Identification of Anti-Aggregatibacter actinomycetemcomitans (Aa) Factors from Healthy Human Saliva

Objectives: Aa is a Gram-negative, non-motile coccobacillus that colonizes the human oral cavity (King and Tatum 1962). Aa is strongly implicated in the etiology of severe forms of Localized Aggressive Periodontitis (LAP). We have shown that those subjects who are free of Aa have a factor(s) in their saliva that prevents attachment and survival of Aa. Several subjects were tested and appear to have a factor in their saliva that inhibits the growth of Aa. The objective of this study is to identify, purify and characterize the Anti-Aa factor(s) from healthy saliva. Methods: Whole saliva obtained from a healthy subject that inhibited Aa growth was clarified by centrifugation at 3000Xg for 10 min and then purified using affinity column chromatography with bound Aa cells. The fractions were eluted from the column by Glycine-HCl (pH 2.3) and 3 M KSCN and were assessed by measuring the optical density of each fraction at 280 nm. The positive fractions were analyzed by SDS-PAGE. Results: Our results show that seven proteins were visualized from the positive fractions by silver stain and Lactoferrin (Lf) is one of the proteins was identified using Western Blot analysis. Further, we plan to identify and characterize the other six proteins and determine their effect on binding of Aa to Buccal Epithelial Cells (BECs) and killing assay individually. Conclusions: We have isolated seven proteins from healthy saliva, one or more of which may influence the ability of Aa to survive in the oral cavity of humans.