metabolismo energético de bacterias orales

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Critical Reviews in Oral Biology & Medicine

IntroductionOral biofilm is a thin microbial film that covers the surfaces of the human oral cavity. It consists of a tremendous number of microorganisms from a broad range of species. It is commonly considered that oral biofilm plays a role in oral health and dis-ease. Recent advances in molecular biological techniques, such as next-generation DNA sequencing, have made it possi-ble to analyze the oral microbiome more precisely and effi-ciently and to answer the question “Who are they?”, which has resulted in the suggestion that an association exists between the microbial profile and oral health and disease. However, the functions of oral biofilm are poorly understood (i.e., “What are they doing?”), since they are based on its metabolic properties, which are complex and have not been fully elucidated.

Carlsson and his colleagues pioneered metabolic studies of caries-associated bacteria in the 1970s and 1980s (Carlsson 1986), while molecular biological analyses of microbial com-position became common in biofilm research. In the 1990s, Marsh (1994) proposed the “ecological plaque hypothesis,” in which the microbiota shifts from being healthy toward a patho-genic state through interactions between bacterial activity and the environment. He also stressed the importance of bacterial metabolic activity for oral diseases, such as dental caries and periodontal disease. During these periods, several researchers, including myself, continued to study the biochemical charac-teristics of bacterial metabolism—for example, metabolic

pathways, mutual interactions between bacterial metabolism and environmental factors, and their involvement in oral health and disease (Takahashi 2005). In the 21st century, Kleinberg (2002) explored a mixed-bacteria ecologic approach to under-stand the role of bacterial metabolism in dental caries, and Nyvad and I (Takahashi and Nyvad 2008, 2011) extended Marsh’s hypothesis and proposed the “extended ecological caries hypothesis,” in which bacterial metabolic activity (acid production) regulates the development of caries through bacte-rial acid adaptation and selection in the oral microbial ecosys-tem. These findings suggest that bacteria-mediated oral diseases—such as dental caries, periodontal disease, and oral malodor, which occur at the interface between oral surfaces and indigenous microbiota—possess an etiology different from that of infectious diseases based on Koch’s postulates.

In this review, I describe 1) the fundamental metabolic framework of oral bacteria and its relationship with oral dis-eases, such as caries, periodontal disease, and oral malodor;

606045 JDRXXX10.1177/0022034515606045Journal of Dental ResearchOral Microbiome Metabolismresearch-article2015

1Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan

Corresponding Author:N. Takahashi, Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan. Email: [email protected]; [email protected]

Oral Microbiome Metabolism: From “Who Are They?” to “What Are They Doing?”

N. Takahashi1

AbstractRecent advances in molecular biology have facilitated analyses of the oral microbiome (“Who are they?”); however, its functions (e.g., metabolic activities) are poorly understood (“What are they doing?”). This review aims to summarize our current understanding of the metabolism of the oral microbiome. Saccharolytic bacteria—including Streptococcus, Actinomyces, and Lactobacillus species—degrade carbohydrates into organic acids via the Embden-Meyerhof-Parnas pathway and several of its branch pathways, resulting in dental caries, while alkalization and acid neutralization via the arginine deiminase system, urease, and so on, counteract acidification. Proteolytic/amino acid–degrading bacteria, including Prevotella and Porphyromonas species, break down proteins and peptides into amino acids and degrade them further via specific pathways to produce short-chain fatty acids, ammonia, sulfur compounds, and indole/skatole, which act as virulent and modifying factors in periodontitis and oral malodor. Furthermore, it is suggested that ethanol-derived acetaldehyde can cause oral cancer, while nitrate-derived nitrite can aid caries prevention and systemic health. Microbial metabolic activity is influenced by the oral environment; however, it can also modify the oral environment, enhance the pathogenicity of bacteria, and induce microbial selection to create more pathogenic microbiome. Taking a metabolomic approach to analyzing the oral microbiome is crucial to improving our understanding of the functions of the oral microbiome.

Keywords: amino acid, carbohydrate, dental caries, oral biofilm, oral malodor, periodontal diseases

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2 Journal of Dental Research

2) the detailed metabolic pathways of representative oral bac-teria; and 3) the role of bacterial metabolic activity in modi-fying the oral environment, which may result in bacterial adaptation and selection in oral biofilm. Furthermore, I dis-cuss 4) future metabolomics-based functional analyses of the oral microbiome.

Metabolic Framework of Oral Bacteria and Its Relationship with Oral Health and Disease

Bacterial Metabolism in Supragingival Plaque and Dental Caries

Saliva is the main source of nutrients for supragingival plaque (Table), and it contains glycoproteins, such as mucins, pep-tides, and amino acids. Glycoproteins can be degraded into sugar molecules and proteins by bacterial and human glycosi-dases, and proteins can be broken down into peptides and amino acids by bacterial and human proteases. Sugars are metabolized to acids by supragingival saccharolytic bacteria, including Streptococcus, Actinomyces, and Lactobacillus, while amino acids can also be metabolized by these bacteria, mainly into acids and ammonia. Overall, the production of acidic and alkaline molecules, together with the continuous flow of saliva, maintains an almost neutral supragingival pH (Fig. 1).

