Submitted to: Journal of Bacteriology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/8/2013
Publication Date: 5/8/2013
Citation: Childress, C.J., Feuerbacher, L.A., Phillips, L., Burgum, A., Kolodrubetz, D. 2013. 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. Journal of Bacteriology. 195(10):2284-2297. Interpretive Summary: About 80% of adults in the US have some form of periodontal disease that ranges from gum inflammation to serious disease that can include both loss of gum tissue and bone tissue. During the course of periodontitis the gums can pull away from the teeth forming a pocket. One periodontal bacteria that can thrive in this pocket and exasperate this condition is Aggregatibacter actinomycetemcomitans (Aa). This particular pathogen can produce and release a toxin that destroys the immune cells that are trying to rid the mouth of this and other harmful bacteria. Aa can live and grow in two different environments, oxygenated and low-oxygen surroundings, similar to the environment found in the periodontal pocket. Aa's surroundings have a profound effect on the levels of toxin it produces, which increases in low-oxygenated conditions and decreases in higher-oxygenated conditions. We wanted to know how this bacteria increases or decreases toxin levels in response to environmental conditions at the genetic level. Previous work identified two proteins in the bacterial cell (IHF, CRP) that directly affect toxin level by either directly or indirectly binding to the cellular DNA code responsible for toxin production. Our goals were to identify other proteins that affect bacterial toxin production and to elucidate how these proteins work together. As a result, we identified a third protein (Mlc) that is required for toxin synthesis by binding to the toxin's DNA. Furthermore, in low-oxygenated conditions, the previously identified CRP protein works through this newly identified Mlc protein to increase toxin synthesis. Our results were surprising and significant since in another bacteria associated with human health, E. coli, the protein Mlc has the opposite function. In addition to demonstrating different interactions between CRP and Mlc and their effects on periodontal toxin production, we also discovered how the IHF protein decreases toxin production by blocking Mlc binding to the toxin DNA. Therefore, we conclude that Aggregatibacter actinomycetemcomitans (Aa) toxin production does not solely rely on the proteins previously identified but requires additional factors, as well.
Technical 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 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 -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 E. 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 knockout genes mlc, ihf and double knockout ihf/mlc strains demonstrate that Mlc can increase RNAP activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a double knockout 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.