INFECTqON AND IMMUNITY, Dec. 1981, p. 871-8790019-9567/81/120871-09$02.00/0

Vol. 34, No. 3

Effect of Human Saliva on the Fluoride Sensitivity of GlucoseUptake by Streptococcus mutansGREG R. GERMAINE' 2* AND LOIS M. TELLEFSON'

Department of Oral Biology, School of Dentistry,' and Department ofMicrobiology, Medical School,2University ofMinnesota, Minneapolis, Minnesota 55455

Received 23 April 1981/Accepted 21 July 1981

The fluoride (F) sensitivity of glucose uptake by whole cell suspensions ofStreptococcus mutans in the presence and absence of human whole salivarysupernatant was studied. It was observed that dithiothreitol (DTT) and otherthiols markedly reduced the F sensitivity of cells when saliva (50%, vol/vol) waspresent during glucose uptake. In the absence of saliva, cells were sensitive to 2to 2.5 mM F regardless of the presence of thiols. Supplementation of cells inphosphate or tris(hydroxymethyl)aminomethane-hydrochloride buffers withphysiological concentrations of calcium or phosphate had no effect on the Fsensitivity of the organism. Experiments with permeabilized cells suggested thatthiols themselves had no direct effect on the F sensitivity of enolase (a principalF target). Cells pretreated with DTT subsequently exhibited decreased F sensi-tivity when examined in the presence of saliva but not in the absence of saliva.Cells pretreated with whole salivary supernatant were found to be subsequentlyless sensitive to F in the absence of saliva during glucose uptake. Furthermore, incases where cells were pretreated with saliva, subsequent additions ofDTT wereunnecessary to obtain maximal reduction in the F sensitivity of glucose uptake.It was concluded that the saliva-dependent reduction in F sensitivity of glucoseuptake was not due to sequesteration of available F by salivary constituents. Thedata suggest that a salivary component(s) interacts directly with the microorgan-ism in some manner which results in reduced F sensitivity of the process understudy. Possible mechanisms of saliva action are discussed.

In a previous report (10), we described theeffects of saliva on the uptake of glucose bywhole cell suspensions of Streptococcus mutansand several other oral microorganisms. It wasfound that saliva decreased the rate of glucoseuptake by S. mutans, and we suggested that thesalivary lactoperoxidase-SCN-H202 system wasresponsible for the inhibitory effect. Part of theevidence which implicated this system derivedfrom the demonstration that a thiol-reducingagent, dithothreitol (DTT), abolished the inhib-itory effect of saliva specimens. We also notedthat glucose uptake by S. mutans suspended insaliva was inhibited by fluoride (F). During thecourse of additional studies with saliva-sus-pended S. mutans, we made the chance obser-vation that DTT markedly reduced F inhibitionof glucose uptake. DTT had no effect, however,on the F inhibition of buffer-suspended cells.Because of the cariostatic activity of F and in-terest in its possible effects on the cariogenicactivities of dental plaque microbes, we pursuedthe initial observation and report some proper-ties of the system here.

MATERIALS AND METHODSBacteria, media, saliva, and glucose uptake.

The microorganisms used were S. mutans BHT andS. mitis 9811. Growth conditions, harvesting, and prep-aration of cells for estimation of glucose uptake, andcollection and preparation of pooled human wholesalivary supernatant have all been described in detail(10). Estimation of glucose uptake was performed onwhole cells suspended in 50 mM potassium phosphatebuffer (unless noted otherwise), pH 7.0. Estimation ofglucose phosphorylation by toluene-acetone-treatedS. mutans has also been described (9). [U-'4C]glucose(1.5 mCi/mmol) was used at a final concentration of200 ytM. Desired supplements (saliva, NaF, thiols,salts, etc.) were included in the glucose uptake mix-tures.

