Bast D J, Brunton J L, Karmali M A, Richardson S E
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Infect Immun. 1997 Jun;65(6):2019-28. doi: 10.1128/iai.65.6.2019-2028.1997.
The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome. To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits. The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro. This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer. In this study, purified 125I-labeled Phe30Ala was administered i.v. to rabbits to determine its specific distribution in rabbit tissues. In contrast to the rapid elimination of i.v. administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS). Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days. Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed. Rabbits were immunized with Phe30Ala and challenged i.v. with either 125I-VT1 or 125I-VT2. The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits. Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2. From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit. The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies. A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E. coli infections.
肠出血性大肠杆菌菌株产生的志贺毒素（VT1和VT2）与出血性结肠炎和溶血尿毒综合征的发病机制有关。为了更好地理解志贺毒素与球三糖神经酰胺（Gb3）受体结合在疾病发生中的作用，我们研究了静脉注射志贺毒素1突变体全毒素（Phe30Ala）对家兔的临床病理影响。先前已表明，VT1 B亚基中第30位苯丙氨酸被丙氨酸取代可降低其在体外与Gb3的结合亲和力和结合能力。受体结合的这种降低对应于VT1对Vero细胞单层毒性活性降低10^5倍。在本研究中，将纯化的125I标记的Phe30Ala静脉注射到家兔体内，以确定其在兔组织中的特异性分布。与静脉注射的125I-VT1从血液中快速清除不同，125I-Phe30Ala在血清中的半衰期延长了52倍，并且没有优先定位于胃肠道和中枢神经系统（CNS）。用相当于VT1半数致死剂量（LD50）10倍的剂量的Phe30Ala攻击的家兔在7天后未表现出典型的VT效应的明显临床症状。然而，用相当于VT1 LD50 100倍的剂量的Phe30Ala给药，尽管症状出现延迟，但导致了与家兔VT1毒血症难以区分的临床和组织病理学特征。用Phe30Ala免疫家兔，然后静脉注射125I-VT1或125I-VT2进行攻击。在Phe30Ala免疫的家兔中，125I-VT1在胃肠道和CNS中的特异性摄取被完全抑制。在用125I-VT2攻击的Phe30Ala免疫家兔中，仅观察到靶器官摄取部分减少。从本研究中，我们得出结论，Gb3结合负责VT1在靶器官的定位以及家兔中的疾病发生。Phe30Ala诱导针对VT1的强抗体和保护性反应的能力表明，受体结合特性降低的VT突变体可能在疫苗策略中有用。为了将其用作天然类毒素以预防人类产志贺毒素大肠杆菌感染，需要进一步降低Phe30Ala的毒性。