When dietary sugars are supplied, bacteria such as those mentioned above initiate efficient acid production, resulting in the demineralization of tooth surfaces. The tooth surfaces are subsequently remineralized, mainly by salivary washing, acid neutralization, and the supply of calcium and phosphate ions. In addition, bacterial alkali production and acid neutralization

can contribute to remineralization. For example, lactate can be converted into weaker acids, such as acetate and propionate, by Veillonella, Lactobacillus, and Actinomyces (Takahashi and Yamada 1999b), which contributes to acid neutralization. However, when demineralization occurs at a greater rate and frequency than that of remineralization, dental caries can develop (Fig. 1).

Bacterial Metabolism in Subgingival Plaque and Periodontal Disease

Gingival crevicular fluid and desquamated epithelia are the main sources of nutrients for subgingival plaque (Table), and they contain glycoproteins, proteins, peptides, and amino acids. Nitrogenous compounds can be degraded into short-chain fatty acids, ammonia, sulfur compounds, and indole/ skatole by subgingival bacteria, including Fusobacterium, Prevotella, and Porphyromonas. Overall, the production of acids and alkalis, with the continuous flow of gingival crevicu-lar fluid, results in the maintenance of an almost neutral/weakly alkaline subgingival pH. Short-chain fatty acids, ammonia, and sulfur compounds are known to be cytotoxic to induce tissue inflammation by modulating immune responses (Niederman et al. 1997) and to promote apoptosis (Kurita-Ochiai et al. 2008). These processes contribute to the initiation and promo-tion of periodontal diseases (Fig. 1).

Bacterial Metabolism in the Tongue Coating and Oral Malodor

Saliva and desquamated epithelia are the main nutrient sources for the tongue surface coating (Table), and they contain glyco-proteins, proteins, peptides, and amino acids. These nutrients

Carbo-hydrates

Sugar-chainedproteins

Aminoacids

Proteins/Peptides NO3

- EthanolPoly-saccharides

Mono- , di-, oligo-saccharides

Acids

Proteins

glyc

osid

ases

glycosidases

proteasespeptidases

Short-chain fatty acidsAmmonia

Sulfur compoundsIndole/Skatole

proteasespeptidases

NO2-

Caries Periodontal diseaseOral malodor

Urea

Ammonia Acet-aldehyde

CarcinogenesisCaries prevention

Systemic health

alkalization/acid-neutralization

O2

H2O2

H2O OSCN-

SCN-

Aerobiosis/Anaerobiosis

oxid

ases

Per-

oxid

ases

Saliv

ary

pero

xida

se

Nitr

ate

redu

ctas

e

Alc

ohol

deh

ydro

gena

ses

urea

se

Weakeracids

Sucrose

gluc

osyl

tran

sfer

ase

fruc

tosy

ltran

sfer

ase

Extracellularpolysaccharides

Figure 1. Bacterial metabolic properties and their relationships with oral diseases.




Oral Microbiome Metabolism 3

can be degraded into short-chain fatty acids, ammonia, sulfur compounds, and indole/skatole by tongue-coating bacteria, including Actinomyces, Veillonella, and Fusobacterium (Persson et al. 1990; Codipilly and Kleinberg 2008) (Fig. 1). The production of molecules associated with oral malodor from glycoproteins is considered to be enhanced by a combina-tion of sugar chain removal by glycosidases and core protein degradation by proteases (Sterer and Rosenberg 2006). Salivary β-galactosidase activity is reported to be associated with the degree of oral malodor (Sterer et al. 2002). Malodorous molecules are also found as cytotoxic compounds in subgingi-val plaque, especially in lesions associated with periodontitis; hence, a positive relationship exists between periodontitis and oral malodor (Ratcliff and Johnson 1999).

Oxygen Metabolism and Anaerobic Conditions

Bacteria can reduce oxygen to hydrogen peroxide and/or water by NAD(P)H oxidases using reducing power derived from their metabolism. Hydrogen peroxide can be further reduced to water by host and bacterial peroxidases or cleaved to oxygen and water by host catalases and catalase-positive bacteria such as Actinomyces oris. Hydrogen peroxide can also oxidize saliva-secreted SCN- to OSCN- via salivary peroxidase. OSCN- inhibits bacterial glycolysis by inactivating glyceraldehyde 3-phosphate (G3P), a bacterial glycolytic enzyme (Carlsson et al. 1983). These oxygen-reducing reactions cause the oxy-gen concentration of oral biofilm to fall, resulting in the cre-ation of a gradient from aerobic through microaerophilic to anaerobic conditions in oral biofilm (Marquis 1995) (Fig. 1).

Roles of Bacterial Metabolic Activity in Carcinogenesis, Caries Prevention, and Systemic Health

In the oral cavity, ethanol from alcoholic drinks is converted by bacterial alcohol dehydrogenase into acetaldehyde, a carcino-gen, which suggests the epidemiologic link between poor oral hygiene and the increased risk of oral cancer associated with alcohol consumption (Homann et al. 2001) (Fig. 1).

