Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans

D. Kolodrubetz, T. Dailey, J. Ebersole, E. Kraig

Research output: Contribution to journalArticle

91 Scopus citations

Abstract

The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analyis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene.

Original languageEnglish (US)
Pages (from-to)1465-1469
Number of pages5
JournalInfection and Immunity
Volume57
Issue number5
StatePublished - Jan 1 1989

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Fingerprint Dive into the research topics of 'Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans'. Together they form a unique fingerprint.

  • Cite this