18. Biofilm Matrix Flashcards
Components of the biofilm matrix
- macromolecules like polysaccharides, proteins and nucleic acids
- smaller ones like nutrients, metals and signalling molecules
What macromolecules are present in biofilm matrix?
- polysaccharides
- proteins
- nucleic acids
Explain exopolysaccharide synthesis
- first discovered as polymer of glucose (made by strep mutans, called mutan)
- formed outside the cell using sucrose as substrate
- complex structures with many types
- some soluble, some insoluble
2 basic types of exopolysaccharide with example
- glucans like glucosyltransferase
- fructans like fructosyltransferase
Most exopolysaccharides are made inside/outside the cell?
- inside
then exported out
Why are exopolysaccharides made inside cells then exported out?
- need energy to build
- no energy for this so comes from bonds in sucrose found inside cells
What do exopolysaccharides make a colony look like?
- cystalline colony appearance
- on a sucrose-containing agar
How is sucrose a good substrate for glucan synthesis?
- glycosidic bond formation needs 13.5 kJ/mol
- free energy available from glycosidic bond in maltase is 16.7 kJ/mol
- free energy in glycosidic bond of sucrose is 29 kJ/mol (equivalent of phosphorylated precursors e.g UDP-glucose)
Note from glucan synthesis - an anomeric carbon is a …
- stereocentre
- if an OH group is down, alpha carbon
- if otherwise it’s beta
Name of fructan polymers
What bond do they have?
- inulins
- beta-2,1 bond type
- levens
- beta 2,6
Inulins are an … polymer
Explain them
- insoluble
- almost identical to fructose polymer in veg
- inulin from beg has terminal residual residue found in dandelion, burdock, chicory, onion, garlic
- long term storage carb instead of starch
How are inulins made?
- only by strep. mutans and some strep, salivarius
- make large amounts
- 95% 2,1 bonds
- 5% 2,6 bonds
Inulins are a … carbohydrate
storage
Levens are …
water soluble
Why are levens named so?
- rotates polarized light to the leftLev
Levens are made by …
- strep. sanguinis
- strep. salivarius
- actinomyces naeslundii
- some strep. sobrinus
NOT strep.mutans
Role of levens
- spores can last years
- once activated, produce calcium carbonate strong enough to glue concrete
What is ‘BacillaFilla’?
- a biological approach to repairing concrete
- with calcium carbonate and cells
- glued together by levan
Which survives better in the biofilm? Glucans or fructans?
glucans
2 basic types of glucans
- water insoluble mutans (alpha 1,3 linked)
- water soluble dextrans (alpha 1,6 linked)
Explain mutans
- insoluble glucose polymer from strep, sobrinus 6715
- almost completely alpha 1,3 bonds
- 70% of plaque exopolysacc is mutan (primary glucan polymer)
Some strains of mutan make 85-90% alpha-1,3 with the remainder being …
Explain
- alpha-1,6
- for example strep mutans includes small and approx equal number of alpha 1,6 and 1,3,6 bonds
- ratio of alpha-1,3 linked backbone residues to the other 2 types is around 12:1:1
- possible structure is polymer with approx every 12th residue containing alpha-1,6 branch which is alpha-1,3 linked to terminal glucose
Strcuture of dextran
- almost all alpha-1,6 glycosidic bonds
- synthesised by strep salivarius
- rotates polarized light to the right
What variations of dextran are present?
- numerous side chains of alpha-1,3 linked glucose
- synthesised by strep sobrinus
- actual structure needs defining but likely to be similar to typical
- side branch is short compared to backbone
- one side branch for around every 15 backbone residues
What similar dextran-like polymers are made?
- one by strep mutans
- side chains twice as numerous but shorter
- comprises just a single glucose residue linked to alpha-1,3 to branch residue
How does environment impact glucans?
- soem form polymers of alpha-1,3 and 1,6 bonds
- e.g GTFG of strep gordonii (produces ratio of 1,3 to 1,6 between 25 to 40% )
- doesn’t need a primer unlike glycogen formation
- exogenous glucan accelerates reaction (binds to site remote from catalytic site and conformational change occurs)
How do enzymes impact glucans?
- GTFs are extracellular (secreted into complex env like plaque and many bacterial species secrete own enzymes (GTFs and others))
- glucans can act as acceptors for unrelated GTF
- reaction is semi-processive
- GTF glucan soup
- proteases can modify GTF and dextranases
What are
- GTF
- FTF?
- gllucosyltransferase
- fructosyltransferase
Types of GTFs
- dextransucrase
- mutansucrase
Types of FTFs
- inulosucrase
- levansucrase
GTFs and FTFs are classfied early on what?
water solubility
GTF-S enzymes produce what?
- water soluble dextran-like glucans with alpha-1,6 linked backbone
GTF-I enzymes produce what?
- insoluble alpha-1,3 backbone polymer
GTF-SI enzymes produce what?
- produce partially soluble alpha-1,3 backbone polymer
Is there lots of consensus on GTF and FTF?
- no general agreement at all
- confusion
- primer independent GTF was called GTFSi
- is this the same as GTF-SI?
