Fusobacterium nucleatum is a gram-negative anaerobe prevalent in many forms of human infections. It is particularly abundant in the oral cavity, playing an important role in the oral ecology and the pathogenesis of periodontal disease. It is also highly prevalent in intrauterine infections, associated with adverse pregnancy outcomes. Studies of this organism have been hindered by the lack of effective genetic tools. A novel genetic tool, sonoporation, was recently developed and used to construct the first allelic exchange mutant, US1 (fadA::ermF-ermAM), in F. nucleatum 12230, a strain known to be refractory to genetic manipulations. Sonoporation utilizes ultrasound to transiently increase the cell membrane permeability. This technology is becoming increasingly useful for drug and gene delivery into mammalian and plant cells, but had never been applied to bacteria until now. Even more interesting is that sonoporation using intact suicide plasmids produced double-crossover allelic exchanges, rather than single crossovers as would by electroporation. Thus, this technology has the potential to be developed into a powerful tool to streamline mutant construction. The focus of this exploratory study is to use F. nucleatum as a model organism to investigate the mechanism of sonoporation in bacteria. We hypothesize that sonoporation can be used for effective DNA delivery into F. nucleatum and for one-step double-crossover mutant construction.
Two specific aims are proposed.
In aim 1, different sonoporation conditions will be tested to identify those optimal for plasmid transformation and/or double-crossover mutagenesis in F. nucleatum.
In aim 2, the mechanism of sonoporation-mediated DNA delivery into F. nucleatum will be investigated. Our long term goal is to develop sonoporation into a robust tool for genetic manipulation of microorganisms ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DE017165-01A2
Application #
7303961
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2007-08-01
Project End
2009-07-31
Budget Start
2007-08-01
Budget End
2008-07-31
Support Year
1
Fiscal Year
2007
Total Cost
$220,850
Indirect Cost
Name
Case Western Reserve University
Department
Dentistry
Type
Schools of Dentistry
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Témoin, Stéphanie; Wu, Karen L; Wu, Vivian et al. (2012) Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament's length and width. FEBS Lett 586:1-6
Temoin, Stephanie; Chakaki, Alia; Askari, Ali et al. (2012) Identification of oral bacterial DNA in synovial fluid of patients with arthritis with native and failed prosthetic joints. J Clin Rheumatol 18:117-21
Han, Y W (2011) Oral health and adverse pregnancy outcomes - what's next? J Dent Res 90:289-93
Fardini, Yann; Wang, Xiaowei; Temoin, Stephanie et al. (2011) Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity. Mol Microbiol 82:1468-80
Fardini, Yann; Chung, Peter; Dumm, Rochelle et al. (2010) Transmission of diverse oral bacteria to murine placenta: evidence for the oral microbiome as a potential source of intrauterine infection. Infect Immun 78:1789-96
Han, Yiping W; Fardini, Yann; Chen, Casey et al. (2010) Term stillbirth caused by oral Fusobacterium nucleatum. Obstet Gynecol 115:442-5
Han, Yiping W; Shen, Tao; Chung, Peter et al. (2009) Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth. J Clin Microbiol 47:38-47
Ortiz, P; Bissada, N F; Palomo, L et al. (2009) Periodontal therapy reduces the severity of active rheumatoid arthritis in patients treated with or without tumor necrosis factor inhibitors. J Periodontol 80:535-40
Buhimschi, Catalin S; Bhandari, Vineet; Han, Yiping W et al. (2009) Using proteomics in perinatal and neonatal sepsis: hopes and challenges for the future. Curr Opin Infect Dis 22:235-43
Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori et al. (2009) Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. J Biol Chem 284:3865-72

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