Fusobacterium nucleatum is an important pathogen that is commonly involved in periodontal disease and other common human infections. Little is known about the virulence mechanisms of this pathogen and investigation has been hampered by a lack of systems for genetic manipulation. The importance of F. nucleatum in oral and systemic diseases has led to interest in genomic sequencing within the next few years. Systems for molecular analysis will be required to effectively utilize sequence data in the study of virulence properties. The Principal Investigator's laboratory has recently isolated and characterized a F. nucleatum plasmid, pFN1. In this proposal, the hypothesis to be tested is that pFN1 can be used to develop a system of gene transfer as well as a host vector system for the application of molecular techniques in F. nucleatum.
In Specific Aim 1 a gene transfer system for F. nucleatum will be developed using electroporation with pFN1-based plasmids that encode a tetracycline selectable marker. Transformation studies will identify a """"""""first generation"""""""" shuttle plasmid, a F. nucleatum strain with high transformation efficiency, and additional antibiotic resistance determinants that confer a selectable phenotype in F. nucleatum. The gene transfer system will be optimized in Specific Aim 2 through refinement of both the shuttle plasmid and the transformation procedures. Characterization of the pFN1-based plasmids will include confirmation of the theta mechanisms of replication, and determination of segregational and structural stability, the pFN1 minimal replicon, and the plasmid copy number. A """"""""second generation"""""""" plasmid will be developed by eliminating nonessential regions of the pFN1 DNA. The efficiency of transformation will be optimized with the second generation shuttle plasmid and evaluated in representative F. nucleatum strains. A chromosomal integration plasmid for F. nucleatum, which lacks a F. nucleatum replicon but contains F. nucleatum genomic DNA sequences and a tetM determinant, will be developed in Specific Aim 3. The genomic organization of transformants will be evaluated by Southern and PCR analyses to confirm the chromosomal integration, and to characterize the site and mechanism of integration. The results of these studies will create the foundation for genetic analysis in F. nucleatum by developing molecular techniques of mutagenesis and complementation. Because systems of gene transfer are not currently available for F. nucleatum, these results will have significant impact on our understanding of F. nucleatum virulence and potentially in the prevention of human infectious diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29DE012639-03
Application #
6176059
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Mangan, Dennis F
Project Start
1998-06-01
Project End
2003-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
3
Fiscal Year
2000
Total Cost
$106,113
Indirect Cost
Name
University of California Los Angeles
Department
Dentistry
Type
Schools of Dentistry
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Bachrach, Gilad; Haake, Susan Kinder; Glick, Alon et al. (2004) Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system. Appl Environ Microbiol 70:6957-62
Gerardo, S Hunt; Yoder, S C; Citron, D M et al. (2002) Sequence conservation and distribution of the fusobacterial immunosuppressive protein gene, fipA. Oral Microbiol Immunol 17:315-20
Haake, S K; Yoder, S C; Attarian, G et al. (2000) Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems. J Bacteriol 182:1176-80