Sponsored by the National Institute of Dental and Craniofacial Research, National Institutes of Health
Project Period: December 1, 2002 – April 30, 2014
This study is an in-depth investigation of the novel FadA adhesin from oral bacterium Fusobacterium nucleatum, which is associated with periodontal disease and preterm birth. FadA plays an important role in the bacterial colonization in the host and in causing preterm birth. Continued structure-function analysis of FadA will facilitate therapeutic development to inhibit the bacterial colonization in the host and to reduce the incidence of preterm birth. Identification of host components and pathways affected by FadA will facilitate therapeutic development of modulate specific cellular processes and targeted drug delivery.
Fusobacterium nucleatum is a Gram-negative anaerobe implicated in various forms of periodontal diseases. It is also associated with infections in other parts of the body and is one of the most prevalent species in intraamniotic infection, causing preterm birth. F. nucleatum binds to and invades host epithelial and endothelial cells, a mechanism allowing colonization at different host sites. Previous studies have shown that F. nucleatum can translocate to the pregnant mouse placenta via haematogenous transmission, followed by activation of localized placental inflammatory responses, leading to adverse pregnancy outcomes. Invasion of mouse placental endothelial cells by F. nucleatum has also been observed in vivo. So far, only one adhesin, FadA (for Fusobacterium adhesin A), has been identified to be required for bacterial binding and invasion of host cells in both tissue-culture and animal models. FadA is a unique adhesin consisting of two forms: the intact non-secreted form (pre-FadA) composed of 129 amino-acid (aa) residues, and the mature secreted form (mFadA) of 111 aa. The crystal structure of mFadA reveals two anti-parallel alpha-helices connected by an 8-aa loop. The crystal structure of mFadA suggests oligomerization in a head-to-tail pattern via a novel "leucine chain" motif. Filament formation and binding to host cells require both pre-FadA and mFadA. A FadA adhesion model has been proposed, with pre-FadA anchored in the inner membrane and a chain of mFadA on top of pre-FadA protruding through the outer membrane. We hypothesize that the receptor-binding site may be located in the loop region, only fully exposed at the tip of the filament. Furthermore, several of the FadA filaments may bundle together to form a cluster of loops, which may be required for binding. One focus of this proposed study is to test the FadA adhesin model. Using a yeast-two-hybrid system, several putative receptors have been identified to interact with FadA. Thus, a second focus of this study is to validate the interactions between the putative receptors and FadA and to investigate the host responses to FadA. Our specific aims are: Aim I. Further characterization of the FadA adhesin in F. nucleatum. Five sub-aims are proposed: (i) investigating fadA expression under different conditions, (ii) investigating the spatial arrangement of FadA in F. nucleatum, (iii) investigating the involvement of the loop region in host-cell binding, (iv) investigating the role of the signal peptide in FadA complex formation, and (v) investigating possible accessory molecules in F. nucleatum associated with FadA. Aim II. Investigation of FadA and host cell interactions. Two sub-aims are proposed: (i) continued identification and characterization of the FadA receptor, and (ii) investigating the effect of FadA on cellular processes. From this study, we hope to identify (i) potential therapeutic targets for inhibiting F. nucleatum colonization in the host, and (ii) host components and pathways affected by FadA, which will facilitate modulation of respective cellular processes and targeted drug delivery.
Han, Y. W., Redline, R. W., Li, M., Yin, L., Hill, G. B., and McCormick, T. S. (2004) F. nucleatum induces premature and term stillbirths in pregnant mice: implication of oral bacteria in preterm birth. Infect. Immun. Vol. 72:2272-2279.
Han, Y. W., Ikegami, A., Rajanna, C, Kawsar, H. I., Zhou. Y., Li, M., Sojar, H., Genco, R. J., Kuramitsu, H. K., and Deng, C. X. (2005) Identification and characterization of a novel adhesin unique to oral fusobacteria. J. Bacteriol. 187:5330-5340.
Han, Y. W. (2006) Laboratory maintenance of fusobacteria. In R. Coico, T. Kowalik, J. Quarles, B. Stevenson, and R. Taylor (ed.), Current Protocols in Microbiology, John Wiley & Sons, Inc.
Han, Y. W., Ikegami, A., Bissada, N., Herbst, M., Redline, R. W. and Ashmead, G. G. (2006) Transmission of uncultivated Bergeyella sp. from the oral cavity to amniotic fluid in preterm birth. J. Clin. Microbiol. 44:1475-1483.
Nithianantham, S., Xu, M., Wu, N., Han, Y. W., and Shoham, M. (2006) Crystallization and preliminary Xray data of the FadA adhesin from Fusobacterium nucleatum. Acta Cryst. F62: 1215-1217.
