Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins.
Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.
Lally
E T
ET
Research Center for Oral Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6002.
Golub
E E
EE
Kieba
I R
IR
Taichman
N S
NS
Rosenbloom
J
J
Rosenbloom
J C
JC
Gibson
C W
CW
Demuth
D R
DR
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005098:Exotoxins; C018579:leukotoxin