Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

Abstract

Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions ؊69 to ؊35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the ؊68 to ؊40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of ⌬mlc, ⌬ihf, and ⌬ihf ⌬mlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a ⌬ihf ⌬mlc mutant still induces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.

Increased leukotoxin production: Characterization of 100 base pairs within the 530 base pair leukotoxin promoter region of Aggregatibacter actinomycetemcomitans

“…In addition, the consensus binding sites are also very similar 20 . In agreement with previous results we also conclude that the Mlc protein acts as an activator for the expression of ltx A 11 . In silico computational analysis suggested the presence of a binding site for the NagC, a potential transcriptional modulator.…”

“…Analysis of terminators and terminator strength showed the presence of a possible weak terminator in the region between 298–530 that can influence ltx A expression. Moreover, our results confirmed the observation that Mlc controls leukotoxin expression in our wild type RhAa3 strain as has been shown in the JP2 strain 11 . Further analysis of the exact region of the terminator and other possible regulatory elements in the leukotoxin promoter region is required.…”

“…Further, the THP-1 cell killing assay showed significantly reduced killing by RhAa-VS6 strain (the nag C deletion mutant). These results suggested that the Mlc did regulate leukotoxin expression in the monkey derived WT strain RhAa3 confirming previous results 11 . We also suggest that the Mlc activates ltxA expression in the RhAa3 strain with a similar mechanism as seen in the JP2-like strains.…”

“…Regulatory proteins such as CRP, (cAMP Receptor Protein) 9 , IHF (Integration Host Factor) 12 were recognized as either positive and/or negative regulators of ltx A expression respectively. Further, Mlc was identified as an activator of ltx A 11 .…”