Identification of RTX toxin target cell specificity domains by use of hybrid genes.
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.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison 53706.
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Forestier
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Welch
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D001483:Base Sequence; D016923:Cell Death; D002478:Cells, Cultured; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000472:pathogenicity*; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D016978:Mannheimia haemolytica / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D003603:Cytotoxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; C018579:leukotoxin