Aggregatibacter actinomycetemcomitans (Aa) is a facultative anaerobe that has been implicated in a variety of periodontal diseases. Its presence is most closely associated with localized aggressive periodontitis (LAP), a disease which primarily affects medically-underserved children in both in the United States and throughout the world. Tooth loss is the end stage of periodontal disease and children with LAP have both a cosmetic deformity since the front teeth are lost and a functional defect with a loss of their first molars, the keystone that maintains the Dental arch. The organism is vertically transmitted within families with a mother with LAP infecting her children with Aa in their infancy with the disease manifesting itself around puberty. These children then go on to infect their children in a self-perpetuating cycle that is LAP. The manner in which organism causes LAP is not known, however, it does produce a number of virulence factors. Our laboratory has concentrated on an RTX toxin that selectively kills human immune cells (leukotoxin;LtxA), the bodies first line of defense. All strains of Aa have the genes for encoding the leukotoxin, Aa strains found in the children with LAP are able to produce more of this virulence factor which suggests its importance in LAP. The current application focuses on an enigma of LtxA of being able to remain soluble in aqueous solutions yet interact with and ultimately damage the target cell membrane. The ability of LtxA to exist in these two opposite environments appears to be counterintuitive if the toxin exists as a rigid, three-dimensional tertiary structure. One explanation for the }one protein-two structures} that we observe with LtxA is that the toxin is an intrinsically disordered protein or contains disordered segments that lack well-structured, three-dimensional folding. Contact with the cell membrane and/or LFA-1 would then enforce folding of disordered regions of LtxA into stable secondary or tertiary structures. Our experimental plan will use model membrane systems to demonstrate the structural reorganization of LtxA (Specific Aim 1) and determine selectivity in the interaction of LtxA with lipid ordered and disordered membranes (Specific Aim 2) and to use electron microscopy and single particle reconstruction to visualize the aqueous and membrane inserted phases of LtxA (Specific Aim 3). Understanding the mechanism of LtxA action will provide insights into pathogenesis and therapeutic interventions in the treatment of LAP. Localized aggressive (juvenile) periodontitis is a form of periodontal disease which primarily effects the medically underserved population in the United States. The etiologic agent is believed to be Aggregatibacter actinomycetemcomitans, a Gram negative. This project will test the hypothesis that a bacterial protein toxin of the periodontal pathogen, A. actinomycetemcomitans undergoes structural reorganization as the molecule transitions from an aqueous to the membrane environment in order to kill human immune cells. To test this hypothesis, we will use liposomal bilayer phospholipid membranes in combination with wild type and mutant forms of the bacterial toxin produced by molecular biology techniques. The outcomes of toxin/membrane interactions will be assessed by biochemical, biophysical and morphological assays.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-MOSS-E (02))
Program Officer
Lunsford, Dwayne
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pennsylvania
Schools of Dentistry
United States
Zip Code
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
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
Fong, Karen P; Pacheco, Cinthia M F; Otis, Linda L et al. (2006) Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity. Cell Microbiol 8:1753-67
Hersh, Elliot V; Lally, Edward T; Moore, Paul A (2005) Update on cyclooxygenase inhibitors: has a third COX isoform entered the fray? Curr Med Res Opin 21:1217-26
Murakami, Y; Xu, T; Helmerhorst, E J et al. (2002) Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol 17:143-9
Haraszthy, V I; Lally, E T; Haraszthy, G G et al. (2002) Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans. Infect Immun 70:3170-9
Guthmiller, J M; Lally, E T; Korostoff, J (2001) Beyond the specific plaque hypothesis: are highly leukotoxic strains of Actinobacillus actinomycetemcomitans a paradigm for periodontal pathogenesis? Crit Rev Oral Biol Med 12:116-24
Jeyaseelan, S; Hsuan, S L; Kannan, M S et al. (2000) Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes. Infect Immun 68:72-9

Showing the most recent 10 out of 26 publications