Jpn. J. Infect. Dis., 53, I-5, 2000

Invited Review

Cu汀ent Understanding of the Cause of Dental Caries

Nobuhiro Hanada*

Department of Oral Science, National Imtitute ofInfectious Diseases 7Toyama 1-23-1, Shinjuku-ku, Too,0162-8640, Japan

(Received March 3, 2000)

CONTENTS:

1. 1m廿oduction 2. Cariogenic 3･ Infection route of the mutans streptococci 4. and dental caries 5･ Development ofa vaccine 6. Conclusion

StJMMARY: Dental caries-associated oral streptococciare called the mutans streptococci, withStTTePtOCOCCuS mutansand sobrL'nus being the most prevalent caries-associated organismsinhumanS. Strains of the mutans streptococciare highly conserved withinnot only mothersand their children but also racial groups, suggesting vertical transmission of this organism withinhumanpopulations. It has been found thatthe mother- child infection route of the mutans streptococci canbe prevented by simply reducing the amount of the mutans streptococci containedinthe mothers'saliva. Moreover, a chlorhexidine vanish reduces the salivary mutans streptococci byanaverage of 3 logs (99.9%),and moved them below detectable levels. We should attempt to eliminate theinfection with the mutans streptococci among Japanese people by attempting to break theinfec- tious chain&om mothers to children.

Althoughadults have decayed teeth,inmost cases it isan 1. Introduction iatrogenic secondary dental caries (around restorationsand Human beings have been exposed to various kinds of easily developsinto pulpitis), which is a sequela of pediatric infectious diseases since ancient times, which have threat- dental caries during school age. Since pediatric dental caries enedtheir very existence. Many attempts have been made to is closely related to the mutans streptococci infection, it is prevent new and recurrent infectious diseases, which may appropriate to call it a mutrs.streptococcal disease･ Although endanger human1ives. However, it is difncult to monitor each theincidence ofdentalcarleS mCreaSed aRerthe Second World infectious disease. Therefore, much attention has not been War, it is a disease that has annoyed humanbeings since paid to commonplace infectious diseases. Dental caries is ancient times; in血is sense, there has been a recu汀ent Out- perhaps one of such disePes･ Previous reports (I-6) have break of the disease. Dental caries of ancient times, however, revealed that dental caries ISaninfectious disease caused by is considered to be different舟om that encountered today. domesticinfection withspecific streptococci. Dentalcaries- Firstly, dental caries was encountered onlyinadultsinancient associated oralstreptococciare calledthe mutans streptococci, times, whereas today it is a disease predominantly foundin with Streptococcus mutaMand Streptococcus sobrinus being children (14). In Japan, dental caries exclusively in adults the most prevalent caries-associated organismsinhumanS. was fわund in the people of the Jomon era (approximately Strains of the mutans streptococci are highly conserved 10,000 B.C. tothe 5th14thcentury B.C.) (15). Secondly, dental within not only mothers and their chil血en but also racial caries in ancient times was localized to the cement-enamel groups, suggesting a vertical transmission of this organism Junction of the , whereas, dental caries today is localized within humanpopulations (6-1 I ). Clinical trials conducted to the enamel of the crown. 0'Sullivan, et al. examined a inEurOpe have shownthat it is possible to preventthe mutans total of I ,974 primary molar teethfromthe skeletal remains streptococciinfectionfrom mother to child (12, 13). Dental of373 childrenfrom prehistoric times to the 18th century for caries that has flourished in Japanafter the Second World caries prevalenceand site characteristics･ Their results showed War is considered to be caused bythe mother-childinfection that caries in prlmary teeth was initiated more often at the withthe mutans streptococci resultingindental caries of the cement-enamel junctionthanatthe contact point of the crown enamel in children. (16). A look at the history of dental caries reveals that the

