IADR Abstract Archives
Effects of Aggregatibacter actinomycetemcomitans leukotoxin on binding to soft and hard tissues
Objectives: Aggregatibacter actinomycetemcomitans (Aa), a Gram-negative, periodonto-pathogen produces leukotoxin (Ltx) that can kill lymphocytes. Our previous non-human primate Aa colonization study showed that the ltxA knockout strain (ΔltxA) did not colonize the tooth surface of the monkeys compared to the wild-type (WT) strain. The objective of this study is to determine the binding ability of these two strains to the hard and soft tissues in vitro and to further assess the differential expression of genes that may contribute to binding and colonization of Aa.
Methods: The binding abilities of WT and ΔltxA to soft and hard tissues were assessed by the buccal epithelial cell (BEC) and hydroxyapatite (HA) binding assays. In both methods, Aa was treated with BEC or HA for 10-15 minutes and the number of bound CFUs were enumerated. Microscopically, variation in colony size and biofilm forming ability were observed. Further, we analyzed the differential gene expression between these two Aa strains in response to ItxA deletion using Real-Time PCR.
Results: Binding assays showed that the WT strain binds significantly (p<0.05) higher to the soft (BEC) and hard (HA) tissues (3-fold) in vitro. The WT colony is two times smaller in size compared to ΔItxA colony after 48hrs of growth in solid media. Further, the ΔItxA strain showed significantly less biofilm forming ability compared to WT strain. Under different conditions, these two strains showed significant (p<0.05) differences in expression of genes related to binding and colonization.
Conclusions: Ltx seems to play an important role in colonization and survival of Aa in vivo. Characterization of the ΔItxA strain in relation to its growth, binding and differential gene expression will help us understand the effect of Ltx on colonization and survival in the oral cavity.
Methods: The binding abilities of WT and ΔltxA to soft and hard tissues were assessed by the buccal epithelial cell (BEC) and hydroxyapatite (HA) binding assays. In both methods, Aa was treated with BEC or HA for 10-15 minutes and the number of bound CFUs were enumerated. Microscopically, variation in colony size and biofilm forming ability were observed. Further, we analyzed the differential gene expression between these two Aa strains in response to ItxA deletion using Real-Time PCR.
Results: Binding assays showed that the WT strain binds significantly (p<0.05) higher to the soft (BEC) and hard (HA) tissues (3-fold) in vitro. The WT colony is two times smaller in size compared to ΔItxA colony after 48hrs of growth in solid media. Further, the ΔItxA strain showed significantly less biofilm forming ability compared to WT strain. Under different conditions, these two strains showed significant (p<0.05) differences in expression of genes related to binding and colonization.
Conclusions: Ltx seems to play an important role in colonization and survival of Aa in vivo. Characterization of the ΔItxA strain in relation to its growth, binding and differential gene expression will help us understand the effect of Ltx on colonization and survival in the oral cavity.