菌（ETEC）是低收入至中等收入国家儿童中的主要肠道病原体。先前的研究已经确定，产热稳定性肠毒素（ST）的ETEC是导致5岁以下儿童腹泻的主要病原体之一。在这项研究中，我们研究了铁和锌结合的人和猪ST的变体，并确定如何主机金属硫蛋白可以解毒ST。我们发现，ST纯化ETEC培养上清液洗脱作为一个双联体在C-18反相色谱。ST双峰的前缘分数被发现是缺乏铁，而ST双峰的后缘分数被发现含有可测量的铁。接下来，我们发现纯化的ST可以在还原和厌氧条件下用铁重建，并且铁结合的ST减弱了T84上皮细胞中cGMP的诱导。此外，我们证明了在铁浓度增加的情况下生长的ETEC 214-4上清液仅能在铁浓度大于5 μ M时诱导cGMP。体外研究还表明，ST结合锌，一旦结合，从ST中除去锌需要变性条件。锌结合ST也未能诱导cGMP。我们发现，ST有助于二硫键的感知氧化型谷胱甘肽池，增加锌从金属硫蛋白的释放速率，并可以被金属硫蛋白解毒。最后，我们发现ST诱导先前显示受去铁胺调控的基因的转录变化。这些研究表明，ST ETEC的发病机制可能与宿主粘膜金属状态密切相关。重要信息肠毒素大肠杆菌（ETEC）是低收入至中等收入国家儿童、部署军事人员和前往流行地区旅行者的主要腹泻病原体。热稳定毒素（ST）是一种具有3个二硫键的非免疫原性分泌性小肽。人们已经认识到，膳食二硫化物调节肠道氧化还原电位，ST可以使用外源性还原剂解毒。使用生物化学和光谱学方法，我们证明了ST可以在还原条件下分别结合铁和锌，从而降低ST的毒性。此外，我们表明，ST调节谷胱甘肽（GSH）/氧化型谷胱甘肽（GSSG）的比例，ST应被视为一种毒素氧化剂。ST可以通过氧化负载锌的金属硫化合物来解毒，导致游离锌释放。这些研究有助于为了解肠道病原体如何调节肠道氧化还原电位奠定基础，并可能影响我们如何为产生它们的病原体设计治疗方法和/或疫苗。
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heatstable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C-18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 mu M. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.