Actinobacillus actinomycetemcomitans (Aa) is an important pathogen in the human oral cavity associated with localized juvenile periodontitis. The bacterium expresses several factors that may contribute to its virulence, one of which is a leukotoxin that kills human white blood cells. This might impair the immune response against Aa. Our studies suggest that the toxicity of Aa dramatically increased when a small DNA sequence was deleted from the promoter sequence of leukotoxin (ltx) operon or when an insertion element is present upstream of the operon. Both events result in a 10-fold increase in leukocyte expression. In the present application, we propose to determine how these events influence the overall expression of the ltx operon in Aa. This will be accomplished by evaluating the specific sequences that control ltx expression and determining if two promoters are functional in highly toxic Aa strains. We will also determine if the deletion removes a sequence that normally functions to repress ltx expression in Aa. Loss of repression may contribute to the constitutively high ltx expression found in highly toxic Aa strains. We will also investigate the mechanism whereby the acquisition of a transposable element stimulate ltx expression. We will determine if a new fusion promoter forms upon insertion of the IS element and/or if the IS element disrupts an existing regulatory sequence that controls ltx expression. Finally, we will determine if toxin-containing vesicles can function to deliver virulence factors (i.e., leukotoxin) to the target cell if concentrated form and if the elaboration of these vesicles is dependent upon the levels of leukotoxin expression. These studies will clarify the mechanism lead to the over-expression of leukotoxin in some strains of Aa and may identify potential new targets for anti-microbial therapy. This is clearly important in light of the increasing incidence of multiple drug resistant bacterial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE010729-06
Application #
6350580
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Mangan, Dennis F
Project Start
1994-04-01
Project End
2003-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
6
Fiscal Year
2001
Total Cost
$242,206
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Rabin, Shira D P; Flitton, Jared G; Demuth, Donald R (2009) Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces apoptosis in nonproliferating macrophages by a phosphatase-independent mechanism. Infect Immun 77:3161-9
Schaeffer, Lyndsay M; Schmidt, M Lee; Demuth, Donald R (2008) Induction of Aggregatibacter actinomycetemcomitans leukotoxin expression by IS1301 and orfA. Microbiology 154:528-38
Kinane, Denis F; Demuth, Donald R; Gorr, Sven-Ulrik et al. (2007) Human variability in innate immunity. Periodontol 2000 45:14-34
Mitchell, Christine; Gao, Ling; Demuth, Donald R (2003) Positive and negative cis-acting regulatory sequences control expression of leukotoxin in Actinobacillus actinomycetemcomitans 652. Infect Immun 71:5640-9
Fong, Karen P; Gao, Ling; Demuth, Donald R (2003) luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation. Infect Immun 71:298-308
Demuth, Donald R; James, Deanna; Kowashi, Yusuke et al. (2003) Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin. Cell Microbiol 5:111-21
Kato, Satsuki; Kowashi, Yusuke; Demuth, Donald R (2002) Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin. Microb Pathog 32:1-13
Fong, K P; Chung, W O; Lamont, R J et al. (2001) Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. Infect Immun 69:7625-34
He, T; Nishihara, T; Demuth, D R et al. (1999) A novel insertion sequence increases the expression of leukotoxicity in Actinobacillus actinomycetemcomitans clinical isolates. J Periodontol 70:1261-8
Hritz, M; Fisher, E; Demuth, D R (1996) Differential regulation of the leukotoxin operon in highly leukotoxic and minimally leukotoxic strains of Actinobacillus actinomycetemcomitans. Infect Immun 64:2724-9

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