Nitrate supplied from green vegetables is converted into nitrite by oral bacteria, including Actinomyces and Veillonella (Doel et al. 2005) (Fig. 1). Nitrite inhibits bacterial acid pro-duction and might contribute to caries prevention (Doel et al. 2004). Nitrite is nonenzymatically reduced to nitric acid under acidic conditions in the stomach. It is then absorbed through

the intestines and reoxidized to nitrate in the blood, before being secreted into saliva. This process is known as enterosali-vary nitrate circulation (Lundberg et al. 2008). It was recently reported that nitrite contributes to systemic health by stimulat-ing the circulatory system (Lundberg et al. 2008).

Metabolic Pathways of Oral Bacteria

Carbohydrates and Sugar Alcohols

Polysaccharides can be hydrolyzed into oligosaccharides, disaccharides, and monosaccharides by host and bacterial gly-cosidases. For example, host α-amylase hydrolyzes cooked starch into carbohydrates, which can then be incorporated and metabolized by oral bacteria (Fig. 1).

Oral bacteria possess 2 types of sugar transport systems, the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and the binding-protein transport system (BPTS). The PEP-PTS consists of a sugar-specific sugar transport domain and a nonspecific sugar phosphorylation domain. Sugars that are transported by this system are immediately phosphorylated by PEP, a high-energy phosphoryl metabolite derived from glycoly-sis (the Embden-Meyerhof-Parnas pathway; Meadow et al. 1990). On the other hand, the BPTS is an ATP-mediated sugar transporter (Russell et al. 1992), and sugars transported by this system are phosphorylated by intracellular kinases (, ).

Oral bacteria are capable of utilizing most dietary carbohy-drates and some sugar alcohols. While they possess constitutive glucose transport systems, they have to induce transport systems for other carbohydrates and sugar alcohols when such molecules are more readily available than glucose. Incorporated carbohy-drates and sugar alcohols are subjected to glycolysis via meta-bolic reactions catalyzed by constitutive and inducible enzymes.

Glycolysis starts with glucose 6-phosphate (G6P) and pro-duces pyruvate, ATP, and reducing power ( to ). In addition, it creates anaerobic conditions by consuming oxygen (Fig. 1). Pyruvate can be further converted into lactate, acetate, ethanol, and formate through various branched pathways (Fig. 2). Most saccharolytic oral bacteria, including Streptococcus, Actinomyces, and Lactobacillus, share these pathways. Actinomyces also assimilates CO

2 to PEP and produces oxaloacetate, which can be

converted into succinate ( to ; Takahashi and Yamada 1999b). This pathway is also shared by Propionibacterium and Prevotella. Protons are expelled by H+-ATPase ().

The bacterial sugar metabolism is known to be regulated by environmental factors, such as the availability of sugar and oxygen that change dynamically in the oral cavity (Table).

Table. Ecological Niches in the Oral Cavity.

Supragingival Plaque Subgingival Plaque Tongue Coating

Surfaces for microbial adhesion Saliva-coated teeth GCF-coated teeth and epithelia Saliva-coated epitheliaNutrient sources Saliva, carbohydrates GCF, desquamated epithelia Saliva, carbohydrates, desquamated epitheliapH Neutral, acidic Neutral, weakly alkaline Neutral, acidicOxygen concentration High, low Low High, lowMetabolic properties Saccharolytic Proteolytic, amino acid–degrading Saccharolytic, proteolytic, amino acid–degrading

GCF, gingival crevicular fluid.





When sugar is in excess supply, as during mealtime, the intra-cellular level of fructose 1,6-bisphosphate (FBP) is increased, and lactate dehydrogenase () is activated in streptococcal cells, resulting in the dominant production of lactate, while increases in the levels of other metabolic intermediates (i.e., G3P and dihydroxyacetone phosphate [DHAP]) inhibit pyru-vate formate lyase (PFL; ), which is responsible for the anaerobic production of formate, acetate, and ethanol (Abbe et al. 1982). Increases in the levels of FBP (in Streptococcus sanguinis and Streptococcus mitis) or G6P (in Streptococcus mutans and Streptococcus salivarius) activate pyruvate kinase (), resulting in the acceleration of glycolysis and lactate pro-duction (Abbe et al. 1983). Increases in the FBP level also

activate ADP-glucose synthase () and subsequently promote the accumulation of intracellular polysaccharides. When the extracellular sugar supply is limited, as in between meals, the levels of FBP, G3P, and DHAP fall, resulting in the inactivation of lactate dehydrogenase and the release of PFL from inhibi-tion. This causes a metabolic shift from lactate-dominant to mixed-acid production, as found in the resting subgingival plaque. The resultant increase in intracellular inorganic phos-phate (caused by the dephosphorylation of phosphorylated sugars) promotes the degradation of intracellular polysaccha-rides into glucose 1-phosphate ().

