Molecular Analysis of Aggregatibacter actinomycetemcomitans ApiA, a Multi-Functional Protein.

Abstract

Aggregatibacter actinomycetemcomitans ApiA is a trimeric autotransporter outer membrane protein (Omp) that participates in multiple functions, enabling A. actinomycetemcomitans to adapt to a variety of environments. The goal of this study is to identify regions in the apiA gene responsible for three of these functions: auto-aggregation, buccal epithelial cell binding, and complement resistance. Initially, apiA was expressed in Escherichia coli. Finally, wild-type A. actinomycetemcomitans and an apiA-deleted version were tested for their expression in the presence and absence of serum and genes related to stress adaptation, such as oxygen regulation, catalase activity, and Omp proteins. Sequential deletions in specific regions in the apiA gene as expressed in E. coli were examined for membrane proteins, which were confirmed by microscopy. The functional activity of epithelial cell binding, auto-aggregation, and complement resistance were then assessed, and regions in the apiA gene responsible for these functions were identified. A region spanning amino acids 186-217, when deleted, abrogated complement resistance and Factor H (FH) binding, while a region spanning amino acids 28-33 was related to epithelial cell binding. A 13-amino-acid peptide responsible for FH binding was shown to promote serum resistance. An apiA deletion in a clinical isolate (IDH781) was created and tested in the presence and/or absence of active and inactive serum and genes deemed responsible for prominent functional activity related to A. actinomycetemcomitans survival using qRT-PCR. These experiments suggested that apiA expression in IDH781 is involved in global regulatory mechanisms that are serum-dependent and show complement resistance. This is the first study to identify specific apiA regions in A. actinomycetemcomitans responsible for FH binding, complement resistance, and other stress-related functions. Moreover, the role of apiA in overall gene regulation was observed.