Pellino Christine A, Karve Sayali S, Pradhan Suman, Weiss Alison A
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, USA.
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, USA
J Bacteriol. 2016 May 13;198(11):1621-1630. doi: 10.1128/JB.00918-15. Print 2016 Jun 1.
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5 toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5 complex is not required for cellular toxicity or in vivo toxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenic Escherichia coli is considered to be an AB5 heterohexamer; however, no known mechanisms ensure AB5 assembly. Stx released by E. coli is not in the AB5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5 assembly is a potential treatment for Stx-associated illnesses.
Complications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.
产志贺毒素（Stx）的大肠杆菌（STEC）是食源性疾病的主要病因，包括危及生命的并发症溶血尿毒综合征。2011年德国爆发的疫情导致近4000例感染，54人死亡。Stx有两种形式，Stx1和Stx2，其效力不同，Stx2a亚型最常与人类致命疾病相关。Stx被认为是一种AB5毒素。单个A（酶活性）亚基通过从真核生物rRNA上切割催化腺嘌呤来抑制蛋白质合成。B（结合）亚基形成同五聚体，并通过与糖脂球三糖神经酰胺（Gb3）结合介导细胞结合和毒素内化。两个亚基对毒性都是必需的。我们在此报告，与其他AB5毒素家族成员不同，Stx由STEC以未组装的A和B亚基形式产生。细胞毒性或对小鼠的体内毒性并不需要预先形成的AB5复合物，毒素组装可能发生在细胞膜上。我们证明，使用缺乏酶活性的A亚基肽破坏A和B亚基的结合可以保护小鼠免受致死剂量毒素的侵害。目前，尚无已被证明对溶血尿毒综合征有效的治疗方法。我们的研究表明，干扰A和B亚基组装的药物可能具有治疗潜力。致病性大肠杆菌产生的志贺毒素（Stx）被认为是一种AB5异源六聚体；然而，尚无已知机制确保AB5组装。大肠杆菌释放的Stx不是AB5构象，而是在受体界面组装。因此，未组装的Stx可产生毒性。这一发现表明，预防AB5组装是治疗与Stx相关疾病的一种潜在方法。
志贺毒素引起的并发症通常是致命的，目前，支持性护理是唯一的治疗选择。此外，由于抗生素能够增强细菌志贺毒素的表达，因此抗生素治疗是禁忌的。我们报告，与普遍的假设相反，STEC产生的志贺毒素以未组装的A和B亚基形式循环，其浓度对小鼠是致命的。与炭疽毒素的情况类似，组装发生在哺乳动物靶细胞表面表达的受体上。使用缺乏酶活性的A亚基肽破坏志贺毒素组装可保护小鼠免受志贺毒素的致命攻击，这为开发治疗方法提出了一种新途径。
