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

肠出血性大肠杆菌可引起婴儿腹泻、出血性结肠炎和人类溶血性尿毒综合征，其致病机制被认为与其产生的Vero毒素密切相关。据报道，两种类型的Vero毒素（VT 1和VT 2）都显示RNA N-糖苷酶活性，并切割真核细胞60 S核糖体亚基的28 S核糖体RNA N-末端4324位的腺苷糖苷键。这种酶活性导致EF-u依赖性tRNA结合的抑制，从而抑制蛋白质合成。通过比较VT 1、VT 2的A亚基和蓖麻毒素A链的一级结构，发现VT 1的N端有3个保守区（51-55、167-171和202-207位氨基酸残基）。蓖麻毒素A链的所有三个区域对应的位置蓖麻毒素的活性位点提出的X-射线晶体衍射分析。为了确定保守的氨基酸残基的毒素活性的VT 1的相对重要性，我们制备了VT 1突变体与单氨基酸取代的阿托西肽定向定点突变。制备了22个突变体，以检查14个保守残基及其对Vero细胞的细胞毒性，并将其对兔网织红细胞裂解物中蛋白质合成的抑制活性与野生型VT 1进行比较。用谷氨酰胺取代167位的谷氨酸，用亮氨酸取代170位的精氨酸，这两种活性都大大降低。这些结果表明，位置167的谷氨酸和位置170的精氨酸在VT 1的毒素活性中起重要作用。这些突变毒素可能是肠出血性大肠杆菌的保护性抗原。大肠杆菌感染有待进一步研究。

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.