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The Oral Ecosystem with Microbial Biofilms
Research theme at the Faculty of Odontology, Malmö University
NEED FOR RESEARCH
Clinical research
In situ models
Research within
the ’Oral health’
profile
Oral health care
In vitro models
Implementation
Identification of new surface proteins
M
r
ScaA lipoprotein
Ssp
A Ssp
B
SGO_1487
Sg
c
Sg
c
Sg
c
SGO_1247
SGO_0060
SGO_0060
SGO_0890
p
5.
6.
5,
6.
I
0
0
The
figure0 shows how a0 strain of Streptococcus
gordonii,
lacking the protein linking adhesins to the cell wall, secretes
them in the culture medium. (2D-gel electrophoresis).
AgI/II
Oral
streptococci
Transmembrane enzymes
e.g. 5’-nucleotidase
Anchorless proteins e.g.
DnaK, EF-Tu, EF-g
Hsa
CshA/B
SGO_148
7
Oral streptococci express many
proteins, i.e. adhesins and enzymes on
their cell surface. We have identified
two new adhesins (SGO_0707 and
SGO_1487) that are covalently bound
to the cell wall via the LPXTG-motif.
Imbalance in the structure of
the salivary film can lead to
mechanical wear, xerostomia,
lesions and plaque
accumulation. Saliva works
as a lubricant by elastic
deformation over time. We
are developing methods to
measure and describe the
properties of saliva.
Davies JR, Svensäter G, Herzberg MC. (2009) Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii. Microbiology. 155, 1977-88.
How do oral mucosae react to the presence of bacteria?
© Faculty of Odontology, Mah
Saliva covers oral surfaces and interacts with bacteria
Secreted enzymes
e.g. challisin
FbpA
CONDITIONING
FILM
SUBSTRATE SURFACE
AbpA/B
SGO_0707
7
6
AgI/II
CshA/B
SGO_070
7
Csh
A
Csh
B
25
5
Rheological methods are used to describe the
viscosity and viscoelastic properties of saliva.
Submandibular-sublingual saliva has a significantly
higher viscoelasticity than parotid saliva,
specifically when secretion is stimulated.
Mats Stading, Daniel Johansson, Christina Diogo-Löfgren and Cecilia Christersson (2009) Viscoelastic properties
of saliva from different glands ANNUAL TRANSACTIONS OF THE NORDIC RHEOLOGY SOCIETY Vol.
17.
Bacteria degrade salivary mucins
Evaluation
Minor salivary gland secretions in children
and adults are investigated with respect to
flow and content. Saliva is collected using
Sialopaper, placed at different mucosal
locations. The flow is measured using the
Periotron 8000 (Proflow™) and content is
investigated using different biochemical
analytical methods. Changes in salivary
composition in relation to age have been
observed.
Mucins are large
glycoproteins that
form a protective
network on all oral
surfaces while at the
same time providing
initial attachment
sites for oral
bacteria. Through
cooperation bacteria
can break down both
the carbohydrateand protein part of
the mucins.
Wickström, C. & Svensäter, G. (2008) Salivary gel-forming mucin MUC5B – a nutrient for dental plaque bacteria Oral Microbiol Immun 23,
Wickström, C., Herzberg, M. C., Beighton, D. & Svensäter, G. (2009) Proteolytic degradation of human salivary MUC5B by dental biofilms
Microbiology 155, 2866-2872.
Mucins induce expression
p
of degradative
g
eenzymes
Sonesson M, Eliasson L, Matsson L. (2003) Minor salivary gland secretion in children and adults. Arch Oral Biol. 48: 535-539.
Sonesson M, Wickström C, Kinnby B, Ericson D, Matsson L. (2008) Mucins MUC5B and MUC7 in minor salivary gland secretion of
children and adults. Arch Oral Biol. 53: 523-527.
Interactions between salivary proteins and bacteria lead to changes in protein
expression (phenotypic changes) in the bacteria. An example is that mucins induce
an up-regulation of degradative enzymes within bacteria that interact with them. The
bacteria use these enzymes to degrade the mucins and then use the degradative
products as nutrients. Not all bacteria express these enzymes simultaneously, rather
focal points within the biofilm exhibit the degradative activity (see Figure).
