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Microbiology for Dentists > 18. Biofilm Matrix > Flashcards

18. Biofilm Matrix Flashcards

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1
Q

Components of the biofilm matrix

A
  • macromolecules like polysaccharides, proteins and nucleic acids
  • smaller ones like nutrients, metals and signalling molecules
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2
Q

What macromolecules are present in biofilm matrix?

A
  • polysaccharides
  • proteins
  • nucleic acids
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3
Q

Explain exopolysaccharide synthesis

A
  • 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
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4
Q

2 basic types of exopolysaccharide with example

A
  • glucans like glucosyltransferase
  • fructans like fructosyltransferase
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5
Q

Most exopolysaccharides are made inside/outside the cell?

A
  • inside
    then exported out
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6
Q

Why are exopolysaccharides made inside cells then exported out?

A
  • need energy to build
  • no energy for this so comes from bonds in sucrose found inside cells
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7
Q

What do exopolysaccharides make a colony look like?

A
  • cystalline colony appearance
  • on a sucrose-containing agar
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8
Q

How is sucrose a good substrate for glucan synthesis?

A
  • 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)
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9
Q

Note from glucan synthesis - an anomeric carbon is a …

A
  • stereocentre
  • if an OH group is down, alpha carbon
  • if otherwise it’s beta
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10
Q

Name of fructan polymers
What bond do they have?

A
  • inulins
  • beta-2,1 bond type
  • levens
  • beta 2,6
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11
Q

Inulins are an … polymer
Explain them

A
  • 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
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12
Q

How are inulins made?

A
  • only by strep. mutans and some strep, salivarius
  • make large amounts
  • 95% 2,1 bonds
  • 5% 2,6 bonds
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13
Q

Inulins are a … carbohydrate

A

storage

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14
Q

Levens are …

A

water soluble

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15
Q

Why are levens named so?

A
  • rotates polarized light to the leftLev
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16
Q

Levens are made by …

A
  • strep. sanguinis
  • strep. salivarius
  • actinomyces naeslundii
  • some strep. sobrinus
    NOT strep.mutans
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17
Q

Role of levens

A
  • spores can last years
  • once activated, produce calcium carbonate strong enough to glue concrete
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18
Q

What is ‘BacillaFilla’?

A
  • a biological approach to repairing concrete
  • with calcium carbonate and cells
  • glued together by levan
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19
Q

Which survives better in the biofilm? Glucans or fructans?

A

glucans

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20
Q

2 basic types of glucans

A
  • water insoluble mutans (alpha 1,3 linked)
  • water soluble dextrans (alpha 1,6 linked)
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21
Q

Explain mutans

A
  • insoluble glucose polymer from strep, sobrinus 6715
  • almost completely alpha 1,3 bonds
  • 70% of plaque exopolysacc is mutan (primary glucan polymer)
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22
Q

Some strains of mutan make 85-90% alpha-1,3 with the remainder being …
Explain

A
  • 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
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23
Q

Strcuture of dextran

A
  • almost all alpha-1,6 glycosidic bonds
  • synthesised by strep salivarius
  • rotates polarized light to the right
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24
Q

What variations of dextran are present?

A
  • 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
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25
Q

What similar dextran-like polymers are made?

A
  • 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
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26
Q

How does environment impact glucans?

A
  • 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)
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27
Q

How do enzymes impact glucans?

A
  • 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
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28
Q

What are
- GTF
- FTF?

A
  • gllucosyltransferase
  • fructosyltransferase
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29
Q

Types of GTFs

A
  • dextransucrase
  • mutansucrase
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30
Q

Types of FTFs

A
  • inulosucrase
  • levansucrase
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31
Q

GTFs and FTFs are classfied early on what?

A

water solubility

32
Q

GTF-S enzymes produce what?

A
  • water soluble dextran-like glucans with alpha-1,6 linked backbone
33
Q

GTF-I enzymes produce what?

A
  • insoluble alpha-1,3 backbone polymer
34
Q

GTF-SI enzymes produce what?

A
  • produce partially soluble alpha-1,3 backbone polymer
35
Q

Is there lots of consensus on GTF and FTF?

A
  • 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
36
Q

Absolute identification of discrete enzymes is best done by …

A

referring to them as a product of particular genes

37
Q

GTF-I, SI and S are enzymes found in …

A

S.mutans

38
Q

GTF-I is a product of gene …

A

gtfB

39
Q

GTF-SI is a product of gene …

A

gtfC

39
Q

GTF-S is a product of gene …

A

gtfD

40
Q

How is the gene identified and named for the GTF enzymes?