*Corresponding author: Tel: +81-3-5285-1 1 I 1, Fax: +81-3-5285- incidence of dental caries has nuctuated &omancient times to today･ For example, theincidenceincreased rapidly during 1 I 72, E-mail: [email protected] the Yayoi era (the 5th-4th century B･C･ to the 3rd century A.D.); and is considered to have been higher during this than ThisarticleisanlnvitedReviewbasedonalecturepresentedat the Muromachi (1392 to 1573 A.D.) Or Kamakura (11$5 to the9thSymposlumOftheNationalInstituteofInfectious 1333 A.D.) eras (14). In modem Japan,theincidence of dental Diseases,Tokyo,21May1999. cariesinboth primaryand permanent teeth has alsoincreased sharply after the Second World War (17). the biofi.lm, thereby predisposlng the tooth surface to dental In this paper, the etiology of dental caries, which has caries. Although, there are many oral streptococci whose changed dramatically, lS Outlined;inaddition, progressinthe prlmary final metabolite is , the metabolic mecha- development of an anticaries vaccine and other useful nism in the mutans streptococci seems to be different from bioactive molecules are discussed. that in other bacteria. The mutans streptococci continuously produce lactic acid under acidic conditions, such as at pH 4, under which conditions dental caries may be readily produced 2･ Cariogenic bacteria (24, 25). On the other hand, while noncariogenic streptococci With the development of carlOgenic bacteriology, it has (S. sanguis, S. salivarius, and S. mitis) other thanthe mutans been demonstrated that current dental caries is an infectious streptococcl produce lactic acid, they stop production under disease caused by the mutans streptococci. The mutans strep- acidic conditions (26). Studies of the relationship between tococcus is a generic name fわr seven streptococcal groups the concentration and synthesis of lactic acid revealed that as that exist in the oral cavityand show a unique reaction with the concentration of lactic acidinthe envkonmentincreases, the sucrose･ Among these, two , namely, S. mutans and S. mutans streptococci continue to produce lactic acid. However, sobrinus have been detected in the oral cavities of Japanese inthe case ofS. sanguis, one of the noncanogenic streptococci, people (6,18,19). The adhesion ofS. mutans to the tooth lactic acid production declines when the concentration of surface takes place following a series of steps: 1)initial adhe- lactic acid in the environment increases, and the lactic acid sion, 2) aggregation of bacteria (formation of a microcolony), di軌ses back into the bacterial cell (26). and 3) formation due to the function of sucrose. If an Clinically, it can be said that the fate of teeth is decided individual infected with the mutans streptococci does not when the primary teeth erupt (6). If the mutans streptococci mgest sucrose o洗en, the bacterial microcolonies of血e tooth are elevated in the saliva during the tooth emptlOn, dental surface can be easily removed. The adhesive force of the caries of the teeth readily develops, while if nonpathogenic mutans streptococci to the tooth surface depends on the static bacteria (S. sanguis, S. salivarius, and S. mitis) are dominant, electrical and hydrophobic ptOPertyof the bacterial surface and the bonding between lectlnS and hydrogen. On the other hand, the microcolonies of those who o洗en Ingest Sucrose (A) adhere firmly to the tooth surface because sticky water- insoluble glucan is synthesized from sucroseinthe presence 垣S&mNuetnatni steSi5) gtfD of glucosyltransferase (GTF) se.cretedfrom the mutans streptococci. The oral microcolonleS gradually develop Into a mature biofilm through the formation of water-insoluble k一一 一ト gtLBmtltant glucanuslng dietary sucrose. The glucanpromotes adhesion gtfC gtfD of the mutans streptococci to the tooth surface. In addition, the water-insoluble glucan serves as a barrier that prevents gtLIC mutant the buffering action of saliva. Acids, which are metabolic products of bacteria, then accumulateinthe biofilm. Several gがgenes participate in the synthesis of water一

戸soluble glucan(Table)･ Inthe case of S･ mutans,three genes, 1.e., g押, gyC,and gyp, have been identified on its chromo- some (20-22). It is observed that gtj8- or gtfC-deficient mutants produce the microcoloniesinthe laboratory, but lack gtD mutant the ability to formbiofilm to the wall of test tubes (Fig.). gtfB gtfC Thus, the gtjB and gtfC genes are considered to be closely related to the formation of biofilmand pathogenicityof this 匿国The erythromycin resistance (Emr) gene organism. In the case ofS. sobrinus, fourGTFs were purified, 匿ヨThe tetracycline resistance (Tcr) gene and these four genes, i.e., got, g昨gqand gdU, have been identified on its chromosome (23). (B) The sucrose consumed by theindividual is I) converted to glucaヮoutside the bacteria by GTF or 2) taken up by the bacterla Via mediation of the scrPTS system and metabolized to pymvic acid, which is converted to lactic acid by a group of glycolytic . The lactic acid decreases the pH of

Table. GTFs derived舟om the mutans streptococci

spe c ie s gがgene glucan

S. mulans gijB GTF-I water insoluble glucan

gくjC GTF-SI water soluble and insoluble glucans

g昨) GTF-S water soluble glucan

S. sobrt'nus g好 GTFII water insoluble glucan gyS GTF-S water soluble glucaJl gdT GTF-S water soluble glucan Figure･ Insertional inactivation of the gqgenes of S･ mutaw(A), Biofllm gdU GTF-S water soluble glucan formation of various stralnS OfS. mutaW inthe test tubes (B).

