Download Fusobacterium Nucleatum

Identification of Genes Involved
in the Synthesis of Sialic Acid
from Fusobacterium Nucleatum.
Hatem Abdelhadi
California State University Long
Beach
Layout
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Introduction
Materials and Methods
Results
Conclusions
Future Experiments
Acknowledgements
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
Introduction
• F.nucleatum:
-Anaerobic, gramnegative, non sporeforming
-Long slender rods, with
tapering ends
-Many different virulence
factors: metabolic
products, proteins, and
lipopolysaccharides (LPS)
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
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
Materials and Methods
• Growth Conditions:
-F.nucleatum on BHI media at 370C in an
anaerobic glove box
• DNA prep:
- performed using DNA-prep kit
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 Nacetylneuraminic acid antibodies
-Ran on gel to see if sialic acid (Nacetylneuraminic acid) was synthesized
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
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
Results- LPS-prep Output Data
Con
10953
(-)Con
Figure 1. LPS output shows presence of LPS
in F10953 vs. controls.
Results- Immunoblot/ LPS-prep
Con
10953
(-)Con
Figure 2. Immunoblot assay of LPS presence in F10953
vs. controls.
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.
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
Future Experiments
• Study the effects that sialic acid has on the
virulence of F.nucleatum
Acknowlegdements
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Dr. Clifton Franklund
David Nolan
Dr. Archie and Dr. Mason
HHMI
References
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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.
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