Transcript F. nucleatum - California State University, Long Beach

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 Cortez
Department of Microbiology
California State University, Long Beach
Gingivitis
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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:
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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 2150 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
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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
Structure of Lipopolysaccharide
(LPS)
O-Antigen
Outer Core
Inner Core
waaE
waaF
waaC
waaQ
Lipid A
P
P
waaY
KDO
He p
He p
P
KDO
He p
KDO
P
Ra Rb1 Rb2 Rb3Rc Rd1 Rd2 Re
P
Sialic Acid (Neu5Ac)
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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.
The Putative Sialic acid (Neu5Ac)
Operon
NeuT(Lst)
Wzx
NeuD
NeuB
NeuA
NeuC
Proposed function of Neu5Ac genes
OH
O
HO
HO
OH
N
H
O
N-Acetylglucosamine
(GlcNAc)
HO
OH
N-Acetylglucosamine 2-epimerase
N
H
O
OH
OH
O
O
O
O
O
HO
O
O
H
N
H
HO
(NeuC)
OH
O
H
N-Acetylneuraminic Acid-Containing Lipopolysaccharide
(LPS)
H2C
O
HO
HO
OH
+
N-Acetylmannosamine
(ManNAc)
O
O
P
O-
O-
Phosphenolpyruvate
(PEP)
Sialyltransferase
(NeuT)
N-Acetylneuraminic Acid Synthetase
(NeuB)
NH2
HO
HO
OH
H
HO
N
O
H
O
N
OH
O
H
OH
O
H
H
HO
OH
N
HO
N-Acetylneuraminic Acid
(NeuAc)
CMP N-Acetylneruaminic Acid Synthetase
(NeuA)
O
H
O
HO
OH
O
O
P
N
O
O
OOH
OH
CMP N-Acetylneuraminic Acid
(CMP-NeuAc)
O
Aims
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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
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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
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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
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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.
Nested PCR
NeuT
Wzx
NeuD
NeuB
NeuA
NeuC
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
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The sialic acid content of whole F.
nucleatum cells will be evaluated using a
standard colorimetric technique on a UV/
Visible spectrophotometer at 549nm.
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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.
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Research Design and Methods: DNA
sequencing and phylogenetic placement
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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
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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
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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
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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
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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
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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
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