Distribution of Sialic Acid Operon Genes Among Clinical Isolates of Fusobacterium nucleatum, and the Correlation of Lipopolysaccharide Siaylation to the Severity Of the Gingival Disease.

Ana CortezDepartment of Microbiology California State University, Long Beach

Gingivitis• The mildest form of

periodontal disease• Plaque and tartar build

up is present at the gum line 

• Gums are red, swollen, and bleed easily

• There is usually little or no discomfort at this stage

• Gingivitis is reversible with professional treatment and good at home oral care.

Periodontitis Signs and symptoms of periodontitis may include:

Swollen and recessed gums Drainage or pus around

one or more teeth Plaque and tartar build up Bad breath An unpleasant taste in your

mouth Pain in one of your teeth,

especially when eating hot, cold or sweet foods

Loose teeth A change in your bite

Causes of Periodontal Diseases

• Periodontal diseases are serious bacterial infections that affect 15 % of adults between 21-50 and 30% of adults over 50 have this disease.

•Plaque (sticky, bacterial biofilm) and tartar (calcified plaque) build up along the gingiva cause inflammation.

•Pockets form between teeth and gums, making an anaerobic region where the presence of bacteria cause the destruction of tissue and bone that supporting teeth.

Fusobacterium nucleatum• Among the 500 species of

oral bacteria, F. nucleatum is the dominant species.

• One of the primary bacterium responsible for tooth and gum decay.

• Produces tissue irritants that inhibit fibroblast cell division and the wound healing process.

• Gram negative, anaerobic rods 5-10µm

P

P

P

P P

ReRdRdRcRbRbRbRa 211 2 3

waaFwaaE

waaQwaaC

waaY

KDO

KDO

KDO

HepHep

Hep

Lipid AInner CoreOuter CoreO-Antigen

Structure of Lipopolysaccharide (LPS)

Sialic Acid (Neu5Ac)

• Attenuates the complement cascade • Human host cells commonly decorate glycoproteins

with sialic acid: a self recognition strategy.• The sialylated capsules and Lipooligosaccharides of

bacteria have been shown to render them resistant to complement cascade mediated killing

• The putative sialic acid operon consist of six genes: Lst (NeuT), Wzk, NeuD, NeuB, NeuA and NeuC .

• spp. vincetii and spp. polymorphum have the putative sialic acid operon and spp. nucleatum does not.

NeuT(Lst) Wzx NeuD NeuB NeuA NeuC

The Putative Sialic acid (Neu5Ac) Operon

O OH

HO

N

HO

HH

OH

N-Acetylneuraminic Acid(NeuAc)

HO

OOH

O H

O

HO

N

HOHH

OH

CMP N-Acetylneuraminic Acid(CMP-NeuAc)

HO

OOH

O H

N

NH2

ON

O

OHOH

OPO

O-

O

OO

HO

N

HO

HH

OHHO

OOH

O H

O

OO

N-Acetylneuraminic Acid-Containing Lipopolysaccharide(LPS)O

HOHO

N

OH

OHO

H

N-Acetylmannosamine(ManNAc)

O

H2C

O

OH

PO O-

O-

Phosphenolpyruvate(PEP)

+

Sialyltransferase

(NeuT)

CMP N-Acetylneruaminic Acid Synthetase

(NeuA)

N-Acetylneuraminic Acid Synthetase

(NeuB)

N-Acetylglucosamine 2-epimerase

(NeuC)

OHO

HON

OH

OH

O

H

N-Acetylglucosamine(GlcNAc)

Proposed function of Neu5Ac genes

Aims

• We intend to determine the incidence of lipopolysaccharide siaylation and the presence of the putative sialic acid operon genes among 25 F. nucleatum strains.

• Among the strains with Neu5ac operon we will determine which genes compose each strains intrinsic operon and what order the genes are in.

• The incidence of Neu5Ac will be correlated to the severity of the gingival disease from which the strain was isolated.

Hypothesis

• We hypothesize that siaylation of F.nucleatum LPS will vary with the more severe forms of gingival diseases induced by strains that exhibit the phenotype and a less severe disease induced by strains that lack this phenotype.

