1. Identification of Genes Involved in the Synthesis of Sialic Acid from Fusobacterium Nucleatum. Hatem Abdelhadi California State University Long Beach

2. Layout Introduction Materials and Methods Results Conclusions Future Experiments Acknowledgements

3. Introduction. Periodontal disease is a variety of inflammatory lesions affecting the periodontum The plaque-associated lesion is the predominant in the general population Plaque from patients w/periodontal disease has been isolated; over 400 different types of bacteria present Fusobacterium nucleatum has been consistently present among these patients

4. Introduction F.nucleatum: -Anaerobic, gram- negative, non spore- forming -Long slender rods, with tapering ends -Many different virulence factors: metabolic products, proteins, and lipopolysaccharides (LPS)

5. Introduction LPS consists of three domains: Lipid A region, core oligosaccharide, and an O-side chain or O antigen Variety of eukaryotic cells and pathogenic bacteria (including F.nucleatum) consist of a nine carbon sugar acid N-acetylneuraminic acid, or sialic acid Sialic acid residues located on the O antigen of the LPS Sialyation on the surface of bacterial cells can enable the bacterium to pass through host cell defenses and avoid complement and opsonophagocytosis

6. Introduction NeuA and NeuD (N-acetylneuraminic transferase encoding sequences) are the genes believed to be involved in the synthesis of sialic acid in F.nucleatum -Located on the distal end of the 0 antigen Our experiment will be to isolate LPS from F.nucleatum (10953) detect for sialic acid knockout NeuA and NeuD detect presence or absence of sialic acid

7. Materials and Methods Growth Conditions: -F.nucleatum on BHI media at 37 0 C in an anaerobic glove box DNA prep: - performed using DNA-prep kit

8. Materials and Methods LPS isolation and purification: -LPS-prep kit -Samples treated with neuraminidase to cleave from O antigen for detection Immunoblot: -Resulting LPS incubated with enzyme-linked N- acetylneuraminic acid antibodies -Ran on gel to see if sialic acid (N- acetylneuraminic acid) was synthesized

9. Materials and Methods Knockout PCR: - In-frame deletions of the genes targeted for deletion - Two asymmetric PCRs were used to generate fragments to the left and right of the sequences targeted for deletion - left and right fragments were annealed at overlapping region and amplified by PCR as a single fragment using the outer primers

10. Materials and Methods Knockout NeuA and D gene is then transferred to bacterial plasmid lacking origin of replication Introduced to Fusobacterium samples by way of Lambda phage kit Forced into bacterial chromosome –Plasmid contains Clandomycin resistance cassette, survival based on those bacteria that successfully incorporate plasmid into genome. Isolate LPS from mutant strain Perform another Immunoblot to detect the presence or absence of sialic acid in the mutant strain

11. Results- LPS-prep Output Data Con 10953 (-)Con Figure 1. LPS output shows presence of LPS in F10953 vs. controls.

12. Results- Immunoblot/ LPS-prep Con 10953 (-)Con Figure 2. Immunoblot assay of LPS presence in F10953 vs. controls.

13. Results- Immunoblot/Knockout LPS prep Con 10953 (-)Con Figure 3. Immunoblot assay of LPS presence of mutant F10953 vs. controls. Sialic acid not synthesized in mutant strain.

14. Conclusions NeuA and NeuD were observed to be the genes involved in the synthesis of sialic acid from Fusobacterium nucleatum The observations gathered from this study can be very important for the progression in understanding how F.nucleatum (and other periodontal bacteria) invade the tissue of the periodontum and cause inflammation and disease

15. Future Experiments Study the effects that sialic acid has on the virulence of F.nucleatum

16. Acknowlegdements Dr. Clifton Franklund David Nolan Dr. Archie and Dr. Mason HHMI

17. References Bolstad, A.I., H.B. Jensen, and V. Bakken. 1996. Taxonomy, Biology, and Periodontal Aspects of F.nucleatum. Clinical Microbiology Reviews. 9: 55-71. Bradshaw, D.J., and P.D. Marsh. 1998. Role of F.nucleatum and Coaggregation In Anaerobe Survival in Planktonic and Biofilm Oral Microbial Communities During Aeration. Infection and Immunity. 66:4729-4732. www.cellsalive.com