2 dental caries is not likely to occur (6). The glycoproteinS such the 1980S､by researchersinNorthem Europe and published as mucin contained in the saliva first adhere to the tooth inthe 1 990S (1 7),and atbTaCted attention丘om all overthe world. surface when the tootherupts. The surface coating by mucins According to them, if the number of the mutans streptococci is called the pellicle. Since both the pellicle and bacteria containedinthe saliva of mothers is reduced to half through possess negative charges, bacteria camot adhere easily to the education regarding oral hygiene,infection &om mothers to pellicle･ However, S. sanguisand S. mitis adhere to the pellicle children can be effectively prevented. Their resultsindicate withthe aid ofbivalent cations such as Ca2+. Furthermore,in that if the mother-child infection can be prevented by artifi- the presence of Actinomyces viscosus and Actinomyces cial measures such as bytheuse ofa vaccine, complete elimi- naeslundii, which have a tendency to agglutinate with oral nation of the mutans streptococci from the oral cavities of streptococci, healthy bacterial plaques whose dental-Caries- humans may be achievedinseveral generations. Fortunately, induction potential is loware formed･When noncar10genic it has not been reported that the mutans streptococci can be oral bacteria, such as S. sanguisand S. mitis, with low dental- transmitted through the birth canal; therefore, it is reasonable caries-induction capacities are attached to the tooth surfaces, to assume that childrenare notinfected with the mutans strep- a continual decrease in血e pH can be prevented. Therefわre, tococci at the time oferuptlOn Ofthe pr皿ary teeth. Based on dental caries is less likely to occur than when the mutans the results of research on the mother-child infection of the streptococci colonize the tooth surface, as is likely with the mutans streptococci, children areinfected with the mutans