RESULTSS. mutans suspended in either phosphate

buffer alone or buffer supplemented with DTTexhibited identical glucose uptake profiles (Fig.la). Marked inhibition of glucose uptake wasnoted in the presence of 2.5 mM F. DTT had noeffect on the extent of F inhibition. In contrast,saliva-suspended cells were refractory to the

871

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

872 GERMAINE AND TELLEFSON

presence of F when DTT was also present (Fig.lb). Similar results were obtained if 50 mMtris(hydroxymethyl)aminomethane-hydrochlo-ride buffer (pH 7.0) was used in place of phos-phate buffer. In this case, the rate of glucoseuptake by saliva-suspended cells in the presenceof 2.5 mM F was only 16% of the rate obtainedin the presence of 1 mM DTT. With both DTTand F present, the rate was 72% of the DTTrate. With tris(hydroxymethyl)aminomethane-hydrochloride buffer-suspended cells, the ratesof uptake in the presence of F with or withoutDTT were identical and were only 4% of the rateobtained in the presence of DTT alone. Thus,the combination ofDTT plus saliva, but neithersubstance alone, rendered the organism refrac-tory to the inhibitory action of 2.5 mM F.One possible explanation for these data is that

DTT promoted a release of calcium ions fromsalivary ligands which, perhaps together withphosphate ions, complexed F so that the concen-

tration of available F was reduced. If true, one

could eliminate the requirement for saliva andDTT by simply supplementing buffers with cal-cium and phosphate ions. To test this idea,

(a) (b)I

DTT±F

30

cn

0 4Z~~~~~~E~~~~~~~~

20

000

10 F±DrTF

0

0 2 4 0 2 4

minutes

FIG. 1. Effect ofDTT and saliva on glucose uptakeby S. mutans in the presence of NaF. Washed cellswere preincubated (at a final absorbance at 540 nmof 1.0) for 10 min in (a) 50 mMpotassium phosphatebuffer (pH 7.0), or in (b) 50%o (vol/vol) whole salivarysupernatant in 50 mM potassium phosphate buffer(pH 7.0). Preincubation mixtures were supplementedwith 1 mM DTT (0), 2.5 mM NaF (A), 1 mM DTTplus 2.5 mM NaF (A), or no additions (0). Afterpreincubation, ['4C]glucose was added to give 200ztM (final specific activity of 1.5 mCi/mmol) and glu-cose uptake was estimated as before (10). See refer-ence 10 for details of the characteristics of glucoseuptake by S. mutans in the presence of saliva.

phosphate buffers were supplemented with cal-cium, and Tris buffers were supplemented withcalcium and phosphate (Table 1). In all cases Fwas inhibitory regardless of the presence of theadded salts or DTT. These observations indicatethat the requirement for saliva demonstrated inFig. 1 is not simply due to the contribution ofcalcium and phosphate by saliva. This notion isfurther supported by results obtained when di-alyzed saliva was examined for its ability topromote reduced F sensitivity of S. mutans glu-cose uptake (Table 2). In this experiment a

TABLE 1. Effect of calcium, phosphate, and DTT onF inhibition ofglucose uptake by buffer-suspended

S. mutansRate of glucose up-takea (nmol/ml per

Buffer (50 mM) min)No sup- Supple-plement mented

Phosphate:No additions 22.8b 22.8CDTTd 230b 22.8cFe 1.6b 1.0CDTT + F 1.6b 1.6c

Tris-hydrochloride:No additions 8.7f 7.79Ca 10.5" 8.8Ca + F 0.4f 0.4Ca + F + DTT 1.Of 0.4' Determined from slope of glucose uptake over 0

to 4 min.b No Ca added.'Ca added as CaCl2 to give 2 mM Ca.dDTT at 1 mM.eNaF at 2.5 mM.fNo phosphate buffer added.g Phosphate buffer added to give 10 mM.

TABLE 2. Effect of dialysis of saliva on itspromotion of reduced F sensitivity ofglucose uptake

by S. mutansRate of glucose uptake (nmol/ml

per min)"Saliva specimen

DTTb Fb DTT +F

Freshc 11.5 0 7.6Storedd 8.4 0 5.4Dialyzede 7.6 0.5 3.4

"Determined from slope of glucose uptake over 0to 2 min.

b Final concentrations: DTT, 1 mM; NaF, 2.5 mM.c Collected just before experiment.d Stored for 18 h at 4°C.'Dialyzed for 18 h at 4°C against 200 volumes of

distilled water.

INFECT. IMMUN.

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALIVA, FLUORIDE, AND S. MUTANS 873

freshly collected saliva specimen was includedas a positive control. Glucose uptake in thept'esence of DTT plus F was 66% of the control(i.e., DTT alone). To control for the time ofdialysis (18 h), a portion of the saliva specimento be dialyzed was simply stored at 4°C for 18 h.In this case glucose uptake in the presence ofDTT plus F was 64% of the control rate. Di-alyzed saliva also promoted reduced F sensitivity(45% of control rate) at an efficiency of 70% ofthe stored saliva specimen.

