Cytotoxic mechanisms of Aggregatibacter actinomycetemcomitans leukotoxin

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DESCRIPTION (provided by applicant): Aggregatibacter ctinomycetemcomitans is a Gram-negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP) and other systemic infections, including infective endocarditis. LAP, which affects medically-underserved children in both the United States and throughout the world, is characterized by a breakdown of the periodontal ligament and alveolar bone structure that holds the teeth in place. Without intervention, loss of teeth occurs, causing both a cosmetic deformity and a functional defect. The manner in which A. actinomycetemcomitans causes LAP is not known; however, it is known that it produces several putative virulence factors, including a leukotoxin (LtxA), a member of the repeats-in-toxin (RTX) family. Based on experimental and clinical data, LtxA is believed to be a primary virulence factor for the bacterium. Thus, preventing or interfering with LtxA activity may be one option for treatment of disease. The toxin kills human and primate white blood cells and likely plays a role in A. actinomycetemcomitans evasion of the immune response during infection. It has been demonstrated that in its initial response with the host cell membrane, LtxA does not form a pore, but rather destabilizes the membrane. Additionally, LtxA is internalized within the cell, where it binds to the intracellular region of its receptor. In the mentored phase of this study, we will investigate the mechanisms of these two findings. In our first aim, we will identify the structural domains of LtxA that are responsible for membrane destabilization. The second aim will allow us to define the mechanism of internalization and determine the structural domains of LtxA that are responsible for internalization. In the independent phase of the study, these mechanisms and structural domains will be exploited in the design of therapeutic devices. In the third aim, a device to block LtxA activity by interfering with the cellular targets of the toxin will be developed. In the fourth aim, the internalization mechanism of LtxA will be utilized in the design of a drug-delivery device to carry a drug directly to the cytosol of a cell. The research will answer vital questions about the mechanisms by which LtxA kills host cells. In addition, the work will lead to the development of therapeutic agents, on with specific activity against LtxA and RTX toxicity and another with more general applications. During this work, the principal investigator will be trained in the necessary biological techniques during the mentored phase of the research that will be vital to her planned career as an independent investigator in microbial pathogenesis. Other elements of the proposed training plan, such as coursework, grant-writing, and a mentored faculty position search, will allow the principal investigator to transition to an independent career in which she applies her engineering background to the study of microbial pathogenesis.