Aggregatibacter actinomycetemcomitans (Aa) has been strongly associated with localized aggressive periodontitis (1-3). At the anaerobic environment of the sub-gingival pocket Aa is exposed to changes in homeostasis, yet little is known about the processes used by Aa to acquire the necessary nutrients such as: iron, in order to adjust to changes and persist. The study the mechanism used by Aa to sense and respond to environmental stress is essential in order to to develop therapeutic treatments for periodontal disease and further the knowledge in the field of microbial endocrinology. A two component system (TCS), known as QseBC, is expressed by Aa and it plays an important role in the process of adaptation and survival in the anaerobic environment. The sensor molecule QseC was found to play a role in growth and virulence of Aa (1). Further, the signals responsible for the activation of this TCS were identified as being catecholamines and iron (CAT-Fe) (Weigel 2015). The protein YgiW, has no known function and it is co-expressed with the QseBC TCS. The regulation of the enterobactin operon genes were found to remain unchanged in the presence of CAT-Fe (Weigel 2015), when other genes involved in iron uptake were downregulated, suggesting that it must play an important role for Aa persistence in the anaerobic environment. Among the four genes encoded in the enterobactin operon there is an outer membrane enterobactin receptor, FepA (4), yet Aa does not produce siderophores (5). Localized aggressive periodontitis promotes increased infiltration of polymorphonuclear (PMNs) leukocytes or neutrophils (6). PMNs have been shown to release catecholamines (CAT), a type of siderophore, suggesting that the inflamed sub-gingival pocket may serve as a CAT-Fe rich environment. Therefore, the proposed hypothesis is that Aa utilizes catecholamines from the host environment and via QseBC, priming metabolism for growth in the anaerobic sub-gingival environment, and then acquiring iron through the uptake of CAT-Fe via the enterobactin complex. The hypothesis will be tested by addressing the following aims: 1) to characterize the synthesis, storage and release of CAT by PMNs stimulated with Aa, 2) to define the interaction of CAT-Fe with QseC and/or YigiW, and 3) determine the contribution of YgiW on QseC activation. The research design will consist of exposing human PMNs directly to wild type and virulence factors mutants of Aa and measuring CAT levels by ELISA to confirm their ability to release CAT (Aim 1a) and identify the signals required for their release (Aim 1b). The granules of PMNs will be isolated and screened using CAT antibodies, to identify where CAT are stored (Aim 1c). Next, biding kinetic analysis will be used to characterize the interaction of CAT-Fe to QseC and/or YgiW (Aim 2a) and surface plasmon resonance will be used to define YgiW contribution to QseC activation (Aim 2b). Lastly, binding kinetic analysis will be employed to determine the binding interaction of CAT-Fe to FepA (Aim 3a) and the chronic periodontitis animal model will be use to asses the role of the enterobactin operon in Aa virulence (Aim 3b).

Public Health Relevance

The mechanisms of Aggregatibacter actinomycetemcomitans (Aa) to sense and adjust to environmental changes will expand the knowledge and serve as a guide to work with similar organisms found in comparable niches. In addition, findings will further the field of microbial endocrinology and iron acquisition systems. The study of the proposed mechanism by which Aa exploits the host response to acquire iron presents the opportunity for future therapeutic targets for periodontal disease.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Predoctoral Individual National Research Service Award (F31)
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NIDR Special Grants Review Committee (DSR)
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Frieden, Leslie A
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University of Louisville
Schools of Medicine
United States
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