Under aerobic conditions, the active form of PFL is irre-versibly inactivated by oxygen. Instead, pyruvate oxidase ()

Glucose

G6P

F6P

FBP

DHAPG3P

1,3BPG

3PG

2PG

PEP

Pyruvate

PEP PYR

GluPEP

PYR

Glucose

Sucrose

Suc6PFruATPADP

ATPADP

Suc

G1P Sucrose

Pi

ATP

ADP

ADP

ATP

ADP

ATP

PEP-PTS

PEP-PTS

BPTS

BPTS

Sorbitol

Sor6P

Sorbitol

Sor Fru

Xylitol

Xyl5P

PEP

PYR

PEP

PYR

XylXylitol

PEP-PTS

PEP-PTS

BPTS

2H

2H

2H

Pi

2H

Lactate Formate

Acetate Ethanol

OAA MAL FUM Succinate2H 2HCO2

Acetyl-PAcetyl-CoA

Acetaldehyde

TCA cycle

Pentose-phosphate pathway

2H

O2Pi

CO2H2O2

ADP

ATP

CoA

CO22H

CoA

Pi

CoA

2H

CoA

2H

ATP ADP

G1P ADPG

Glun

Glun+1

ATP PPi

PDAiP

Glun/Frun

Glun+1/Frun+1

Fru/GluH2O

Lactate

Propionate

Glu/Fru

ADP

ATPH+

H+

StreptococcusActinomycesLactobacillusothers

Veillonella

MAL

FUM

OAA

Pyruvate

Formate

AcetateSuccinate

H2O

Succinyl-CoA

Propionyl-CoAADP

ATPCO2

CO22H

CO2 2H

Pi

2H

2H

Figure 2. Bacterial metabolic pathways for carbohydrates and sugar alcohols. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; Acetyl-P, acetyl-phosphate; ADPG, ADP-glucose; DHAP, dihydroxyacetone phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; Fru, fructose; Fru

n, fructan consisting of n molecules of fructose; FUM, fumarate; G1P, glucose 1-phosphate; G3P, glyceraldehyde

3-phosphate; G6P, glucose 6-phosphate; Glu, glucose; Glun, glucan consisting of n molecules of glucose; MAL, malate; OAA, oxaloacetate; PEP,

phosphoenolpyruvate; Sor, sorbitol; Sor6P, sorbitol 6-phosphate; Suc, sucrose; Suc6P, sucrose 6-phosphate; Xyl, xylitol; Xyl5P, xylitol 5-phosphate; , glucose-phosphate isomerase; , phosphofructokinase; , fructose-bisphosphate aldolase; , triose phosphate isomerase; , NAD-linked glyceraldehyde 3-phosphate dehydrogenase; , phosphoglycerate kinase; , phosphoglyceromutase; , enolase; , pyruvate kinase; , NADP-linked glyceraldehyde 3-phosphate dehydrogenase; , FBP-dependent lactate dehydrogenase; , pyruvate oxidase; , acetate kinase; , pyruvate dehydrogenase; , phosphate acetyltransferase; , acetaldehyde dehydrogenase; , alcohol dehydrogenase; , pyruvate formate-lyase; , FBP-independent lactate dehydrogenase; , H+-ATPase; , phosphoenolpyruvate carboxylase/carboxykinase; , malate dehydrogenase; , fumarase; , fumarate reductase; , glucokinase; , sucrose 6-phosphate hydrolase; , fructokinase; , sucrose phosphorylase; , phosphoglucomutase; , sorbitol 6-phosphate dehydrogenase; , sorbitol dehydrogenase; , phosphatase; , ADP-glucose synthase; , glycogen synthase; , phosphorylase; , glucosyltransferase/fructosyltransferase; , glucanase/fructanase; , malate-lactate transhydrogenase; , malic enzyme; , pyruvate carboxylase.





and pyruvate dehydrogenase () convert pyruvate into acetate (Abbe et al. 1991). Pyruvate oxidase is found in S. sanguinis and Lactobacillus species, while S. mutans and Actinomyces species possess pyruvate dehydrogenase. Lactate can also be converted back into pyruvate by another type of lactate dehy-drogenase () and then into acetate by pyruvate oxidase (in Lactobacillus) or pyruvate dehydrogenase (in Actinomyces; Takahashi and Yamada 1999b). When the supply of sugar is limited, PFL is gradually converted into an oxygen-tolerant inactive form, which can be reactivated by sugar metabolism under anaerobic conditions. S. sanguinis possesses this mecha-nism, which protects PFL from oxygen inactivation (Takahashi et al. 1987).

These sugar metabolic pathways are also shared by peri-odontal disease– and oral malodor–associated saccharolytic bacteria, such as Fusobacterium and Prevotella, which can sometimes cause acidification, similar to caries-associated bacteria (Takahashi et al. 1997).

Lactose can be transported by the PEP-PTS, and the resultant lactose 6-phosphate is cleaved into glucose and galactose 6-phos-phate by phospho-β-galactosidase. Galactose can also be trans-ported by the PEP-PTS, which results in the production of galactose 6-phosphate. Galactose 6-phosphate is degraded into G3P and DHAP via the sequential formation of tagatose 6-phos-phate and tagatose 1,6-bisphosphate (the tagatose 6-phosphate pathway). On the other hand, BPTS-transported lactose is cleaved into glucose and galactose, and galactose, as well as BPTS-transported galactose, can be phosphorylated and converted to G6P via the sequential formation of galactose 1-phosphate and glucose 1-phosphate (the Leloir pathway; Crow et al. 1983).

