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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE009517-21A1
Application #
8704630
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
1990-07-01
Project End
2019-01-31
Budget Start
2014-03-14
Budget End
2015-01-31
Support Year
21
Fiscal Year
2014
Total Cost
$414,550
Indirect Cost
$138,000
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Dentistry
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Shahabuddin, Nishat; Boesze-Battaglia, Kathleen; Lally, Edward T (2016) Trends in Susceptibility to Aggressive Periodontal Disease. Int J Dent Oral Health 2:
Kaplan, Jeffrey B; Sampathkumar, Vandana; Bendaoud, Meriem et al. (2016) In vitro characterization of biofilms formed by Kingella kingae. Mol Oral Microbiol :
Balashova, N; Dhingra, A; Boesze-Battaglia, K et al. (2016) Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells. Mol Oral Microbiol 31:106-14
Brown, A C; Koufos, E; Balashova, N V et al. (2016) Inhibition of LtxA toxicity by blocking cholesterol binding with peptides. Mol Oral Microbiol 31:94-105
Bárcena-Uribarri, Iván; Benz, Roland; Winterhalter, Mathias et al. (2015) Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members. Biochim Biophys Acta 1848:1536-44
Brown, Angela C; Balashova, Nataliya V; Epand, Richard M et al. (2013) Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association. J Biol Chem 288:23607-21
Walters, M J; Brown, A C; Edrington, T C et al. (2013) Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures. Mol Oral Microbiol 28:342-53
Brown, Angela C; Boesze-Battaglia, Kathleen; Du, Yurong et al. (2012) Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization. Cell Microbiol 14:869-81
Fong, K P; Tang, H-Y; Brown, A C et al. (2011) Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains. Mol Oral Microbiol 26:262-76
Kieba, Irene R; Fong, Karen P; Tang, Hsin-Yao et al. (2007) Aggregatibacter actinomycetemcomitans leukotoxin requires beta-sheets 1 and 2 of the human CD11a beta-propeller for cytotoxicity. Cell Microbiol 9:2689-99

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