Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains

Abstract

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated as a virulence determinant in various human infections and is encoded by a multigene operon consisting of four known genes, designated ltxC, ltxA, ltxB, and ltxD. The ltx operon appears to be present in all A. actinomycetemcomitans strains, but levels of toxin expression vary greatly among strains. Thus, to gain a better understanding of the expression and regulation of the ltx operon, we have analyzed the ltx promoters of a highly toxic (JP2) and a minimally toxic (652) strain of A. actinomycetemcomitans. The nucleotide sequence of the JP2 ltx promoter contains -10 and -35 elements situated 350 bases upstream of ltxC, and primer extension of JP2 RNA confirmed that they are functional in vivo. However, a second primer extension product of 40 bases was present, and analysis of a series of truncated JP2 promoters fused to lacZ suggested that the region immediately upstream of ltxC also promotes transcription in Escherichia coli. These results suggest that two promoters may direct ltx expression in JP2. In addition, a small open reading frame capable of encoding a peptide of 78 amino acids was identified upstream of ltxC. Northern blots showed that this open reading frame is transcribed as part of a 4.2-kb mRNA, a transcript not previously identified as being derived from the ltx operon. In contrast, strain 652 expresses low steady-state levels of ltx mRNA, and its intact ltx promoter was inefficient in transcribing lacZ in E. coli. The nucleotide sequence of the 652 promoter is similar to that of the JP2 promoter but contains a region of 530 bp that is not present in JP2. Of 15 additional strains of A. actinomycetemcomitans that were analyzed, 13 contained promoters resembling the 652 sequence and 2 possessed JP2-like promoters. Both strains possessing the JP2-like promoter expressed 10- to 20-fold-higher levels of leukotoxin than did the strains possessing promoters resembling the 652 promoter. These results suggest that high levels of leukotoxin expression may correlate with the presence of the JP2-like promoter.

Characterization of a quinol peroxidase mutant inAggregatibacter actinomycetemcomitans

“…Hybridizations were carried out with ULTRAhyb (Ambion, Austin, TX) at 42 1C. Blots were probed with a 2.5-kbp fragment amplified from ltxA spanning nucleotides 644-3199 in the previously published ltxCA sequence (Brogan et al, 1994). Signal detection was carried out using anti-digoxigenin (DIG) alkaline phosphatase and CDP-Star (Roche Applied science, Indianapolis, IN) according to the manufacturer's instructions.…”

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

“…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].…”

“…The leukotoxin is encoded by an operon consisting of four genes, ItxC, ltxA, ItxB and ItxD. The ItxA gene encodes the leukotoxin itself, the ItxC gene may encode for an acyl carrier protein involved in acylation of the protein while the remaining two genes are probably involved in the transport of the toxin and its insertion into the membrane [51]. Nonleukotoxic strains have been found to contain all four genes of the Itx gene cluster.…”

“…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].…”

Leukotoxin fromAggregatibacter actinomycetemcomitanscauses shrinkage and P2X receptor-dependent lysis of human erythrocytes

“…Clinical data indicate that LtxA is a major virulence factor in A. actinomycetemcomitans-mediated periodontitis (Haraszthy et al, 2000a;Haubek et al, 2001;. Thus, a particular clone of the bacterium, termed the JP2 clone, is strongly associated with aggressive periodontitis in adolescents of west-or north-African descent, and members of the JP2 clone have a characteristic 530 bp deletion in the promoter region of the ltx gene operon leading to highly increased production of LtxA (Brogan et al, 1994). LtxA is a member of the repeat-in-toxin (RTX) family of bacterial exotoxins, which molecular characteristics include the presence of glycine-rich nonapeptide repeats.…”