Sugar alcohols such as sorbitol can be transported, dehydro-genated (, ), and metabolized through the Embden-Meyerhof-Parnas pathway (Fig. 2). Since sugar alcohols generate more reducing power, their metabolic flow tends to result in ethanol production, which involves efficient usage of reducing power (, ; Abbe et al. 1991). Xylitol can also be phosphorylated and incorporated via a fructose-specific PEP-PTS in mutans streptococci; however, xylitol 5-phosphate can-not be metabolized further. Instead, xylitol 5-phosphate can be turned into xylitol by dephosphorylation () and excreted (Trahan et al. 1991). This process is considered to be a futile cycle, as PEP, a high-energy phosphoryl metabolite, is wasted (Hausman et al. 1984).

Veillonella species utilize lactate as an essential carbon and energy source (Fig. 2). They possess a unique pathway in which lactate and oxaloacetate are converted to pyruvate and succinate, respectively, by a series of enzymatic reactions start-ing with malate-lactate transhydrogenase (; Ng and Hamilton 1971). Pyruvate is further converted into acetate and formate, while succinate is converted into propionate.

Various food webs are found among bacterial species in the oral biofilm, although details are omitted in this review.

Sucrose can also be converted extracellularly into glucan and fructan by glucosyltransferase and fructosyltransferase (), respectively, and act as sources of biofilm matrix mole-cules. Furthermore, glucan and fructan can be hydrolyzed () to give glucose and fructose, respectively.

Proteins, Peptides, and Amino Acids

Proteins can be degraded into peptides and amino acids by bac-terial and host proteases and peptidases (Fig. 3). Several excel-lent review articles about proteases and peptidases have already been published elsewhere. In general, bacteria utilize amino acids for biosynthesis, but periodontal disease– and oral malodor–associated bacteria also ferment amino acids to pro-duce energy. In the latter process, amino acids are deaminated and then converted into short-chain fatty acids with the pro-duction of ATP. Although various amino acids are reported to be transported into oral bacteria, only a few have been demon-strated to be fermentative substrates with specific metabolic pathways. This is probably due to the fact that the metabolic pathways for amino acids are unique and complex and many bacteria, such as Porphyromonas gingivalis, prefer peptides to amino acids, which are not always commercially available (Takahashi and Sato 2001).

Glutamate is one of the most abundant amino acids in human bodily fluids; thus, many bacteria utilize it (Fig. 3). In P. gingivalis, glutamate is deaminated, decarboxylated, and reduced to succinyl-CoA (, ) and then degraded to butyrate ( to b) and propionate (, p) with the production of acetyl-CoA (Takahashi et al. 2000), depending on its redox state (Takahashi and Sato 2001). Acetyl-CoA can turn to ace-tate with the production of ATP (, ). Recently, it is proposed that butyrate can also be produced from acetyl-CoA (; Hendrickson et al. 2009). In Fusobacterium species, these pathways are modified; most species convert 2-oxoglutarate into crotonyl-CoA via 2-hydroxyglutarate and vinylacetyl-CoA (), while some species convert glutamate into succinate-semialdehyde via 4-aminobutyrate () (Gharbia and Shah 1991).

In P. gingivalis and Prevotella intermedia, aspartate is deaminated, reduced, and decarboxylated into pyruvate, which is then further degraded into acetate, producing ATP ( to ; Takahashi et al., 2000; Takahashi and Yamada 2000). P. gingi-valis converts fumarate to succinyl-CoA via succinate (, s) and subsequently produces propionate and butyrate, while P. intermedia also produces formate from pyruvate (). These enzymes for glutamate and aspartate fermentation have been confirmed at gene levels (Nelson et al. 2003).

Valine and leucine are branched amino acids and can be converted into isobutyrate and isovalerate, respectively, through sequential reactions of deamination, decarboxylation, and reduction with the production of acetyl-CoA (, , i). As mentioned above, acetate can be synthesized from acetyl-CoA with the production of ATP (, ). These pathways are shared by P. gingivalis and P. intermedia (Takahashi et al. 2000; Takahashi and Yamada 2000).

Peptostreptococcus micros utilizes serine from oligopep-tides to produce acetate and formate (, , to ; Uematsu et al. 2007), while Eubacterium species metabolize arginine and lysine into butyrate via ornithine, although the detailed meta-bolic pathways have not been identified (Uematsu et al. 2003).

Cysteine is degraded by cystathionine γ-lyase (cysteine desulfhydrase; ) into pyruvate, producing hydrogen sulfide





and ammonia, while methionine is degraded by methionine γ-lyase (methioninase; ) into 2-oxobutyrate, producing methyl mercaptan (methanetiol) and ammonia. Pyruvate and 2-oxobutyrate can be metabolized further, leading to the pro-duction of acetate and propionate, respectively, with ATP pro-duction ( to , , p). These pathways are shared by Fusobacterium, Prevotella, Porphyromonas, and Treponema (Pianotti et al. 1986; Yoshimura et al. 2000; Fukamachi et al. 2005). Streptococcus, Actinomyces, and Veillonella also pro-duce hydrogen sulfide from cysteine (Persson et al. 1990; Washio et al. 2005).

Indole and skatole are synthesized from tryptophan (Codipilly and Kleinberg 2008). Indole is produced by tryptophanase (; Yoshida et al. 2009) and can be further metabolized to acetate

with ATP production. Skatole is synthesized via indolepyruvate and indoleacetate, but the responsible enzymes have not been determined.