Han, Y. W., Ikegami, A., Chung, P., Zhang, L., Zhou, Y., and Deng, D. X. (2007) Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum. Appl. Environ. Microbiol. 73:3677-3683.
Xu, M., Yamada, M., Li, M., and Chen, S. G., and Han, Y. W. (2007) FadA from Fusobacterium nucleatum utilizes both secreted and non-secreted forms for functional oligomerization for attachment and invasion of host cells. J. Biol. Chem. 282:25000-25009.
Liu, H., Redline, R., and Han, Y. W. (2007) Fusobacterium nucleatum induces fetal death in mice by stimulating TLR4-mediated inflammatory response. J. Immunol. 179:2501-2508.
Nithianantham, S., Xu, M., Yamada, M., Ikegami, A., Shoham, M., and Han, Y. W. (2009) The crystal structure of the FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif: The leucine chain. J. Biol. Chem. 284:3865-3872.
Han, Y. W., Shen, T., Chung, P., Buhimschi, I. A., and Buhimschi, C. S. (2009) Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth. J. Clin. Microbiol. 47 (1)
Chung, P., Ahn, S. H., Ikegami, A., Shen, T., and Han, Y. W. (2009) FadA adhesin is required for Fusobacterium nucleatum colonization in mouse placentas. Infect. Immun. Vol. 77: 3075-3079
Fardini, Y., Chung, P., Joshi, N., and Han, Y. W. (2009) Transmission of diverse oral bacteria to murine placenta: evidence of oral microbiome as a source of intrauterine infection. Infect. Immun. Vol. 78:1789-1796.
Han, Y. W., Fardini, Y., Chen, C., Iacampo, K. G., Peraino, V. A., Shamonki, J. M., and Redline, R. W. (2009) Term stillbirth caused by oral Fusobacteriun nucleatum. Obstet. Gynecol. 115 (2):442-445.
Ikegami, A., Chung, P., and Han, Y. W.* (2009) Complementation of the fadA mutation in Fusobacterium nucleatum: the surface exposed adhesin promotes cellular invasion and placental colonization. Infect. Immun. 77(7):3075-3079.
Buhimschi, C. S., Bhandari, V., Han, Y. W., Dulay, A. T., Baumbusch, M. A., Madri, J. A., and Buhimschi, I. A. (2009) Using proteomics in perinatal and neonatal sepsis. Hopes and challenges for the future. Curr. Opin. Infect. Dis. 22(3):235-243.
Vernon, L. T., Demko, C. A., Whalen, C., Lederman, M., Toossi, Z., Wu, M., Han, Y. W., and Weinberg, A. (2009) Characterizing periodontal disease in HIV+ adults. Comm. Dent. Oral Epidemiol. Vol. 37: 427-437.
Fardini, Y., Temoin, S., Wang, X., Nithianantham, S., Lee, D., and Han Y. W.* (2011) Fusobacterium nucleatum adhesin FadA binds vascular-endothelial cadherin and increases endothelial permeability. Mol. Microbiol. doi. 10.1111/j. 1365-2958.2011.07905x.
Morsani, J. M., Amnioshariae, A., Han, Y. W., Montagnese, T. A., and Mickel, A. (2011) Genetic predisposition to persistent apical periodontitis. J. Endodon. 37(4):455-459.
Han, Y. W. * (2011) Oral health and adverse pregnancy outcome – what's next? J. Dent. Res. 90(3):289-293.
Fardini, Y., Chung, P., Dumm, R., Joshi, N., Redline, R. W., and Han, Y. W.* (2010) Implication of oral bacteria in placental infections. Placenta 31(8):A42.
Redline, R. W., Han, Y. W., Lu, C. Y., and Krishnan, L. (2010) Murine fetoplacental infection models. Infect. Immun. 78(11):4965.
Sun, Y., Karmakar, M., Sanhita Roy, S., Ramadan, R., Williams, S., Howell, S., Shive, C., Han, Y. W., Stopford, C. M., Rietsch, A., and Pearlman, E. (2010) Pseudomonas aeruginosa is regulated by TLR4/MD-2 and TLR5 on corneal macrophages, and is dependent on TRIF, Mal/TIRAP, and MyD88. J. Immunol. 185(7):4272-83.
Fardini, Y., Chung, P., Joshi, N., and Han, Y. W.* (2010) Transmission of diverse oral bacteria to murine placenta: evidence of oral microbiome as a source of intrauterine infection. Infect. Immun. 78 (4):1789-1796.
Han, Y. W.* "Fusobacterium nucleatum interaction with host cells" in Genomic Inquiries into Oral Bacterial Communities Edited by P. Kolenbrander, American Society of Microbiology Press (in press)