Links
Part 1 Some basic sugar chemistry
Part 2 The structure of the polymers (this page)
Part 3 The reactions catalysed by FTFs and GTFs
Part 4 The enzymes themselves
Part 5 The role of EPS in dental plaque and caries
Part 2 - The polymers
There are two basic types of bacterial extracellular polysaccharides (EPS).
Heteropolysaccharides. These are polymers comprising a number of different sugars and require several enzymes for their synthesis.
Homopolysaccharides. These are polymers comprising just one sugar type, requiring just a single enzyme for their synthesis.
Homopolysaccharides are synthesised by many bacterial species but this tutorial will be limited to the bacteria belonging to the group known as lactic acid bacteria (LAB) and the substrate is limited to sucrose. Not all LAB synthesise EPS but those that do have been the subject of considerable research because of the industrial and medical importance of the EPS they synthesise.
Included in the LAB group are lactobacilli and oral streptococci and the EPS they synthesise has been implicated in the pathology of dental caries.
Glucose and fructose polymers.
Because sucrose is the substrate, only two basic homopolymers can be formed ie those composed either entirely of glucose or entirely of fructose. However, there is scope for variety because different glycosidic bonds are produced by the different enzymes responsible and these different bonds confer different physical and chemical properties on the resultant EPS.
Making matters slightly more complex is the fact that these enzmes can modify the product of another which leads to some complex polymers (this is described in more detail in Part 2).
The two main types of enzymes responsible are those which polymerise the glucose moiety of sucrose, known as glucosyltransferases (GTFs), and those which polymerise the fructose moiety, known as fructosyltransferases (FTFs).
In the mouth
Dental plaque comprises as many as 12 or more different species of streptococci many of which synthesise EPS. Of these, some produce a single FTF or GTF, some produce both and some produce more than one type of each. Given that some of these enzymes will modify the product of another which may be from a different species and that also present are degradative enzymes such as dextranase, it is no wonder that the situation in the mouth is so complicated.
The aim
The aim is to try and make some sense of it all. In this section we will look at the various polymers formed, next we will look at the enzymes which produce them and finally we will look at the functions we think these polymers subserve in the mouth and, in particular, their role in plaque and, ultimately, caries formation.
This is a generic term for polymers of fructose. In the mouth two types are synthesised from the fructose moiety of sucrose. The evidence so far suggests that fructans of both types are used as storage products.
β-2,1 Fructan
This is an insoluble polymer almost identical to a fructose polymer found in vegetables called inulin. The only difference is that inulin has a terminal glucosyl residue. Inulin is synthesised by a number of plants including dandelions, burdock, Jerusalem artichoke, chicory, onion and garlic where it is used as a long-term storage carbohydrate instead of starch.
It used to be thought that, the only bacterial species known to synthesise β-2,1 Fructan (not inulin) was Strep. mutans but we now know that a few other species are also capable including some strains of Strep. salivarius.
The function of fructan is described in Part 4 but, briefly, the finding that it is rapidly turned over suggests it is used as a short-term storage compound. Yes, I know inulin is a long-term storage compound but this is the mouth not a Jerusalem artichoke. Since it is extracellular it can be quickly degraded by other bacteria (not found in artichokes) which provides a nice example of food-chain co-operation in the dental plaque community. On the other hand, the ecological advantage of having your external food source used by another cell or, worse, a cell from a different species is open to question.
Although it is widely reported that Strep. mutans synthesises a pure β 2,1 fructan, finding the evidence is proving difficult. If anyone knows of some published work which has analysed the bond structure of a fructan and shown it to be devoid of any links other than β-2,1 I would be pleased to hear about it.
What we do know is that the fructan synthesised by a couple of strains of Strep mutans (JC1 and JC2) is about 95% β 2,1 and 5% β 2,6. The two possible structures are:
- a β-2,1 polymer with every 20th residue substituted at carbon 6 with a fructose
- β-2,1 polymers cross-linked with β-2,6 links.