It might be argued that organic saliva constit-uents bound F and thereby reduced its availa-bility for inhibition of the organism. This model,however, seems especially unlikely. The F bind-ing capacity of whole saliva has been demon-strated (3, 4) to be rather low at near neutral pH(ca. 100 nmol of F per ml of saliva). Accordingly,this capacity for F binding would be able toaccount for a decrease in F of only 4% at theinitial F concentration employed in our studies(2,500 nmol ofF per ml of saliva). This argumentis consistent with results of a previous study inwhich it was shown that, in the mM range of Fconcentrations, no significant F binding by salivaat near neutral pH was evident (2). Thus, al-though saliva may indeed sequester F, its Fbinding capacity is too low to significantly affectthe F concentrations employed in our studies(see below).The ability of DTT plus saliva to interfere

with F inhibition of glucose uptake by S. mutansdecreased with increased F concentration (Fig.2). The effect is clearly discernible at 5 mM Fand is approaching the control (F alone) at 10mM F. Identical results were obtained if theDTT concentration was elevated to 4 mM (datanot shown). With 2.5mM F, maximum reductionin F inhibition required only 100 AM DTT (Fig.3). The increase in the rate of glucose uptakeobserved in the absence of F is most likely dueto the inhibition of the lactoperoxidase systemin saliva (see Fig. 1 and reference 10). The re-quirement for DTT may be satisfied with any ofseveral thiols (Table 3). A comparison of thedose-response relationship of DTT and 2-mer-captoethanol according to the protocol em-ployed in the experiment of Fig. 3 revealed anidentical concentration dependence for interfer-ence with inhibition of glucose uptake by 2.5mM F. Thiols are well known for their metal ionbinding (25). In cases where thiols have beensuspected of exerting their effects via binding ofmetal ions rather than through reduction ofdisulfides, it has been shown that a relationshipexisted between the equilibrium constants forligand-metal ion complex formation and thethiol's efficiency in the system under study (6, 7,

100 F020-

c0

0

000

80 -

60 -

40 F

20

o0 4 8 12

NaF (mM)FIG. 2. Fluoride sensitivity ofglucose uptake by S.

mutans suspended in 50% saliva with and withoutDTT. Saliva was present in all cases. NaF was pres-ent at the concentrations indicated. DTT was addedto 1 mM. The control suspension contained 1 mMDTT to give the maximum possible rate of uptakeunder these conditions (10).

0

0S 4

0:3

010

0

0

. 1 10 100 1000

DTT (,aM)FIG. 3. Effect ofDTT concentration on the F sen-

sitivity of S. mutans glucose uptake in 50% saliva.The data were normalized to the rates obtained withno added NaF orDTT (0) or with 2.5mMNaF alone(0).

11). Even though the collection of thiols used inour studies varied widely (~-106-fold) with re-

spect to metal binding ability, they all exhibitedsimilar activity on the glucose uptake system.

F*DrT

F

I I

.0-

I I I I

VOL. 34, 1981

I

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

874 GERMAINE AND TELLEFSON

TABLE 3. Effect of thiols on saliva-dependentreduction ofF sensitivity ofglucose uptake by S.

mutansRate of glucose uptake

Additiona (nrmol/ml per min)'-F +F +F/-F

None 16.5 6.0 0.36DTT 27.5 22.0 0.80Cysteine 25.5 22.5 0.92Glutathione 27.0 25.5 0.942-Mercaptoethanol 26.5 23.5 0.89a All reagents added to give 1 mM final concentra-

tion.b Determined from slope of glucose uptake over 0

to 2 min.

Thus, it seems unlikely that the requirement forthiols is actually dependent on their metal ionbinding.At this point we considered the possibility

