Induction of Aggregatibacter actinomycetemcomitans leukotoxin expression by IS1301 and orfA

Abstract

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 b-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.

Aggregatibacter actinomycetemcomitans Leukotoxin

“…The promoter region in these strains was 1926 bp in length and contained a DNA transposable element (IS1301) that was required for high leukotoxicity. More recently, Schaeffer et al (2008) characterized the mechanism that leads to overproduction of LtxA in these strains. They reported that both the transposase promoter and a protein encoded by an open reading frame just upstream of ltxC (OrfA) are required for maximal transcriptional activity of the operon.…”

Autoinducer-2 and QseC Control Biofilm Formation and In Vivo Virulence of Aggregatibacter actinomycetemcomitans

“…AI-2 produced by A. actinomycetemcomitans regulates expression of virulence factors, biofilm formation, and iron uptake and also influences the planktonic growth of the organism under conditions of iron limitation (13,38,41). A. actinomycetemcomitans possesses two periplasmic AI-2 receptors, LsrB and RbsB, both of which are linked to ABC transporters (19,40), suggesting that A. actinomycetemcomitans may import AI-2.…”

Differential Transcriptional Regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK Operons by Integration Host Factor Protein

“…This organism expresses LuxS and secretes autoinducer 2 (AI-2), and AI-2-dependent quorum sensing has been shown to regulate the expression of virulence factors, iron acquisition systems, and biofilm formation (17)(18)(19)(20). The complex regulatory network involved in A. actinomycetemcomitans biofilm growth and the role of quorum sensing in this process have begun to be explored in recent years.…”