Research Design and Methods: Gel electrophoresis and Southern blot

• 25 F. nucleatum strains isolated from patients diagnosed with one of these diseases : severe ulcerative periodontal disease, juvenile onset periodontitis, mild periodontitis and gingivitis.

• Use PCR to amplify genes, isolate the chromosomes of each strain using a kit and treat them with restriction enzymes.

• The fragments produced will be separated on an 1% agarose gel and then denatured to single stranded DNA.

• Southern hybridization will be performed using the separate Neu5Ac genes as probes to determine the presence of the Neu5Ac genes in each strain.

Research Design and Methods: Nested PCR

• Determination of the order of the Neu5Ac genes within operon of each strain will be carried out using a series of nested forward and reverse PCR primers synthesized for each gene.

• The orientation of the genes in the southern hybridization positive strains will be determined as illustrated.

NeuT Wzx NeuD NeuB NeuA NeuC

Nested PCR

Figure 1. The black lines are the amplicons of a strain of F. nucleatum. The relative sizes are due to insertions, deletions and rearrangement of base pairs.

Research Design and Methods: Sialic acid determination

• The sialic acid content of whole F. nucleatum cells will be evaluated using a standard colorimetric technique on a UV/ Visible spectrophotometer at 549nm.

• To discriminate between strains able to make sialic acid de novo and those that can incorporate exogenous pools, the strains will be compared after growth in media with or without exogenous sialic acid.

.

Research Design and Methods: DNA sequencing and phylogenetic placement

• DNA sequencing will be performed using universal primers to amplify the 16S rDNA, the intergenic transcribed spacer (ITS) and a short portion of the 23S rDNA.

• Construction of the phylogenetic tree will be executed using MrBayes: Bayesian Inference of Phylogeny software.

Correlation of results

• The hybridization and nested PCR results will be plotted onto our previously constructed phylogenetic tree.

• We hope to determine if Neu5Ac incorporation in F. nucleatum LPS is phylogenetically constrained.

• We will also attempt to find a correlatation with the presence of sialic acid in F. nucleatum LPS with the severity of disease in the individual from which the isolate was obtained.

Discussion: Results

• The results of the proposed study is that each F. nucleatum strain either contains the sialic acid operon or doesn’t.

• It is possible that some F. nucleatum strains have sialylated LPS but do not contain the entire putative Neu5Ac operon in which case the source of Neu5Ac is likely exogeneous.

• Out of the strains that do contain the operon, each can have any combination of six genes of the Neu5Ac operon with the possible addition or subtraction of any number of base pairs.

Discussion: Hypothesis

• We expect that siaylation of F. nucleatum LPS will vary with:

The sialylated strains being isolated from patients diagnosed with the more severe forms of gingival diseases.

The unsialylated strains being isolated from patients diagnosed with the milder gingival diseases.

Discussion: The Hypothesis is supported

If our hypothesis is supported two conclusions can be extrapolated.

The incorporation of Neu5Ac into F. nucleatum LPS can hinder the function of the host defenses via disruption of the complement pathway as has been shown in the case of N. gonorrhoeae, which is resistant to complement activation due to the presence of Neu5Ac in the bacterium LPS.

Siaylation of F. nucleatum LPS can be a mimicry based virulence factor. The siaylated LPS of H. influenzae and related bacteria have epitopes that mimic human antigens possibly allowing the bacteria to evade the immune system .

Discussion: the hypothesis is not supported

• If our hypothesis is not supported by the data collected, other aspects of the F. nucleatum LPS can explain its inhibitive effects to the host immune system.

• Little is known about the role of F. nucleatum LPS in the pathogenicity of the bacterium.

Future experiments

• As the Neu5Ac operon genes in this study were not sequenced, future experiments might include the cloning and sequencing of the sialic acid operon genes of the respective strains to prepare for studies that can determine the definitive function of each of the Neu5Ac enzymes.

Acknowledgements:

HHMI Dr. Clifton Franklund Partricia Amezcua Dr. Mason