Stickland reactions can also occur in the mixtures of amino acids in which bacteria perform coupled redox reactions between pairs of amino acids. Proline is known to be reduced to δ-amino-n-valerate in dental plaque (Curtis et al. 1983). Eubacterium brachy produces phenylpropionate, phenyllactate, and phenylacetate from phenylalanine via redox reactions; it also converts leucine to isovalerate, hydroxyisocaproate, and isocaproate in the same manner (Hamid et al. 1994) (Fig. 3).

The metabolic regulation for amino acid degradation has not yet been clearly elucidated; however, as described in the next section about the ecological shift, some bacteria, such as

zzzzzzzzzzzzzzzz

Peptides/Amino acidsATP-linked transporter (ABC transporter)

Peptides/Amino acids

Glutamate AspartateValine/ Leucine

Methionine

Proteinsproteases and peptidases

Succinyl-CoA

2-Oxoglutarate

Succinate- semialdehyde

Crotonyl-CoA

Butyryl-CoA

Succinate

Fumarate

4-Hydroxybutyrate

Oxaloacetate

Malate

Pyruvate

Acetyl-CoA

Acetyl-phosphate

2-Oxoisovalerate/ 2-Oxoisocaproate

Isobutyryl-CoA/ Isovaleryl-CoA

(R)-Methyl- malonyl-

CoA

Propionyl-CoA

2-Oxobutyrate

Propionyl-CoA

Propionate Butyrate AcetateIsobutyrate/ Isovalerate

Propionate

NH3 2H NH32-Oxo-

glutarate

Glutamate

Formate

ATP

ADP

CoA

CO2 2H

Pi

CoA

Acetyl-CoA

Acetate

Acetyl-CoA

Acetate

Acetyl-CoA

Acetate

CoA

CO2 2H

CoA

Cysteine

H2S NH3 CH3SH

Succinate Formate

Acetyl-CoA

Acetate

CoA CO2 2H

CoA CO2 2H

NH3 2H

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2H

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Fusobacterium Prevotella Porphyromonas others

Serine Tryptophane

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Indole-acetate

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NH3 H2O

NH3

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NH3

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Phenyl- propionate/ Isocaproate

Phenylpyruvate/ 2-Oxoisocaproate

2H

Phenylacetate/ Isovalerate

Phenyllactate/ Hydroxyisocaproate

⑲

⑳

⑱

CO2

Figure 3. Bacterial metabolic pathways for proteins, peptides, and amino acids. , glutamate dehydrogenase; , 2-oxoglutarate oxidoreductase; , succinate-semialdehyde dehydrogenase; , 4-hydroxybutyrate dehydrogenase; , enzyme(s) for the conversion of 4-hydroxybutyrate into crotonyl-CoA; , butyryl-CoA oxidoreductase; , apbsi, acyl-CoA:acetate CoA-transferases, involved in the production of a (acetate), p (propionate), b (butyarate), s (succinyl-CoA), and i (isobutyrate and isovalerate), respectively, with the production of acetyl-CoA; , methylmalonyl-CoA mutase; , aspartate ammonia-lyase; , fumarase; , malate dehydrogenase; , oxaloacetate decarboxylase; , pyruvate oxidoreductase; , pyruvate formate-lyase; , phosphate acetyltransferase; , acetate kinase; , fumarate reductase; , pathway from acetyl-CoA to crotonyl-CoA via acetoacetyl-CoA; , pathway from 2-oxoglutarate to crotonyl-CoA via 2-hydroxyglutarate and vinylacetyl-CoA; , pathway from glutamate to succinate-semialdehyde via 4-aminobutyrate; , branched-chain amino acid aminotransferase; , branched-chain 2-oxoacid oxidoreductase; , serine dehydratase; , cystathionine γ-lyase (cysteine desulfhydrase); , methionine γ-lyase (methioninase); , 2-oxobutyrate oxidoreductase; , tryptophanase.





P. gingivalis, P. intermedia, and Veillonella, are reported to modify their metabolic properties, as well as their peri-odontal pathogenicity, in response to the environmental factors.

Alkalization and Acid Neutralization

Arginine is converted to citrulline by arginine deiminase, releasing ammonia (), and is subsequently degraded to ornithine, ammonia, and carbon dioxide (, ). Ornithine can be further degraded to putrescine and carbon diox-ide () (Fig. 4 [1]). This series of reac-tions is called the arginine deiminase system, which produces alkaline mole-cules and is considered to counter sugar metabolism–based acidification (Burne and Marquis 2000). This pathway is shared by various oral bacteria including S. sanguinis, S. mitis, Streptococcus gor-donii, and certain Lactobacillus and Actinomyces species. Arginine is also metabolized via the agmatine deiminase system, which leads to the production of ammonia, carbon dioxide, and putrescine ( to , ), resulting in alkaline condi-tions (Liu et al. 2012). Urea is the major alkaline-generating substrate in the oral cavity supplied from saliva and can be converted to ammonia and carbon diox-ide by bacterial urease such as S. sali-varius and Actinomyces naeslundii () (Fig. 4 [2]).

