Links

Back to overview

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 (this page)

Part 5 The role of EPS in dental plaque and caries

The enzyme names

One of the problems with some areas of science is that the researchers who discover something have an annoying tendency to add their name to it or have others do it for them. Boyle's Law, Pacini's corpuscles and Chagas' disease, the list is long and few give a clue as to what the law or disease is about. We all, therefore, owe a debt of gratitude to the unsung heros of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NCIUBMB) because one of their jobs is to name enzymes and they do it in a logical and systematic way.

Enzymes are classified (named) on the basis of the reaction catalysed and the product which is formed. In the case of the extracellular polysaccharides (EPS) formed in dental plaque we have, therefore:

1. dextransucrase
2. mutansucrase
3. inulosucrase
4. levansucrase

Other names you may come across are:

1. glucansucrase which includes both dextransucrase and mutansucrase
2. fructansucrase which includes both inulosucrase and levansucrase

Common names

Although dextransucrase and levansucrase are more descriptive and roll off the tongue equally well, the common names for these EPS-forming enzymes are glucosytltransferases (GTFs) and fructosyltransferases (FTFs).

Genes

The convention is that the the gene and the product of the gene have the same acronym. To distinguish them, the gene is normally lower case and written in italics.

Thus the enzyme GTF is the product of the gene gtf.

The early work

In the 1960s studies showed that some mutans streptococci stuck to the sides of glass vessels when they were cultured in the presence of sucrose but not in the presence of other sugars such as glucose. When the culture fluid was analysed it was found to contain a polymer which was similar to dextran. At the time, there was growing evidence for the role of both mutans streptococci and sucrose in caries so this finding suggested a possible link between the formation of this dextran which helped mutans streptococci adhere to surfaces and caries.

Naturally, this stimulated a lot of research which continues to this day. It quickly became apparent that more than one GTF or FTF was being produced by various mutans streptococci. Furthermore, GTF and FTF production was discovered elsewhere among the oral flora.

The research has, in the main, focused on the GTF enzymes because the evidence suggests that their product (various glucans) have a more direct role in caries through their physical interaction with mutans streptococci (adhesion and aggregation) than FTFs. The general view currently is that fructans act as a reserve energy source. This is thought to play a role in caries by prolonging acid production in plaque beyond sucrose utilisation.

Early classification of GTFs

Once it was discovered that there was more than one GTF it was inevitable that they were classified or grouped to introduce some kind of order. This early classification was based on whether the polysaccharide product was soluble or not. Thus we have GTF-S enzymes producing water soluble dextran-like glucans with an α-1,6 linked backbone and GTF-I and GTF-SI enzymes producing an insoluble α-1,3 backbone polymer and a partially soluble polymer respectively.

This all got terribly confusing not least because there was no general agreement on naming these enzymes and some researchers identified a primer-independent GTF which they called GTFSi. Naturally the primer-dependent enzymes became GTFSd. Since GTF-SI is actually primer-independent it is possible that GTFSi and GTF-SI are one and the same but we may never know for sure because the appelation Si and Sd is no longer used. GTF-S, GTF-I and GTF-SI, however, remain in common use as a means of describing the polysaccharide formed.

Absolute identification of discrete enzymes, however, is best done by referring to them as the product of particular genes.

Fructosyltransferases

Only two types of FTF have been shown to exist. One synthesises an inulin-like polymer and one a levan. Little information is currently available concerning the relevant genes which are all simply identified as ftf.

Absence of Strep. sobrinus FTF

References have been made to a levan-producing FTF in Strep. sobrinus but this dates from a single article published in 1979*. The work has not been confirmed and the current consensus view is that FTF is absent from Strep. sobrinus.

* Corrigan & Robyt (1979) Nature of the fructan of Streptococcus sobrinus OMZ 176. Infect. Immun 26:387-389

Glucosyltransferases

It has been known for a very long time that mutans streptococci produced dextran-like polymers but it was the discovery in the 1960s of insoluble mutan production and its ability to promote adherence which sparked such a lot of interest in polysaccharide synthesis in dental plaque. Adherence, aggregation and the role these polymers play in caries aetiology is dealt with in Part 5 which deals with EPS and Caries.

The table opposite lists the oral streptococci which produce EPS, the type produced and the identity of the gene coding for the GTF responsible. It is not exhaustive but will give an idea of the range of oral streptococci which produce them.

It is interesting, and often pointed out, that Strep. salivarius, an organism less noted for a role in caries, has 4 identified genes coding for GTF whereas Strep. mutans, the declared caries pathogen has only 3. This, of course, is not evidence against a role for EPS and Strep mutans in caries aetiology, or, for that matter, evidence for a more important role for Strep. salivarius. It is just an interesting observation which is a good enough reason to investigate further.

By knowing more about the distribution, number and types of these genes and the properties of the various polysaccharides we will get a better understanding of their role in the oral environment. Clearly they seem to be primarily involved in sticking mutans streptococci to surfaces and each other but it has been proposed that they have a wider role in the microbial ecology of the mouth. For further discussion on this see Part 5 which deals with EPS and Caries.

