Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.
The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.
Lally
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Research Center in Oral Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6002.
Kieba
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Demuth
D R
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Rosenbloom
J
J
Golub
E E
EE
Taichman
N S
NS
Gibson
C W
CW
D000188:Actinobacillus / Q000235:genetics*; D001427:Bacterial Toxins; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015245:Deoxyribonuclease BamHI; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics*; D005786:Gene Expression Regulation; D007166:Immunosuppressive Agents; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D001427:Bacterial Toxins; D005098:Exotoxins; D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; C018579:leukotoxin; D015245:Deoxyribonuclease BamHI