In general, bacterial amino acid fermentation—that is, amino acid deamination (ammonia production) and the subse-quent degradation of 2-oxoacid to an organic acid and carbon dioxide (Fig. 3)—produces both acidic and alkaline substances, resulting in acid neutralization (Takahashi 2003) (Fig. 4 [3]). The decarboxylation of amino acids produces carbon oxide and amines, which also lead to acid neutralization (Hayes and Hyatt 1974)—for example, glutamate, arginine, and histidine are decarboxylated into γ-amino butyrate, agmatine, and hista-mine, respectively, some of which are inflammatory mediators (Fig. 4 [4]).

The produced acids can be further degraded or modified to give carbon dioxide and/or weaker acids, resulting in a reduction in the acidity of the environment (Fig. 4 [5]–[8]). Veillonella converts lactate into formate, acetate, and propionate, while Lactobacillus and Actinomyces turn lactate into acetate (Fig. 2). Campylobacter recta and other bacteria utilize formate as an energy source and an electron donor in reactions in which fuma-rate (derived from aspartate and asparagine) acts as an electron acceptor. In these reactions, formate is converted to carbon diox-ide, and fumarate is converted into succinate (Ohta et al. 1991).

Malate can be decarboxylated to lactate by the malolactic enzyme (malate dehydrogenase) found in streptococci, resulting in a reduction in the acidity of the environment. The latter mech-anism is reported to protect streptococci from environmental stresses, such as acidic pH, oxidation, and starvation (Sheng et al. 2010). Some bacteria, including Pseudoramibacter alacto-lyticus, can convert acetate and butyrate into butyrate and capro-ate (weaker acids), respectively, which decreases the acidity of the environment (Gottschalk 1985).

Ecologic Shifts Involving Microbial Adaptation and Selection: Metabolic Activity as an Ecologic Modifying FactorSupragingival plaque mainly consists of non–mutans strepto-cocci and Actinomyces, and it tends to maintain a neutral pH via the production of acidic and alkaline substances and the acid neutralization, with salivary washing, acid neutralization, and supply of calcium and phosphate ions, resulting in the bal-ance between demineralization and remineralization (the dynamic stability stage); however, the balance can tilt toward

Arginine

Arginine

NH3

Citrulline

Carbamoyl-phosphate

Ornithine

CO2

NH3

Putrescine

CO2

NH3, CO2

NH3, CO2

ADP

ATP

Pi

H2O

[1] Arginine/agmatine deiminase system

[2] Urease

Agmatine

Carbamoyl-putrescine

Putrescine

Pi

CO2CO2

NH3NH3

UreaUrea

[3] Amino acid fermentation

[4] Amino acid decarboxylation

Amino acids 2-Oxoacids Acids

Amino acids Amines

Lactate Formate, Acetate, Propionate

Malate Lactate

CO2

CO2

CO2

NH3

CO2

StreptococcusActinomycesLactobacillusothers

Formate CO2SuccinateFumarete

Acetate, Butyrate Butyrate, Caproate

[5] Lactate degradation

[6] Formate degradation

Ornithine

[7] Malolactic reaction

[8] Acid conversion

Figure 4. Bacterial metabolic pathways for alkalization and acid neutralization.





demineralization, usually due to the frequent intake of carbo-hydrates and the subsequent acidification (Fig. 5). This process increases bacterial acidogenicity and acidurance via a series of biochemical adaptive responses, including 1) an increase in the proton impermeability of the cell membrane; 2) the induction of H+-ATPase activity, which expels protons from cells; 3) the induction of metabolic pathways involved in alkalization and acid neutralization (Fig. 4); and 4) the induction of stress pro-teins that protect enzymes and nucleic acids from acid denatur-ation (the acidogenic stage; Takahashi and Yamada 1999a; Takahashi 2005; Takahashi and Nyvad 2008, 2011). These phenomena subsequently encourage the proliferation of more acidogenic bacteria, such as “low pH” non–mutans strepto-cocci and Actinomyces species. Once a prolonged acidic envi-ronment, more aciduric bacteria—such as mutans streptococci and non–mutans aciduric bacteria (e.g., lactobacilli and bifido-bacteria)—can become dominant through acid selection (the aciduric stage), which can enhance plaque cariogenicity. During the development of dental caries, the frequent presence of sugar in the environment and the resultant bacterial acidifi-cation are the main ecologic modifying factors and driving forces (Takahashi and Nyvad 2008, 2011).

