A. actinomycetemcomitans is associated with human periodontal disease and expresses a leukotoxin (ItX) that kills human cells that are required for the immune response against infection. Most A. actinomycetemcomitans strains express low levels of this toxin, but some strains contain alterations in the ltx promoter and exhibit a hyper-leukotoxic phenotype. These strains exhibit altered regulation of the Itx genes and are associated with localized juvenile periodontitis. We will determine how the ltx genes are regulated in minimally leukotoxic organisms and how the regulatory processes are altered in hyper-leukotoxic organisms. We have identified a c/s-acting negative regulator of toxin expression that interacts with a trans-acting protein. This protein will be isolated and we will determine how this interaction reduces ltx expression. We will also determine if the acquisition of an insertion element (IS) leads to increased toxin expression by displacing this c/s-acting negative regulator upstream from the ltx genes. We will also determine if IS introduces an outwardly directed promoter that transcribes the ltx genes. We have also shown that ltx expression is induced during anaerobic growth. A c/s-acting sequence and a trans-acting polypeptide that may play a role in anaerobic regulation have been identified. We will determine if this sequence functions as an UP element that interacts with the ltx-subunit of RNA polymerase. Finally, the ltx operon possesses a fifth gene, orphan, that resides upstream from IXC. Frame shift mutations in orfA reduce ltx expression. Therefore, we will determine if the OrfA polypeptide is involved in the regulation of It expression and whether a naturally occurring deletion in orfA leads to the hyper-leukotoxic phenotype. It is clear that the leukotoxin is an important virulence determinant of A. actinomycetermcornitans and thus it is important to understand the mechanisms that lead to the hyper-leukotoxic phenotype. These studies will determine how/Ix expression is regulated in A. actinomycetemcomitans and will identify mechanisms that contribute to hyper-expression of the toxin. The cis- and/or transacting components that are found to differ in ltx hyper-expressing strains may represent new diagnostic targets to detect hyper-virulent A. Actinomycetemcomitans. It is also possible that the regulatory processes themselves may represent targets that can be exploited for the development of local or systemic therapies Aimed at affecting leukotoxin expression in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE010729-10
Application #
6879100
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Nokta, Mostafa A
Project Start
1994-04-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
10
Fiscal Year
2005
Total Cost
$285,180
Indirect Cost
Name
University of Louisville
Department
Dentistry
Type
Schools of Dentistry
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
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
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
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
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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