Glucan Binding Proteins

Some interesting phenomena were reported by a number of early researchers studying sucrose metabolism by Strep. mutans.

1. Sucrose added to a culture of Strep. mutans caused the cells to stick together (aggregate).
2. The same thing happened if some dextran was added.
3. Strep. mutans cells would adhere to the glass walls of the culture vessel or to wires suspended in the culture medium in the presence of sucrose.

The conclusion drawn from these observations was that the EPS formed from sucrose or the added dextran was binding one cell to another or to a surface. This lead to a search for whatever it was that the EPS or dextran was binding to.

We now know that GTF enzymes possess a Glucan Binding Domain (GBD) located adjacent to the C-Terminus of the protein. This domain is quite distinct from the catalytic domain. It also lead to the discovery of a group of proteins which have no GTF activity but are capable of binding glucan. These became known as the Glucan Binding Proteins (GBP).

Both GTFs and GBPs can be associated with the cell surface or, because they can be cast adrift from the cell, any other surface such as the walls of a glass culture vessel or stainless steel wire they come into contact with and stick to (see opposite). In either case they act to enable dextran to form cross links between cells or between cells and the surface.

They are, in effect, acting as binding sites and if they stick to, or become incorporated into, the tooth pellicle then they promote the adhesion of Strep. mutans to the tooth surface.

GBPs are a heterogeneous group

After these initial findings lots of follow-up experiments were done and it became clear that there was more than one type of GBP. Some were bacterial surface proteins and some were secreted. They also differed in size and the strength of the bond formed with glucan. All these differences in properties translate into different functions which go some way to explaining the various observations which were made. When the amino acid sequences (primary structure) of these different GBPs were worked out and the genes identified it became obvious that they were, in fact, very distinct proteins.

It has also become evident that different species, and even different strains of the same species, make different GBPs.

Types of GBP

Much of the early work on GBPs was done before the taxonomy of what subsequently became known as the mutans streptocci was revised. It transpired that one of the strains (6715) which was widely used in these studies became re-classified as Strep. sobrinus. After this taxonomic revision it was clear that Strep. sobrinus was more readily aggregated than Strep mutans and this, in turn, lead to one of the Strep sobrinus GBP (GBP-4) being designated the glucan-binding-lectin (GBL) - probably to reflect a supposed higher status. GBLs were then defined as GBPs which confer the property of aggregation in the presence of added α-1,6 glucan.

Function

We know that GBPs bind glucans because we can isolate them and measure this binding in the lab. However, it transpires that some of them, at least, have additional functionality. One useful way of studying the function of a particular gene is to delete it and see what difference this makes to the so-called "knock-out" mutant.

When this was done with the gbpB, the gene coding for GBPB in Strep mutans SJ32 (see table opposite) then the knock-out mutants were not viable. Lethal mutations such as this are taken as evidence that the gene is essential for the organism. A more in depth study of this gene and its product GBPB revealed that it also functioned as a peptidoglycan hydrolase. Peptidoglycan is an important component of bacterial cell walls which must be cleaved during cell division. One of the enzymes responsible is peptidoglycan hydrolase.

Similarly, Dei can bind glucan but also functions as a dextranase inhibitor. ie it modulates the activity of the enzyme dextranase, an enzyme which has been shown to be involved in modifying EPS. Dextranase has a role in plaque formation and structure and, therefore, probably caries as well. It will be included in Part 5 which deals with EPS and caries.

The effect of glucan binding

Notwithstanding these other functions, some of which we not yet have knowledge of, these GBPs bind glucan in subtlety different ways which is reflected in their effect on growing cultures of mutans streptococci or isolated cells. While many are known to bind cells together (aggregation) GBPA, for example, exerts its effect on the shape and density of the growing biofilm produced during growth of Strep. mutans Ingbritt. GBPA-deficient mutants have been observed to produce flatter but more even biofilms. On the other hand GBPC-deficient mutants of Strep. mutans 109c produce thicker biofilms than the parent strain.

Quite what the significance of these observations is in terms of dental plaque formation and caries remains in the realm of conjecture and further research. It would be wrong to assume that what we observe when studying isolated strains in the lab translates directly into the highly complex environment which is dental plaque.

At the moment we just do not know enough to draw any firm conclusions - but you have to start somewhere.

A word of explanation & caution

The only way of really knowing if two proteins from different species or strains are really different or are actually just the same protein isolated from different sources is to clone and then sequence the respective genes. If these are the same then the protein is the same. We know that GBPA, GBPB, GBPC and GBPD are distinct because their genes are distinct. However, GBP-1, GBP-2, GBP-3, GBP-4 and GBP-5 have, as yet, not been sequenced. Therefore we can draw no conclusions about their relationship with other GBPs.

Furthermore, looking at these proteins and genes in isolated lab strains tells us little about what is happening in wild strains in dental plaque. The way to find this out is to look for them in freshly isolated strains. These may carry a mixture of different genes which interact in, as yet, unknown ways.

[top]