Subgingival plaque also maintains metabolic homeostasis, with Gram-positive facultative anaerobes being dominant (the dynamic stability stage); however, bacterial cytotoxic end prod-ucts can induce host defense responses and subsequently deepen periodontal crevices and increase the secretion of gingival

crevicular fluid (Fig. 5). This protein-rich environment results in increased numbers of proteolytic and amino acid–degrading bacteria, such as Fusobacterium and Prevotella (the proteo-lytic/amino acid–degrading stage). The increase in the numbers of bacterial cytotoxic end products, as well as bacterial compo-nents, can enhance host inflammation and bleeding, which can introduce more periodontopathogenic bacteria, such as P. gingi-valis and oral Treponema (inflammatory stage), since P. gingi-valis requires the hemin contained in blood hemoglobin, while some oral Treponema requires branched-chain short-chain fatty acids for their growth, which can be produced by P. gingivalis (Takahashi 2005). Relatively acid-tolerant bacteria, such as Fusobacterium and Prevotella, can neutralize acidic environ-ments by acid neutralization activity through amino acid fer-mentation and facilitate the introduction of more periodontopathic bacteria, such as P. gingivalis, which is very acid sensitive and needs a neutral environment to become domi-nant (Takahashi et al. 1997). The proteolytic/amino acid–degrading and saccharolytic bacterium P. intermedia can live in both a protein-rich, neutral to weakly alkaline subgingival site and a sugar-rich, acidic supragingival site, but it increases its protease activity and cytotoxic end product production when sugar is omitted from the culture medium (Saito et al. 2001), suggesting that its pathogenicity might increase after it colo-nizes subgingival sites (where the carbohydrate supply is lim-ited). In addition, a rise in host body temperature can increase the pathogenicity of P. gingivalis (Smalley et al. 2000; Curtis

Dynamic stability stage Dynamic stability stage

Acidogenic stage

Aciduricstage

Proteolytic/amino acid-degrading stage

Inflammatorystage

Mic

robi

alad

apta

tion/

sele

ctio

n

euqalplavignigbuSeuqalplavignigarpuS

Dominance of non-MS and Actinomyces Dominance of Gram-positive facultative anaerobes

Increase in “low-pH” non-MSand Actinomyces

Increase in Fusobacteriumand Prevotella

Increase in MSand non-mutansaciduric bacteria

Increase in Pgand Treponema

Ecological adaptation (sugar-frequent/acidic-pH)Increase in proton-impermeability of cell membraneInduction of H+- ATPaseInduction of alkalization/acid-neutralizationInduction of stress proteins

Ecological adaptation(protein-rich/neutral-to-weakly alkaline-pH)

Induction of proteolytic/amino acid-degrading activityInduction of alkalization/acid-neutralizationInduction of virulent factors

Sugar metabolism (acidic end-products)

Host eating-habit (sugar intake)

Net mineral gain

(lesion regression/arrest)

Net mineral loss

(lesion initiation/progression)

Protei

n/amino ac

id meta

bolism (c

ytotoxic

end-pro

ducts)

Host defe

nse re

sponse

(GCF)

Gingival in

flammati

on

Gingival h

ealth

Figure 5. Ecological shifts associated with microbial adaptation and selection. MS, mutans streptococci; non-MS, non–mutans streptococci (oral streptococci except for mutans streptococci); PG, Porphyromonas gingivalis. Modified, combined, and extended from Takahashi (2005) and Takahashi and Nyvad (2008, 2011).





et al. 2011). During the initiation and development of periodon-tal disease, protein-rich and neutral/weakly alkaline environ-ments are the main ecological modifying factors and driving forces. Needless to say, the host immune system is involved in the etiology of periodontal disease through host-parasite inter-action, but in this review, it is omitted because of limited space.

Oral malodor can also develop through bacterial proteolytic/amino acid–degrading activity. Although the effects of envi-ronmental factors on oral malodor remain unclear, periodontitis-associated bacteria are suggested to be involved in the production of hydrogen sulfide and methyl mercaptan (de Boever et al. 1994). Other indigenous bacteria, including Veillonella and Actinomyces, are also responsible for produc-ing hydrogen sulfide (Persson et al. 1990; Washio et al. 2005). Hydrogen sulfide production from cysteine by Veillonella is enhanced by lactate (Washio et al. 2014), suggesting that the production of malodorous substances is also regulated by bac-terial metabolic activity through environmental modification.

Future Research into the Functions of the Oral MicrobiomeIt was difficult to measure bacterial metabolic activity, particu-larly in the oral microbiome, due to the small sample sizes involved; however, recent advances in metabolomic technology have made it possible to analyze the levels of various types of metabolites in small samples. Comprehensive analyses of metabolites (metabolome analysis or metabolomics) can pro-vide insights into the metabolic functions of the oral microbi-ome (Takahashi et al. 2010; Takahashi and Washio 2011). Together with comprehensive analyses of the mRNA molecules present within the oral microbiome (metatranscriptome analy-sis or metatranscriptomics), the metabolomic approach might make it possible to obtain an overview of the metabolic activity of the oral microbiome and its relationship with oral microbiome–associated oral diseases (Nyvad et al. 2013). This may also facilitate the verification of oral health care protocols based on oral microbiome control, which could be a true “plaque con-trol,” based on the answer to the question “What are they doing?” Furthermore, the omics approach to oral microbiome and human tissues, including immune systems, may provide the paradigm shift of host-parasite cohabitation/interaction from reductionistic to holistic understanding, known as supraorgan-ism or superorganism (Turnbaugh et al. 2007).

Author Contributions

N. Takahashi, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the man-uscript. The author gave final approval and agrees to be account-able for all aspects of the work.

Acknowledgments

This study was partly supported by KAKENHI (no. 60183852) from the Japan Society for the Promotion of Science, Japan. The author declares no potential conflicts of interest with respect to the authorship and/or publication of this article.

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