Forestier C, Welch R A
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison 53706.
Infect Immun. 1991 Nov;59(11):4212-20. doi: 10.1128/iai.59.11.4212-4220.1991.
The Escherichia coli hemolysin (HlyA) and Pasteurella haemolytica leukotoxin (LktA) are cytolytic toxins encoded by genes belonging to the recently described RTX gene family. These cytotoxins are, respectively, 1,023 and 953 amino acids in length and are encoded by genes within identically organized operons. They share 45% amino acid sequence identities but differ in their target cell specificities. In vitro-derived recombinant hybrid genes between hlyA and lktA were constructed by using restriction endonuclease sites created by oligonucleotide site-directed mutagenesis. The cytolytic activity of hybrid proteins was investigated using as targets sheep erythrocytes and two cultured cell lines from different species (BL3, bovine leukemia-derived B lymphocytes; and Raji, human B-cell lymphoma cells). HlyA is cytolytic to all three cell types. LktA lyses only BL3 cells. Among the hybrid proteins displaying cytolytic activity, the striking finding is that the hemolytic activity of several LktA-HlyA hybrids was independent of any cytolytic activity against either cultured cell species. The hemolytic activity was associated with the HlyA region between amino acids 564 and 739. Structures that are critical for HlyA cytolytic activity against BL3 or Raji cells were destroyed when LktA-HlyA and HlyA-LktA hybrids were made, respectively, at amino acid positions 564 and 739 of HlyA. In contrast to HlyA, which lysed the two different cultured cell lines with equal efficiency, Lkt-HlyA hybrids possessing the amino-terminal 169 residues of LktA lysed BL3 cells more efficiently than Raji cells. This suggests that a significant but not exclusive element of the LktA ruminant cell specificity resides in the amino-terminal one-fifth of the protein. A molecular model of the functional domains of HlyA and LktA is presented.
大肠杆菌溶血素（HlyA）和溶血巴氏杆菌白细胞毒素（LktA）是由属于最近描述的RTX基因家族的基因编码的细胞溶解毒素。这些细胞毒素的长度分别为1023和953个氨基酸，由结构相同的操纵子中的基因编码。它们具有45%的氨基酸序列同一性，但靶细胞特异性不同。通过使用寡核苷酸定点诱变产生的限制性内切酶位点构建了hlyA和lktA之间的体外重组杂交基因。使用绵羊红细胞和来自不同物种的两种培养细胞系（BL3，牛白血病衍生的B淋巴细胞；以及Raji，人B细胞淋巴瘤细胞）作为靶标研究杂交蛋白的细胞溶解活性。HlyA对所有三种细胞类型都具有细胞溶解作用。LktA仅裂解BL3细胞。在显示细胞溶解活性的杂交蛋白中，引人注目的发现是几种LktA-HlyA杂交蛋白的溶血活性与对任何一种培养细胞系的细胞溶解活性无关。溶血活性与氨基酸564至739之间的HlyA区域相关。当分别在HlyA的氨基酸位置564和739处制备LktA-HlyA和HlyA-LktA杂交体时，对BL3或Raji细胞具有细胞溶解活性的HlyA结构被破坏。与以相同效率裂解两种不同培养细胞系的HlyA相反，具有LktA氨基末端169个残基的Lkt-HlyA杂交体裂解BL3细胞的效率高于Raji细胞。这表明LktA反刍动物细胞特异性的一个重要但非唯一的元件存在于该蛋白质的氨基末端五分之一处。本文提出了HlyA和LktA功能域的分子模型。
