Study on Structure and Function of Vero Toxins (Shiga-like) Toxins Produced by EscherichiaDB coli

肠出血性大肠埃希菌可引起婴儿腹泻、出血性结肠炎和溶血性尿毒症综合征。发病机制被认为与生物体产生的Vero毒素密切相关。据报道，两种Vero毒素(VT1和VT2)都具有RNA N-糖苷酶活性，并从真核细胞60S核糖体亚基的28S核糖体RNA的N端断裂4324位的腺苷糖苷键。这种酶活性导致抑制EF-u依赖的tRNA结合，从而抑制蛋白质合成。对VT1、VT2的A亚基和蓖麻毒素A链的一级结构进行了比较，发现了三个保守的区域(来自VT1 N端的氨基酸残基51-55、167-171和202-207)。X-射线晶体衍射分析表明，蓖麻毒素A链上的三个区域在位置上都与蓖麻毒素的活性中心相对应。为了确定保守氨基酸残基对VT1毒素活性的相对重要性，我们通过寡核苷酸定向定点突变的方法制备了单一氨基酸取代的VT1突变体。制备了22个突变体，检测了14个保守残基，并比较了它们对Vero细胞的细胞毒性和对兔网织红细胞裂解物中蛋白质合成的抑制活性。将167位的谷氨酸替换为谷氨酰胺，将170位的精氨酸替换为亮氨酸，这两种活性都大大降低。这些结果表明，谷氨酸在167位和精氨酸在170位在VT1的毒素活性中起重要作用。这些突变毒素作为保护性抗原(S)保护肠出血性大肠杆菌感染的可能性有待进一步研究。

Enterohemorrhagic Escherichia coli causes infant diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in man. Pathogenesis is considered to be closely related to Vero toxins produced by the organisms. It has been reported that both two types of Vero toxins (VT1 and VT2) show RNA N-glycosidase activity and cleave a glycosidic bond of adenosine at position 4324 from N-terminus of 28S ribosomal RNA of 60S ribosomal subunit of eukaryotic cells. This enzymatic activity results in the inhibition of EF-u dependent tRNA binding and thus the inhibition of protein synthesis. This mode of action to that of ricin.Comparison of the primary structures of the A subunits of VT1, VT2 and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1). All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis. To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis. Twenty-two mutants were prepared to examine 14 conserved residues and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1. Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically. These results suggest that glutamic acid at position 167 and arginine at position 170 play the important role in the toxin activity of VT1. A possibility of these mutant toxins to be protective antigen (s) to protect enterohemorrhagic E. Coli infection wil be further studied.