Localized aggressive (juvenile) periodontitis is a form of periodontal disease which affects medically underserved children in the United States. The etiologic agent Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium. To establish an ecological niche in the upper aerodigestive tract of man microorganisms have devised various schemes by which they evade or subvert host defense mechanisms designed to eliminate them-the simplest of these is to kill host immune cells. This application is designed to test our hypothesis that a bacterial protein toxin (LtxA) of this organism undergoes structural reorganization as the molecule transitions from an aqueous to the membrane environment to kill human immune cells. We will use liposomal bilayer phospholipid membranes in combination with wild type and mutant forms of LtxA to characterize the host parasite interaction. The outcomes of toxin/membrane interactions will be assessed for membrane binding, lipid raft association, and/or membrane disruption using biochemical, biophysical and morphological assays. The application has three specific aims that are designed to relate LtxA structure to its biologic function.
In Specific Aim #1 w will focus on toxin-induced membrane changes such as raft clustering/cholesterol binding, acylation and HII phase formation/membrane disruption using a panel of mutant LtxA proteins. Experiments proposed in Specific Aim #2 will determine fate of the toxin after an iniial membrane encounter. Does it remain at the cell surface or internalized and routed to lysosomes for breakdown by either the endosomal or autophagosomal pathway.
Specific Aim #3 contains studies of cellugyrin a transport protein and its potential role in the transprt of LtxA into the cell interior.
Localized aggressive (juvenile) periodontitis is a form of periodontal disease which affects medically underserved children in the United States. The etiologic agent Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium. This application is designed to test our hypothesis that a bacterial protein toxin (LtxA) of this organism undergoes structural reorganization as the molecule transitions from an aqueous to the membrane environment to kill human immune cells. We will use model membrane systems in combination with wild type and mutant forms of LtxA to characterize the host parasite interaction. The outcomes of toxin/membrane interactions will be assessed by membrane binding, raft association, and/or membrane disruption using biochemical, biophysical and morphological assays.
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