Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, 1ktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt-strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lkiCABD and IkiCA, respectively. Lkt-strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the Ikt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment.

Can presence or absence of periodontal pathogens distinguish between subjects with chronic and aggressive periodontitis? A systematic review

“…It has been noted, however, that the main difference between clinical groups may not be the prevalence but rather the amount of putative pathogens found in positive samples (Gunsolley et al 1990). Leukotoxin production, a factor considered important for virulence of AA, varies significantly among strains of this species (Zambon et al 1983b, Kolodrubetz et al 1989, Spitznagel et al 1991, Brogan et al 1994). The strain-specific difference in leukotoxin production seems to be regulated at the level of transcription (Spitznagel et al 1991).…”

“…Leukotoxin production, a factor considered important for virulence of AA, varies significantly among strains of this species (Zambon et al 1983b, Kolodrubetz et al 1989, Spitznagel et al 1991, Brogan et al 1994). The strain-specific difference in leukotoxin production seems to be regulated at the level of transcription (Spitznagel et al 1991). Brogan et al (1994) detected a 530 bp deletion in the promoter region of the leukotoxin operon and found that strains with this feature produced 10-20 times more leukotoxin.…”

Virulence factors of Actinobacillus actinomycetemcomitans relevant to the pathogenesis of inflammatory periodontal diseases

“…Nonleukotoxic strains have been found to contain all four genes of the Itx gene cluster. Leukotoxin-positive strains produce two mRNA transcripts encoding Itx-CABD and ItxCA, whereas leukotoxin-negative strains produce lower levels of ltxCA and no ltx-CABD [52]. Therefore, toxin regulation appears to be at the level of mRNA transcription or stability.…”

“…More recently, in an analysis of 17 strains of the organism, Brogan et al [51] have shown that the structure of the Itx promoter region of highly leukotoxic strains is similar and differs from that of non-leukotoxic strains. There is also evidence suggesting that leukotoxin production is under the control of environmental factors as it has been shown that the levels of Itx mRNAs are reduced in cells approaching the stationary phase of growth [52].…”

“…In addition to the leukotoxin, there is evidence to suggest that other components of A. actinomycetemcomitans are able to lyse PMNs. Brogan et al [51,52] have reported that the EDs0 for human PMNs of a 'non-leukotoxic' strain of the organism (NCTC 9710) is lower than that of a leukotoxic strain (Y4). This supports our finding that material obtained by saline extraction of strain NCTC 9710 was highly cytotoxic (EDs0 < 1.0 ng/ml) to human PMNs [57].…”