Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans , an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A . actinomycetemcomitans . Unexpectedly, an A . actinomycetemcomitans leukotoxin ( ltx A) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A . actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltx A mutation in vitro . Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly ( P <0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased ( P <0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [ rcp A ( P = 0.018), rcp B ( P = 0.02), tad A ( P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth ( P = 0.008) and exo-polysaccharide production ( P <0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltx A restored binding to SHA ( P <0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltx A diminished hard tissue binding in vitro , which helps explain the previous in vivo failure of a ltx A knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltx A ) could affect other seemingly unrelated genes (such as rcp A, rcp B tad A etc) , and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.