- nobody knows
- but solution lies in genes
Absolute identification of discrete enzymes is best done by …
referring to them as a product of particular genes
GTF-I, SI and S are enzymes found in …
S.mutans
GTF-I is a product of gene …
gtfB
GTF-SI is a product of gene …
gtfC
GTF-S is a product of gene …
gtfD
How is the gene identified and named for the GTF enzymes?
- appending a non-italicised letter in alphabetical order
- starting wit hthe first to be isolated and sequenced
What happened to gtfA?
- gtfA product is actually a sucrose phosphorylase
- that transfers glucose from sucrose to a phosphate
- bears little resemblence structurally to GTFs
- no longer appears on list of these enzymes
FTFs are less complex than GTFs. Why?
- only two types
- one synthesises an inulin-like polymer
- one’s a levan
Do we know the genes which produce FTFs?
- little info on them
- both just said to come from ftf gene
FTF is absent where?
from strep sobrinus
Can only strep produce glucans and fructans?
- no
- several oral bacteria can produce both
Aside from strep, what bacteria can produce glucans?
- neisseria spp
- lautropia mirabilis
- lactobacillus spp
Aside from strep, what bacteria can produce fructans?
- leuconostoc
- lactobacillus
- actinomyces
Function of exopolysaccharides
- S.mutans biofilms grown in glucose are thin with little to no matrix
- sucrose-grown ones are much thicker
- knockout mutans lacking gtfB or gtfC form thin biofilms
How to reduce sucrose biofilm
treatment with mutanase
Mutan-rich microcolonies are found at …
low pH
How to test for glucan binding proteins
- add sucrose or dextran to suspension of strep mutans cells
- cells will clump
- add sucrose to culture of strep mutans
- cells adhere to wire suspended in culture medium
How did people know to test for glucan binding proteins?
- EPS formed from sucrose or added dextran was binding one cell to another or a surface
- led to search for whatever it was EPS or dextran was binding to
Glucan binding proteins are a … group
heterogenous
There are more than one type of GBP. Some are … and others are …
- bacterial surface proteins
- secreted
GBPs differ in …
- size
- strength of bond formed with glucan
- function in adhesion/aggregation
Different species bind glucan differently. How?
- aggregation/adhesion
- GBPA-deficient mutans produce flatter but more even biofilms
- GBPC-deficient mutans of strep. mutans produce thicker biofilms than parent
FTFs in caries
- very active in plaque more than GTFs
- total amount of fructan in plaque is low, suggesting they’re turned over rapidly - consistent with their function to be a short term energy store
- fructans thought to contribute to caries by extending fermentation time of bacteria
- inconsistent evidence but inactivation of ftf in strep. mutans causes reduced caries (other studies show no change)
- fructan impact may be more subtle by improving survival of strep.mutans in plaque rather than affecting acid production
Fructan degradation is done by …
fructanase
- from gene fruA
GTFs in caries
- glucans help mutans streptococci to stick
- mutans strep don’t adhere well to teeth helped by glucans
- soluble dextrans mediate cell-cell interactions (cell aggregation) and used as energy store
- insoluble glucans contribute to cell-surface interaction/adherance as no oral bacteria produce mutanase (some bacteria in env and fungi do)
- mutans strep haas evolved to exploit sucrose to enhance adhesion
Is S.mutans usually alone in biofilm?
Explain
- never
- other bacteria may adhere to S.mutans via glucan bridges
- cell-cell adhesion enhances interspecies interaction like metabolite cycling, competition and signalling
Define ‘amyloids’
proteins that form robust fibrils with beta strands running perpendicular to length of fibril
Amyloids were first discovered where?
in disease like Alzheimer’s, Parkinson’s, Huntingdon’s
Role of functional amyloids
- biofilm stabilisation
- melanin formation
- initiation of innate antiviral immune response
What bacteria produces large amounts of extracellular DNA?
P.aeruginosa
Treatment of eDNA with what disrupts biofilms
- DNase I
Biofilm is inhibited for up to … hours due to extracellular DNA
60
Why does eDNA inhibit biofilm formation?
- DNA changes conformation in biofilm
- resistant to enzyme
Functions of extracellular DNA
- gene transfer
- chelates metal ions (they can’t penetrate as DNA is negative)
- biofilm formation
- antimicrobial protection
- nutrient uptake
- biofilm stability
Extracellular DNA in dental plaque
- it’s present in subgingival plaque (and in plaque on implants)
How is eDNA in plaque treated?
- DNase enzyme NucB
- inhibits plaque formation
- reduces colonisation by periodontal pathogens
Define ‘plaque fluid’
fluid which fills space between bacteria in dental plaque
Plaque fluid is what percentage of the plaque volume?
30%
Composition of plaque fluid
- saliva (modified)
- bacterial metabolites/waste
- material leeched from tooth
- gingival fluid
Functions of plaque fluid
- buffer between saliva and tooth
- maintains calcium, phosphate and fluoride ions
- retain antimicrobials and other mouthwash components
Ca, P and F ions are reduced when what is present?
sucrose
How does plaque fluid use retained antimicrobials?
- stops plaque maturing into cariogenic or periodontal disease
- gives an argument for using mouthwash to combat plaque in hard to reach areas