We have developed a model in which oral epithelial
cells, growing on a surface, are exposed to bacteria.
Preliminary results suggest that living bacteria
attach to the cell surface and are internalised
without breaking down the epithelial cells.
Wickström, C., Herzberg, M. C., Beighton, D. & Svensäter, G. (2009) Proteolytic degradation of human salivary MUC5B by dental biofilms Microbiology 155, 2866-2872
Changes in bacterial properties are studied by:
Mutans Prediction in Skåne
mRNA-FISH (gene expression)
Markers for specific active genes in
bacteria can be visualized using this
technique. In this way changes in bacterial
physiology can be identified.
Proteomics (protein expression)
By visualizing bacterial proteins with
protein dyes, new or specific proteins can
be cut out and identified using massspectrometry.
When salivary
alivary proteins and bacteria interact, the bacteria develop stress resilient phenotypes. By intervening
interven
in
this process, new “anti-microbial” strategies, which prevent oral disease, can be developed.
Biofilms on implants
Identification by
mass-spectrometry
SDS/PAGE gel showing proteolytic
degradation of the protein
core of mucins
177–182
OVERALL HYPOTHESIS
The causes of certain lichenoid changes are
unknown. We are testing the hypothesis that
microbial biofilms have the ability to initiate
inflammatory changes in the oral mucosa.
The figure shows how the carbohydrate
structures of mucins are broken down
by bacteria (0-8h)
Osseo-integration is necessary for healing and retention of titanium
implants and biological modified titanium surfaces for oral use are
now available. In this project, microbial biofilms growing on these
surfaces are studied. Other types of surfaces studied are, for example,
peritoneal catheters.
Protein expression directly on the bacteria
After the identification of protein of
interest, we develop antibodies and are able
to visualize the proteins directly on the
bacteria using microscopy.
Biofilm bacteria are often acid tolerant
MuPiS is a clinical study comprising 909 5-7 year-old
children. The overall aim is to increase our knowledge of the
genetic contribution to dental caries, with focus on variation in
host immune response in terms of HLA, salivary IgA and
mutans streptococci.
Which bacteria can be found in dentine caries?
Bacteria growing in a biofilm express different properties than bacteria growing
in solution. Biofilm bacteria are more stress tolerant, for example more resilient
to antibiotics and more acid tolerant. Picture A shows a plaque sample, where
most of the bacteria are acid tolerant (green), whereas the sample in Picture B
contains mostly non-acid tolerant bacteria (red).
(Dye: LIVE/DEAD® BacLightTM Viability Stain).
A
B
A section through a dentine caries lesion
Flow cells are used to
study how microbial
biofilms develop.
mRNA-FISH techniques are used to
visualize different bacteria in the
growing biofilm.
Neilands J. (2007). Acid Tolerance of Streptococcus mutans Biofilms. Thesis, Malmö University.
Fluorescence microscopy picture of bacteria
sampled from a dentine caries lesion
Hypothesis: Different bacterial consortia can survive and generate the same net
disease promoting factors in the dentine carious process. The consortia will
differ at various depths in the lesion and between different lesions.
Project: Mapping of the dentine caries microbiota at various depths in the
dentine caries lesion.
Methods: Selective agar plating and molecular methods.
Co-ordinator:
[email protected]
Ph.D. students:
Researchers:
Christina Diogo Löfgren, Marjan Dorkhan, Helena Fransson, Victoria Fröjd, Christian A Kindblom, Liv Kroona, Anders Lager, Maria Pihl, Mikael Sonesson
Gunilla Andersson, Madeleine Blomqvist, Cecilia Christersson, Julia Davies, Kristina Hamberg, Ahmed Al-Hassani, Agnethe Henriksson, Bertil Kinnby, Eva Kreutz, Maaike Plantin,
Zdenka Prgomet, Gunnel Svensäter, Elisabeth Thornqvist, Ulrika Troedsson, Marie-Louise Wallengren, Gunnar Warfvinge