Ingestion of sweets.When noncar10genic bacteria with low streptococci between approximately 19 moれ血s and 3 1 months dental-Caries-induction potentialareinitially attached to the of age (8). This findingindicates thatthe mother-childinfec- tooth surface, subsequent the mutans streptococci coloniza- tion of the mutans streptococci canbe prevented by measures tion of the empted teeth is prevented, since the presence of targeted at mothers whoare weaning babies (1 3). the noncar10genic bacteria prevent contact between the Sandham, et al. (29) reported that a chlorhexidine vamish, car10genic streptococciand the teeth. On the other hand, in covered witheither sealant, reduced the salivary mutans strep- children in whom the mutans streptococci are dominant in tococci by an average of3 logs (99.9%), and moved them the saliva during the eruption of teeth, the bacteria adheres to below detectable levels. Treatment with a slngle prophylaxis the pellicle of the erupted teeth and multiply on the tooth had no effect on mutans streptococcus levels (29). In order to surface, especially within the pits and fissures. These teeth prevent the earlyinfection of children with the mutans strep- are then most likely to develop dental caries (6). tococci, the chlorhexidine vanish method should be applied Similarresults were confmedinanimal tests. Tanzer et al. to mothers whoare weanlng babies. (27,28)investigated theincidence of dental cariesinspecific- -free (SPF) ratsinfected with S. salivarius S. mutans, 4. Sucrose and dental caries and S. sobrinus. Their results indicated that this incidence depends on the sequence of infection with the bacteria. We have isolated the activities of two tandem arranged gtf infected withS. salivarius, or either S. muta7V Or S. sobrinus, genes (gtjBand gtjC) coding for GTF (GTF-I and GTF-SI) Or uninfected rats were transiently co-Gaged so as to allow (21). The third gtfgene (gtjD) coding for GTF-S has been natural fecal transfer of organisms due to coprophagy. Initial isolated from a different locus of the chromos?mal DNA (22)･ oral colonization of rats by S. salivarius inhibited the To determine the role of the GTF in the car10genicityofS. ecological emergence of fecally transmitted S. mutansand S. mutans, we devised a strategy to replace most of the gene of sobrinus. GTF (gtf) with a heterologous DNAfragment coding for Therefore, when the rats were infected first with S. erythromycinresistance (Emr) or tetracycline resistance (Tcr) salivarius (nonpathogenic oralindigenous bacteria) prior to (Fig.). Insertionalinactivation of the gqgenesindicated that the mutans streptococci infection, the incidence of dental gtjB and gtfC genes coding for GTF-Ⅰand GTFISI activities caries was lower than that when the rats were infected first were required for in vitro sucrose-dependent adherence to with the mutans streptococci Prior to S. salivariusinfection. smooth surfaces. Since no vaccine against the mutans streptococci is currently Why does sucrose predisposeindividuals to dental caries available, infection with the mutans streptococci cannot be more thanother dietary sugarS? This could be due to the completely prevented. Under such circumstances, it is substrate-specificityof GTF secretedfromthe mutans strepto- important to obtain data to confim the theory that by only cocci, which is nmw;therefore, GTF camot utilize substances delaylnginfection withthe mutans streptococci would be effec- other thansucrose as a substrate for enzyme activity. In other tiveindecreaslng theincidence of dental cariesinchildren. words, the water-insoluble glucan and oral biofilmcannot be synthesized &om substrates other thansucrose. According to anepidemiological study, 96% of 12-year-Old children were 3･ Infection route of the mutans streptococci infected with the mutans streptococci in an area in Africa Afterthe discovery of the mutans streptococciinthe United where theincidence of dental caries was low (30). The States, its route of infection from mothers to children has incidence of dental cariesinpeople livinginthisarea may be gradually been clarified. However, Since it was not demon- low because of the low consumption of sucrose. strated whether the mother-child infection of the mutans Generally speaking, avoidance of sweets isthe most impor- streptococci could be artiflCially prevented, this problem has tant measureinpreventing dental caries. However, differences not been seriously discussed. In general, the prevention of inthe "dental-caries-induction capacities" of ,血ctose, infection by a pathogen is the option of choiceamong the and sucrose, all of which are sweet, are not widelyknown. measures to preventinfectious diseases. However, the mutans Sucrose is a disaccharide composed of glucoseandfructose streptococci, whichare widespread throughout the world,are containing a high-energy bond. The energy associated with considered to beanindigenous bacteriumin the oral cavity the hydrolysIS Of sucrose to glucose andfructose plays an and the prevention of infection with the mutans streptococci important role in the induction of dental caries. The free is consideredunlikely. The results of clinical tests conductedin energy produced by hydrolysis of the high-･･energy bond in

3 sucrose is 6,600 Gal-mol. The GTF secreted from the mutans prevention of dental caries (37). streptococcI POlymerizes glucose, uslngthe energy obtained Kelly et al. (38) inhibited the binding of a cell-surface throughthe hydrolysis of sucrose to glucose and fructose, adhesin of S. mutans to salivary receptors uslng a Synthetic resultinginthe synthesis of water-insolubleglucan. Therefore, peptide blO25) Corresponding to residues 1025-1044 0fthe if one Ingests glucose or f山ctose instead of sucrose, no adhesin. Two residues withinpl025that contribute to binding hydrolysis energy lS generated, water-insoluble glucanis not were identified by site-directed mutagenesis. In an in vivo synthesized,andthe "dental-caries-induction levels"are low. human streptococcal adhesion model, direct application or Dental caries isaninfectious disease,and whether or not one p1025 to the teeth prevented recolonization of S･ mutans but will develop dental caries strongly depends ontheamount of notActinomyces, as compared witha controlpeptide or saline･ sucrose consumed. Even if infection cannot be prevented, These strategleS may be used against other pathogenic dental caries can be ameliorated by reducing the consump- bacteria in which adhesins mediate colonization of mucosal tion of sucrose. surfaces.

