Extracellular Polysaccharides & Caries

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Part 1 Some basic sugar chemistry

Part 2 The structure of the polymers

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 (this page)

Part 5 - EPS & Caries Aetiology

The aim here is to try and bring together what we know about EPS and in the context of dental plaque and caries.

The first thing that must be emphasised is that while there is a consensus that EPS and mutans streptococci are important in the aetiology of dental caries, they are, by no means, the only factors. So, bearing that in mind, how do EPS contribute?

Lots of things contribute to caries but dental plaque sits at the heart of everything. Strep. mutans or other species (see opposite) have to exist within plaque to be cariogenic, plaque resides on the tooth surface, EPS is formed and exists within plaque and sugar has to diffuse into plaque before the caries is initiated.

Fructans

The two polymers made are based on β-2,1 (inulin-like) or β-2,6 (levan) linked fructose respectively. They are described in more detail in the section dealing exclusively with the structure of extracellular polysaccharides.

There is evidence that the FTFs which synthesise fructans are very active in plaque, more so, indeed, than GTFs. There is also evidence that the total amount of fructan in plaque is low. Taken together this suggests that fructans are turned over rapidly which is consistent with the view that their main function is to act as a short term energy store.

Fructans are thought to contribute to caries by extending the fermentation time of plaque bacteria. Although there is evidence that inactivation of ftf in Strep. mutans leads to reduced caries other studies have shown little difference. The impact of fructans on caries may be more subtle by improving the survival of Strep. mutans in plaque rather than directly affecting acid production.

Fructan degradation is achieved by the enzyme fructanase (the gene is fruA)

Fructanase

This exofructosidase is found free in plaque fluid but, interestingly, it is also found anchored in the cell wall which is quite unusual.

Fructanase hydrolyses fructan releasing individual fructose molecules which can be taken up by nearby bacterial cells and metabolised. Cell-free fructanase has the potential to hydrolyse fructan remote from the cell from which either the fructan or the enzyme was originally associated. The cell-associated fructanase, however, will only be able to hydrolyse fructans immediately adjacent to the cell which, therefore, are more likely to originate from that cell.

Glucans

The polymers made are based on cores which are either α-1,3 linked or α-1,6 linked glucose and which may be variously cross-linked. They are described in more detail in the section dealing exclusively with the structure of extracellular polysaccharides.

It should be remembered, however, that plaque is a very complex community and glucans made by one cell or species may be modified by the enzymes of another. This modification is made possible because a glucan may take part in the acceptor reaction for a different GTF. This is possible because the synthetic reaction is semi-processive. This is discussed more fully in the section dealing with the complex dental plaque environment in the section describing EPS

Glucans help mutans streptococci to stick

Notwithstanding the complexity of the glucan mixture found in dental plaque fluid the consensus view of the function of glucans appears, on the surface, to be relatively straightforward.

In contrast to other oral bacteria, particularly those species which pioneer the colonisation of teeth (Strep. oralis, Strep. mitis and Strep. sanguis), mutans streptococci are not good at adhering to teeth in the absence of sucrose or exogenous glucans. This seems a little odd in light of their role in caries and the fact that teeth (more precisely hard surfaces) are their preferred colonisation site.

A considerable amount of evidence supports the view that both soluble and insoluble glucans, in conjunction with GBPs, promote cell-cell and cell-surface interactions in plaque. Soluble dextrans mediate the cell-cell interactions (cell aggregation) while the insoluble glucans contribute mainly to cell-surface interaction (adherence). Mutans streptococci appear, therefore, to have evolved the means to exploit sucrose to enhance adhesion.

Other functions

Although DexA activity will undoubtedly release some glucose which will be taken up and metabolised, this is not thought to be of major importance in caries. Neither are the other functions which have been suggested such as protection from host defences, bacteriophages and bacteriocins.

There is evidence, however, that as well as promoting adhesion and cell-aggregation glucans bulk out dental plaque and reduce bacterial cell density. This will effectively enhance diffusion of metabolites within plaque leading to greater metabolic activity and thus more acid production.

Putting it in perspective with animal studies

Rat experiments very clearly show GTFs are needed for smooth surface caries which stands to reason since S. mutans has to stick. but most human caries is in fissures where adhesion is less critical but the influence on plaque structure/permeability may be a big deal. The importance of adhesion in the rat model may explain why anti-caries vaccines based on GTF worked in rats but were not successful in monkeys.

EPS can bulk out plaque

This is an electron micrograph of a culture of Strep. mutans cells which had been incubated in the presence of sucrose. Note the cell separation resulting from the presence of EPS* pushing the cells apart. This is consistent with the proposed function of glucan in bulking out dental plaque thereby increasing the rate of diffusion.

* much of this was dextran because it was degraded by added dextranase.

At first glance the advantage of producing EPS is not clear. Synthesising large amounts of polysaccharide and storing it extracellularly has been likened to scattering cash in the street in the hope that some of it will be around when you need to buy a loaf of bread. However, there must be some advantage otherwise the process would not have evolved.

In addressing this, the first thing to remember is that the bacteria have not actually expended large amounts of energy in synthesising EPS. The energy required for the synthetic reaction originates in the sucrose covalent bond so it does not come at the expense of the bacterial cell. Thus the only energy expenditure is that associated with the synthesis of the GTF and FTF enzymes which is considerably less.

Also, the advantage of glucan synthesis is reasonably clear because it is closely associated with maintenance of mutans streptococci in their environment through adhesion.

The case for fructan, however, is less obvious and although the analogy with scattering money around appears relevant, a couple of other factors need bearing in mind.

First, only a small amount of the available sucrose is converted to EPS so it is not as if EPS represents a significant use of the available resource. In the money analogy, this is akin to a multimillionaire scattering a small amount of his wealth in the streets in an act of carelessness, so small it would hardly be missed.

Second, the ability to convert some extracellular sucrose to EPS, especially fructan, at a time of excess when the cellular sucrose uptake mechanisms are fully saturated makes sense (this extracellular synthesis is not subject to cellular control unlike uptake mechanisms). In these circumstances sucrose would otherwise be lost because it would easily diffuse out of plaque which is prevented by trapping it as a large polymer.

Returning to the money analogy this is akin to a multimillionaire leaving money lying around in suitcases because his arms and pockets are already full and he couldn't possibly carry any more. Some may just be left available in a couple of hours - so it's worth the chance.

Clearly mutans streptococci must be advantaged by EPS synthesis as evidenced by the fact that the systems have evolved and are being maintained. What is less easy to understand is why this has happened in environments or societies in which sucrose is absent and has been absent for a very long time. One explanation is that redundant systems are known to be maintained for long periods in some bacteria. Another possibility is that the EPS-synthetic activity of GTFs and FTFs may not be these proteins' prime or only function. This is not as unusual as it may at first sound.

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