Actinobacillus actinomycetemcomitans (Aa) has been implicated in the pathogenesis of juvenile and adult periodontitis. This microorganism also is capable of causing more serious human infections such as endocarditis and soft tissue abscesses, and the likely source of these infections is Aa derived from oral sources. The ability of Aa to cause these disease states clearly depends on its ability to colonize and/or invade oral, cardiac, and soft tissues, and to elaborate various products that contribute to its survival in the host, or to cause damage to these tissues. The identification of Aa virulence traits, and tests for their actual roles in pathogenesis, will depend on the availability of modern genetic and molecular tools, and suitable animal models. With regard to the former, genetic and molecular analyses of Aa and its virulence have been lacking, due in large measure to the paucity of genetic transfer systems for this microorganism, and the total absence of appropriate host-vector systems for the application for recombinant DNA technology, including the delivery of manipulated genetic information for mutagenesis and studies of gene regulation. The applicant has described the isolation of plasmid DNA from two strains of Aa, and the construction of E. coli/Aa shuttle plasmids from one of the Aa replicons (pVT736-1), and the use of one of the shuttles to establish an electroporation-based transformation system for this species. More recently, the applicant has examined the properties of pVT736-1 in greater detail, and obtained preliminary data on the replication in Aa of three additional plasmids, two of Gram-negative and one of Gram- positive bacterial origin. The goal of this proposal is to assess the potential of all four plasmids as molecular and genetic tools for the analysis of Aa.
Two specific aims are: 1) to evaluate the properties of each plasmid relative to its potential as an E. coli/Aa shuttle vector; and 2) to construct a vector with properties that enhance recombinational events between Aa genomic DNA and plasmid DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE012107-02
Application #
2430145
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1996-06-01
Project End
1999-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Dentistry
Type
Schools of Dentistry
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Chen, Jinbiao; Pappas, Donald L; Galli, Dominique M (2010) Mapping of the nick site on conjugative plasmid pVT745 by interrupted mating. Plasmid 63:136-42
Galli, Dominique M; Chen, Jinbiao (2006) Entry exclusion activity on conjugative plasmid pVT745. Plasmid 55:158-63
Permpanich, Piyanuj; Kowolik, Michael J; Galli, Dominique M (2006) Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils. Cell Microbiol 8:72-84
Chen, Jinbiao; Leblanc, Donald J; Galli, Dominique M (2002) DNA inversion on conjugative plasmid pVT745. J Bacteriol 184:5926-34
Galli, Dominique M; Kerr, Micah S; Fair, Amber D et al. (2002) Parameters associated with cloning in Actinobacillus actinomycetemcomitans. Plasmid 47:138-47
Galli, D M; Chen, J; Novak, K F et al. (2001) Nucleotide sequence and analysis of conjugative plasmid pVT745. J Bacteriol 183:1585-94
Galli, D M; LeBlanc, D J (1997) Identification of a maintenance system on rolling circle replicating plasmid pVT736-1. dgalli@iusd.iupui.edu. Mol Microbiol 25:649-59