The structure most commonly attributed is the cross-linked polymers shown opposite. However, this does appear to be an assumption.
β-2,1 Fructan
This is:
β-D-fructofuranosyl 2,1 β-D-fructofuranoside which, because it comprises only a repeating fructose, is simply called β 2,1 fructan.
β-2,1 Fructan polymers cross-linked with β 2,6 glycosidic bonds.
β-2,6 Fructan (levan)
These polymers are water soluble. Opposite is β-D-fructofuranosyl 2,6 β-D-fructofuranoside which is otherwise known as levan because, in solution, it rotates polarized light to the left. Levan is made by a few species of bacteria found in dental plaque including Strep. sanguis, Strep. salivarius and Actinomyces naeslundii but not by Strep. mutans.
There is evidence that a pure β-2,6 fructose polymer is synthesised by strains of Strep sobrinus (OMZ176) and Strep salivarius (ATCC 13419). Other strains of Strep. salivarius (eg strains HHT and 51) have been shown to synthesise β-2,6 backbones cross-linked with β-2,1 links. In this polymer the ratio of β-2,6 to β-2,1 is about 8:1 indicating a quite highly branched structure.
A slightly different structure to this β-2,1 cross linked polymer has been discovered in other strains of Strep. salivarius (eg ATCC 25975). In this structure 70% of the fructose is present as β-2,6 backbone with a further 15% present as terminal fructose and 15% present as β-2,1 link. The proposed structure is a highly branched globular structure. See opposite.
Levan
β-2,6 polymers cross-linked with β-2,1
Highly branched β-2,6 backbone
Originally it was thought that only two types were made. One was water soluble and the other insoluble. We now know that different varieties of each type are made but that all have a core polymer which is either an α-1,3 linked or α-1,6 linked. In nearly all cases an α-1,3 linked core makes the polymer water insoluble and an α-1,6 linked core makes it water soluble.
Water insoluble (mutan)
One of the first to be described was the insoluble glucose polymer from Strep sobrinus 6715. This was called mutan and, as it happens, almost completely comprises α-1,3 glycosidic bonds. It was called mutan because, at the time, this strain of Strep sobrinus was included in the species Strep. mutans. When the EPS in dental plaque was analysed it was discovered that 70% of it consisted of this insoluble variety so mutan was concluded to be the primary glucan polymer formed.
Analysis of the mutan polymers from other strains revealed between 85-90% α-1,3 with the remainder being α-1,6.
For example, Strep mutans GS5 mutan includes a small and approximately equal number of α-1,6 and α-1,3,6 bonds. The ratio of the α-1,3 linked backbone residues to these two other types being about 12:1:1. A possible structure is a polymer with approximately every 12th residue containing an α-1,6 branch which is α-1,3 linked to a terminal glucose. See the diagram opposite.
Mutan
Water soluble (dextran)
A glucan polymer comprising almost exclusively α-1,6 glycosidic bonds is synthesised by Strep salivarius ATCC 25975. It is called dextran because in solution it rotates polarized light to the right.
A version of dextran which includes numerous side chains of α-1,3 linked glucose has been shown to be synthesised by Strep sobrinus OMZ176. As with many of these polymers, the actual structure has not been absolutely defined but it is likely to be similar to that shown opposite. Note that the side branch is short compared with the backbone. and that there is a side branch for about every 15 backbone residues.
The diagram below tries to put these side branches into perspective.
Similar dextran-like polymers are produced by other strains such as Strep. mutans GS5 but in this example the side chains are about twice as numerous but shorter comprising, probably, just a single glucose residue linked α-1,3 to the branch residue.
Strep sobrinus dextran
Further complications
As if the situation was not complicated enough there are a couple of other things that need to be born in mind. One concerns the enzymes themselves, the other concerns the environment in which these polymers are synthesised.