5. Development of a vaccine 6. Conclusion

A cell-surface adhesinofthe mutans streptococci may be Since the clinical data indicatlng that the prevention of involvedinthe binding of bacteria to the tooth surface,and infection with the mutans streptococci is possible were not has long been focused upon as a candidate for a preventive available until the 1990S, no Japanese public health center vaccine for dental caries. The peptide PAc (365-377) was consideredthe prevention ofthisinfection, which is widespread showntomiseanandbodyinmice, which inhibitedthe binding throughout the world. As I have discussed in this paper, in of salivary components to the cell-surface adhesin. Usingthis clinical trials it has been found thatthe mother-childinfection peptide as a unit peptide, two constructs based on multiple route of the mutans streptococci can be prevented by simply antigenic peptides, and several types of tandem repeats of reducingthe amount of the mutans streptococci containedin two orthree copleS Were Synthesized to estimatethe immun0- mothers'saliva. Moreover, a chlorhexidine vanish reduces genicityofthesepeptides (3 1-35).Anincreaseinthe immun0- the salivary mutans streptococci by an average of 3 logs genicitywas observed for all constructs with the use of an (99.9%),and movedthem below detectable levels. We should adjuvant compared to the unit peptide alone. However, the attempt to eliminatetheinfection withthe mutans streptococci tandem repeat constructs generallyinducedantibody produc- by attempting to breaktheinfectious chain from mothers to tioninthe absence ofanadjuvant, whilethe multipleantigenic children. peptide constructs did notinduceantibody production under the same conditions (35). Although such a phenomenon may REFERENCE S be restricted tothis particularpeptide sequence, these results mayinfluencethe strategy for the design ofpeptide vaccines. I. Beighton, D., Manji, F., Baelum, V･, Fejerskov, 0･, One British research group, which has been involvedin Johnson, N. W and Wilton, ∫.M. (1989): Associations the development of a vaccine against dental caries, succeeded between salivary levels of , Strep- inSymthesizinganIgGand secretary IgAantibody against S. tococcus sobrinus, lactobacilli,and caries experiencein mutans cell surface adhesin丘om tobacco (36). Antibodies Kenyanadolescents. J. Dent. Res., 68, 1242-1246･ canbe most effectivelyused to preventthe mother-childinfeC- 2. Firestone, A. R., Graves, C., Caufield, P. W.and Feagln, tion with S. mutans when glVen tO mothers whoare weanlng F. F. (1 987): Root surface caries subsequent to gingivec- babies (36). tomy ln rats inoculated with Streptococcus sobrinus Thusfar, we have tried to synthesizeantibodiesusing higher (mutans)and Actinomyces viscosus･ J･ Dent･ Res･, 66, forms of organisms such as horsesand cows, utilizingthe 1583-1586. serum or milk collectedfromthese animals. With the growth 3. Babaahmady, K. G., Challacombe, S･ J･, M訂Sh, P･ D･and of genetically recombined plants, saferand moreinexpensive Newnan, H. N. (1998): Ecological study ofStreptococcw antibodiesare expected to beintroduced to the marketplacで muta脚, Streptococcus sobrinusand spp･ inthe near future. GTF derived from the mutans streptococci at sub-sites from approximal from children･ playsanimportant role inthe development of dental caries. Caries Res., 32, 51-58. We examinedthe possible presence of self-inhibitory segments 4. K6hler, B., Birkhed, D.and OIsson, S. (1995): Acid pro- within the enzyme molecule for the purpose of developlng duction by human strains of Streptococcus mutansand anticaries measuresthroughGTF inhibition (37). Twenty-two Streptococcus sobrinus. Caries Res･, 29, 402-406･ synthetic peptides derived &om various regions Presumably 5. de Soet, J. J., VanLoveren, C., Lammens, A. J., Pavicic, responsible forinsoluble-glucansynthesis were studied with M. J., Homburg, C. H., ten Cate, J. M･and de Graaff, J･ respect totheir effects on catalytic activity. One of them, which ( 199 1): Differencesincariogenicity between &esh isolates is identical inamino acid sequence to residues 1176-1194, of Streptococcus sobrinusand Streptococcus mutans･ slgniBcantlyand speciGcally inhibited bothsucrose hydrolysis Caries Res., 25, 116-122. and glucosyl transfer toglucanby GTF-I. Double-recIPrOCal 6. Loesche, W. J. (1986): Role of Streptococcus mutansin analysis revealed that this inhibition is noncompetitive. human dental decay. Microbiol. Rev., 50, 353-380･ Furthermore,the peptide is tightly bound to the enzyme once 7. Li, Y.and Caufield, P. W. (1995): The fidelityofinitial complexed, even in the presence of sodium dodecyl sulfate acquisition of mutans streptococci byinfantsfrom their

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