A
  • appending a non-italicised letter in alphabetical order
  • starting wit hthe first to be isolated and sequenced
41
Q

What happened to gtfA?

A
  • 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
42
Q

FTFs are less complex than GTFs. Why?

A
  • only two types
  • one synthesises an inulin-like polymer
  • one’s a levan
43
Q

Do we know the genes which produce FTFs?

A
  • little info on them
  • both just said to come from ftf gene
44
Q

FTF is absent where?

A

from strep sobrinus

45
Q

Can only strep produce glucans and fructans?

A
  • no
  • several oral bacteria can produce both
46
Q

Aside from strep, what bacteria can produce glucans?

A
  • neisseria spp
  • lautropia mirabilis
  • lactobacillus spp
47
Q

Aside from strep, what bacteria can produce fructans?

A
  • leuconostoc
  • lactobacillus
  • actinomyces
48
Q

Function of exopolysaccharides

A
  • 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
49
Q

How to reduce sucrose biofilm

A

treatment with mutanase

50
Q

Mutan-rich microcolonies are found at …

A

low pH

51
Q

How to test for glucan binding proteins

A
  • 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
52
Q

How did people know to test for glucan binding proteins?

A
  • 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
53
Q

Glucan binding proteins are a … group

A

heterogenous

54
Q

There are more than one type of GBP. Some are … and others are …

A
  • bacterial surface proteins
  • secreted
55
Q

GBPs differ in …

A
  • size
  • strength of bond formed with glucan
  • function in adhesion/aggregation
56
Q

Different species bind glucan differently. How?

A
  • aggregation/adhesion
  • GBPA-deficient mutans produce flatter but more even biofilms
  • GBPC-deficient mutans of strep. mutans produce thicker biofilms than parent
57
Q

FTFs in caries

A
  • 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
58
Q

Fructan degradation is done by …

A

fructanase
- from gene fruA

59
Q

GTFs in caries

A
  • 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
60
Q

Is S.mutans usually alone in biofilm?
Explain

A
  • never
  • other bacteria may adhere to S.mutans via glucan bridges
  • cell-cell adhesion enhances interspecies interaction like metabolite cycling, competition and signalling
61
Q

Define ‘amyloids’

A

proteins that form robust fibrils with beta strands running perpendicular to length of fibril

62
Q

Amyloids were first discovered where?

A

in disease like Alzheimer’s, Parkinson’s, Huntingdon’s

63
Q

Role of functional amyloids

A
  • biofilm stabilisation
  • melanin formation
  • initiation of innate antiviral immune response
64
Q

What bacteria produces large amounts of extracellular DNA?

A

P.aeruginosa

65
Q

Treatment of eDNA with what disrupts biofilms

A
  • DNase I
66
Q

Biofilm is inhibited for up to … hours due to extracellular DNA

A

60

67
Q

Why does eDNA inhibit biofilm formation?

A
  • DNA changes conformation in biofilm
  • resistant to enzyme
68
Q

Functions of extracellular DNA

A
  • gene transfer
  • chelates metal ions (they can’t penetrate as DNA is negative)
  • biofilm formation
  • antimicrobial protection
  • nutrient uptake
  • biofilm stability
69
Q

Extracellular DNA in dental plaque

A
  • it’s present in subgingival plaque (and in plaque on implants)
70
Q

How is eDNA in plaque treated?

A
  • DNase enzyme NucB
  • inhibits plaque formation
  • reduces colonisation by periodontal pathogens
71
Q

Define ‘plaque fluid’

A

fluid which fills space between bacteria in dental plaque

72
Q

Plaque fluid is what percentage of the plaque volume?

A

30%

73
Q

Composition of plaque fluid

A
  • saliva (modified)
  • bacterial metabolites/waste
  • material leeched from tooth
  • gingival fluid
74
Q

Functions of plaque fluid

A
  • buffer between saliva and tooth
  • maintains calcium, phosphate and fluoride ions
  • retain antimicrobials and other mouthwash components
75
Q

Ca, P and F ions are reduced when what is present?

A

sucrose

76
Q

How does plaque fluid use retained antimicrobials?

A
  • stops plaque maturing into cariogenic or periodontal disease
  • gives an argument for using mouthwash to combat plaque in hard to reach areas

Microbiology for Dentists (37 decks)

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