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Most Aggregatibacter actinomycetemcomitans strains express relatively low levels of leukotoxin, encoded by the orfA-ltxCABD operon. However, several strains isolated from patients with localized aggressive periodontitis are hyperleukotoxic and transcribe the ltx operon at high levels. These strains possess a copy of IS1301 in the ltx promoter and previous studies have suggested that the presence of the insertion sequence increases ltx transcription by uncoupling a cis-acting negative regulator of ltx expression from the basal elements of the ltx promoter. However, we now report that replacing IS1301 with an equal length of random sequence has little effect on transcriptional activity of the ltx promoter, suggesting that the physical displacement of the negative regulatory element does not contribute to the hyperleukotoxic phenotype of IS1301-containing strains. Instead, we show that a -10-like element upstream of the transposase ORF of IS1301 is required for increased transcriptional activity of the ltx promoter. Site-specific mutation of the -10 sequence, or reversing the orientation of IS1301 relative to the basal ltx promoter elements, reduced transcriptional activity to levels exhibited by the native ltx promoter. However, no increase in transcription was observed when IS1301 was recombinantly inserted into a ltx promoter that contained a truncated copy of orfA, suggesting that an intact orfA may also be required for IS1301-mediated induction of ltxCABD. Therefore, to determine if orfA functions as a regulator of ltx expression, three independent ltx-promoter-lacZ-reporter constructs containing frameshift mutations in orfA were analysed. Each exhibited significantly lower expression of beta-galactosidase than the control reporter with intact orfA. In addition, OrfA protein was shown, by mobility shift electrophoresis, to interact with the ltx promoter at or downstream of the -35 sequence. These results suggest that a potential transposase promoter and the OrfA polypeptide may modulate leukotoxin expression in hyperleukotoxic A. actinomycetemcomitans strains containing IS1301.

Induction of Aggregatibacter actinomycetemcomitans leukotoxin expression by IS1301 and orfA