Sugar metabolism and acid production

Streptococci are the most important organisms in terms of acid production and special features of their metabolism are worth mentioning. They can take up sugars from the surrounding medium by a high-affinity transport mechanism which is energized by phosphoenolpyruvate (page 534) and is known as the phosphotransferase system. It is specific for individual sugars and operates at neutral pH values. This allows the organism to survive at low sugar concentrations and neutral pH values, conditions which are found naturally in the saliva and on the enamel in the absence of food. In addition, streptococci have a second, low affinity sugar uptake system which is activated by a protonmotive force (pmf) (page 221). This operates maximally in S. mutans at pH 5·5 and allows the organism to take advantage of the pH gradient generated when the extracellular pH is low compared to the intracellular pH. Consequently, the cell can continue to transport and metabolize sugars under acidic conditions which are unfavourable to other bacteria. This adaptation to a sugar-rich environment and a low pH make it a dominant organism in cariogenic plaques.

Streptococci do not contain enzymes of the citrate cycle, nor do they possess cytochrome systems but, instead, they rely on anaerobic glycolysis for their energy. The fate of pyruvate produced by this pathway is then determined by the overall availability of sugars. The enzyme lactate dehydrogenase is allosterically activated by the glycolytic intermediate fructose 1,6-bisphosphate, which together with glyceraldehyde 3-phosphate tends to accumulate when sugar is readily available so that lactate formation is favoured (Figure 35.2). The alternative pathway, catalysed by the enzyme pyruvate formate lyase, is inhibited by glyceraldehyde 3-phosphate so that this pathway only becomes operative when glycolytic intermediates are low, that is when sugar is in short supply. This allows the organism to generate an extra molecule of ATP for each glucose utilized, from the intermediate acetyl phosphate.

Streptococcal lactate dehydrogenase exists either as an inactive dimer or active tetramer; the latter configuration is stabilized by binding fructose 1,6-bisphosphate thus increasing the rate of breakdown of pyruvate sevenfold.

The lactic and acetic acids produced by these pathways are transported out of the cell in their protonated forms and accumulate in the external medium, often at very high concentrations. A high external lactate concentration, generated when glucose is readily available, eventually prevents further secretion of lactate and causes a gradual acidification of the bacterial cell interior so that the cell ceases to metabolize. Veillonella, organisms that utilize lactic acid, benefit from this plentiful supply of substrate and aid the streptococci by the removal of an unwanted waste product. Veillonella metabolize lactate by the following reactions:

< ?xml:namespace prefix = "mml" />Lacate+H2O→acetae+CO2+H2Lacate+H2→propionate+H2O

Propionic and acetic acids have higher pK values than lactic acid and so tend to act as buffers at higher pH values, thus raising the pH of the medium.

In practice, it is not possible to equate plaque acid production directly with the presence of any one type of bacterium. Analyses of organic acids in overnight fasting plaque before exposure to dietary sugar showed a predominance of acetic acid with some propionic, butyric and formic acids. The total acid concentration increased immediately after exposure to sugar, mainly due to the formation of lactic acid, while the concentration of the other acids tended to decrease. Fasting plaque has a neutral or slightly alkaline pH, and Stephan in 1940, using a micro pH electrode, demonstrated that the application of a sugar solution caused the intraplaque pH to fall rapidly to about pH 5.5 within 2–3 min, corresponding to the maximum rate of lactic acid production. It took up to 40 min for the pH to return to resting conditions, as lactic acid was slowly replaced by the other organic acids. Modern techniques using indwelling electrodes fixed in the dentition allow plaque pH to be measured continuously and values as low as pH 4·0 have been measured in response to sucrose. Acid is produced from a range of carbohydrate-containing foods, fruits and juices and this technique is used to determine the potential cariogenicity of foodstuffs. pH changes vary from site to site depending on salivary access and plaque thickness and typical Stephan curves are shown in Figure 35.3.

Nitrogen metabolism and base production

The provision of a source of nitrogen is just as important for bacterial growth as the provision of a source of energy and reference of Table 33.1 (page 478) shows that salivary urea is the most readily available source. Plaque possesses a high urease activity and a number of different reactions are known by which microorganisms incorporate ammonia into amino acids, namely:

Subsequent transamination reactions then provide the other amino acids needed for protein synthesis. Glutamine serves as a –NH₂ donor in a number of biosynthetic reactions and Figure 35.4 shows how carbohydrate and nitrogen metabolism may be interrelated in the plaque. An additional source of amino acids within the interior may be provided by hydrolysis of matrix proteins.

Free amino acids, if not required for protein synthesis, can be fermented under anaerobic conditions. Generally, these reactions produce organic acids as well as ammonia and, therefore, are unlikely to have a marked effect on plaque pH. Arginine and arginine-containing salivary peptides are a special case, however, since they act as substrates for base-producing reactions by the arginine deiminase pathway:

Arginine→citrulline+NH3Citrulline+Pi→carbamoyl phosphate+ornithineCarbamoyl phosphate+ADP→ATP+CO2+NH3

This pathway which is widely distributed in bacteria, acts as an energy source under anaerobic conditions, and produces a marked pH-rise effect due to the production of NH₃. It is induced by arginine supplementation. It also acts as a source of ornithine which can subsequently be decarboxylated, particularly at low pH values, to produce an increase in the pH (page 508) and the evil-smelling amine putrescine which occurs in decaying meat.

Ornithine+H+→putrescine+CO2

NADH is produced by the energy-producing anaerobic oxidation of many amino acids according to the scheme:

Ornithine+H+→putrescine+CO2

The NADH can be reoxidized to NAD⁺ either by the reductive deamination of ornithine or by the reductive cleavage of a proline ring to produce 5-aminopentanoic acid CH₂NH₃⁺[CH₂]₃COO⁻.

These reactions were first described by Stickland and this δ-amino acid may account for up to 25% of the total amino acids in plaque fluid. Stickland reactions are usually associated with amino acid-fermenting anaerobic organisms which are commonly found in the region of the gingival crevice and in periodontal pockets. Tissue destruction and proteolytic activity in this region would generate an excess of free amino acids thus favouring 5-aminopentanoic acid accumulation.

In a long-established plaque, when bacterial growth is limited, more ammonia may be produced than is required for amino acid production. Accumulation of either ammonia or organic acids will influence the acid-base ratio and hence the pH of the plaque. However, when the system is an open one and there is a good flow of saliva, pH changes will be resisted since the bacterial waste products are able to diffuse away from their site of origin (Figure 35.5a). This is the situation in the thin loosely aggregated type of plaque which contains little or no extracellular polysaccharide. However, if plaque is allowed to accumulate, through inadequate oral hygiene, formation of extracellular polysaccharides, especially when sucrose is readily available, will lead to the formation of closed plaque which retains organic acids and ammonium salts within its matrix (page 497). The accumulation of these charged metabolites is greatest at sites where access for saliva is poor, resulting in pronounced changes in the pH which only returns slowly to normal after the supply of substrate has been exhausted (Figure 35.5b). Sometimes a new steady state may be established at a higher or lower pH value and this may provoke a long-term modification in the plaque flora which will cause pathological changes in the host.

Certain factors tend to counteract changes in plaque pH.