The enzymes
These are described in more detail separately in Part 3 but, it is clear that some of them can catalyse the formation of polymers comprising both α-1,3 and α-1,6 bonds. A good example of this is the GTFG enzyme produced by Strep. gordonii which has been researched in some detail.
The polymer produced by this enzyme has been looked at by different researchers and the ratio of α-1,3:α-1,6 bonds reported varies between 25% and 40%. This may be due to experimental error or it may be due to the way the experiments were conducted. ie the conditions in the test tube.
In other words, the local environment may affect the enzyme activity. Not exactly a controversial statement bearing in mind what is well known about how enzymes work. So it would be hardly surprising that the local environment could cause GTFG to synthesise different α-1,3:α-1,6 ratios. This is clearly an area worth further study.
Quite apart from the effect the local environment may have on any particular enzyme it has to be remembered that these GTFs are extracellular. Furthermore, they are being secreted into a very complex environment (dental plaque) which comprises a very large number of different bacteria many of which will also be secreting their own particular mix of GTFs and other enzymes.
Glucans as a GTF substrate
It was originally thought that some GTFs require primers in the form of partly or completely formed polymer. More recent evidence, however, does not support this but rather that pre-formed glucan can act to accelerate GTF action by binding to a site remote from the catalytic site.
Glucans can, though, act as the acceptor for the addition of further glucose units by an unrelated GTF. This is even further complicated by the finding that the catalytic action of GTFs is semi-processive. This means that a forming glucan polymer does not remain bound to its GTF until fully completed (whatever that means) but instead repeatedly binds and un-binds from the GTF during synthesis. In a soup of glucans and GTFs such as plaque fluid one could envisage a mixture of different glucans each being worked on by different GTFs one at a time.
Proteases
Among the other enzymes in dental plaque are proteases and dextranases. Proteases will digest proteins and GTFs are proteins. Proteases may also modify rather than simply digest and we know that the GTFG of Strep. gordonii can be modified after secretion. As it happens, the product stays the same as far as we know but the point is that this might not be the case with other GTFs and some proteases are extracellular.
Dextranases
Secreted dextranases such as DexA of Strep mutans and Strep. sobrinus will degrade α-1,6 regions of pre-formed polymers leaving behind a modified glucan and isomaltosaccharides which can be taken up and used as an energy source. Dextranases will thus further complicate the complex glucan mixture resulting from pre-formed glucans acting as glucose acceptors (see above). Strep. sobrinus produces a DexA inhibitor (Dei) specific to Strep. sobrinus DexA which appears to be under the control of carbohydrate concentration although the significance of this control is not clear.
These significance of these complications insofar as they can be deduced is discussed in Part 5 which deals with EPS & Caries.
What is all this fuss about solubility anyway?
Much is made about the solubility of the various polymers formed and found in dental plaque. The reason for this is because these two different polymers appeared to have quite distinct interactions with mutans streptococci.
The soluble polymer (mainly α-1,6 links) causes cells to stick together. This is called aggregation (sometimes called agglutination). Aggregation of Strep. mutans can be caused by adding dextran to a suspension of cells. It can also be caused by adding some sucrose to a suspension of Strep. mutans cells and allowing them to metabolise it for a few minutes. The soluble dextran formed causes the cells to clump together.
The insoluble polymer, on the other hand, promotes the adhesion of cells to surfaces.
Unfortunately most early work was done at a time when the group we now call the mutans streptococci were all lumped together as one species called Strep. mutans and the species used to isolate the soluble and insoluble polymers was subsequently identified as a Strep. sobrinus.
Strep. sobrinus is less common in human mouths than Strep. mutans which casts some doubt on the overall importance of EPS in the pathology of caries. Nevertheless the general conclusions also apply to Strep. mutans though, in general,the phenomena of polymer-dependent aggregation and adhesion are less dramatic than in Strep. sobrinus.
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