that saliva caused some change in the bacterialpermeability barrier such that DTT (and otherthiols) more readily permeated the cell mem-brane and reduced the F sensitivity of enolase.Alteration of the permeability of the membraneof S. mutans by salivary constituents (lysozymeand perhaps other cationic proteins) has beendescribed (24). Thus, we investigated the possi-bility that DTT reduced the F sensitivity of S.mutans enolase. Washed cells were subjected toa freeze-thaw followed by treatment with tolu-ene-acetone as described earlier (9, 21) to de-stroy the selective permeability of the cell mem-brane. Such decryptified cells were used to esti-mate glucose phosphorylation with 2-phospho-glycerate (2PG) as the primary energy source.In this system glucose phosphorylation is de-pendent on the glucose phosphotransferase sys-tem and phosphoenolpyruvate. Since 2PG wasthe only phosphate donor added, bacterial eno-lase must convert the 2PG to phosphoenolpyr-uvate to observe glucose phosphorylation (Fig.4). With this system, no glucose phosphorylation(denoted glucose uptake in Fig. 4) occurred inthe absence of exogenous 2PG. Furthermore,DTT had no effect on glucose phosphorylationin the presence of 2PG. NaF at 10 mM com-pletely inhibited 2PG-dependent glucose phos-phorylation. DTT had no effect on inhibition ofglucose phosphorylation by 10 mM NaF (Fig. 4).A more detailed examination revealed that, overa range of F concentrations from 0.1 to 10 mM,DTT at 1 mM had no effect on the rate of 2PG-dependent glucose phosphorylation (Fig. 5).These results therefore do not support a directrole of DTT in reducing the F sensitivity ofenolase in whole cells of S. mutans.Over the course of several experiments using

bacteria that were not particularly sensitive tothe salivary lactoperoxidase system under theconditions used, we noted that glucose uptakeby saliva-suspended cells was usually less sensi-tive to F inhibition than was uptake by cellssuspended in buffer. These latter observationstogether with the data presented above indi-cated that saliva alone may render cells lesssensitive to F. We therefore decided to comparethe relative effectiveness of saliva alone andsaliva plus DTT for their ability to reduce the Fsensitivity of whole cells. This is most clearlyillustrated with S. mitis 9811 since glucose up-take in this organism is not affected by thesalivary lactoperoxidase-SCN-H202 system un-der the experimental conditions employed here(10). Glucose uptake by the organism was stud-ied under three conditions: (i) cells suspended inphosphate buffer, (ii) cells suspended in 50%saliva (containing phosphate buffer), and (iii)cells suspended in 50% saliva [as in (ii)] plusDTT. In each condition the effect of 0 to 8 mMF on glucose uptake was studied. The resultsclearly indicated that saliva in the absence of

I

50-

E40 -

0E

30 /

20 -

0.

10 _O20

0 6 12 18 24minutes

FIG. 4. Glucose phosphorylation by toluene-ace-tone-treated S. mutans. No saliva was present in thisexperiment. Washed and freeze-thawed cells weretreated with toluene-acetone as described earlier (9).Glucose phosphorylation (denoted as uptake on thefigure) was estimated by an anion-exchange papermethod (9). Additions to the reaction mixtures were:none (0); 1 mM2PG (A); 1 mM2PG plus 1 mMDTT(0); 1 mM 2PG plus 10 mM NaF (A); 1 mM 2PG, 1mM DTT, and 10 mM NaF (0). Final concentrationof bacteria in the reaction mixtures was the same asemployed in the whole cell studies.

INFECT. IMMUN.

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALIVA, FLUORIDE, AND S. MUTANS 875

DTT reduced the F sensitivity of the organismas compared to buffer-suspended cells (Fig. 6).A further increase in the F resistance of S. mitiswas observed when the cells were suspended insaliva plus DTT. Thus, it indeed appears thatsaliva alone may reduce the inhibitory effect of

-

Q

0

0

100

80F

60 F

40

20

2 3log [FJ,,UM

4

FIG. 5. Effect ofDTT on the F sensitivity of2PG-dependent glucose phosphorylation by S. mutans.Cells wereprepared as in Fig. 4. Control refers to therate of glucose phosphorylation (denoted as uptakeon the figure) in the absence of NaF (arrow). Allreaction mixtures contained 1 mM 2PG. Symbols: 0,

no further additions; 0, plus 1 mM DTT.

(a) (b)

25 -

E

o20-

E

CL

0

*100

F. Maximal reduction in F sensitivity, however,still required both DTT and saliva.

In the foregoing experiments, DTT and salivawere both present during glucose uptake by thetest bacteria. In addition to promoting the sa-liva-dependent reduction of F sensitivity of S.mutans and S. mitis, DTT also inhibits theaccumulation of lactoperoxidase-dependentdamage to S. mutans (see Fig. 1 and reference10). To investigate the role of DTT in the pro-motion of the saliva-dependent reduction of Fsensitivity in S. mutans, a means of circumvent-ing the salivary inhibition of S. mutans glucoseuptake which did not employ DTT was neces-sary. A preliminary experiment demonstratedthat a heat treatment (800C, 10 min) of salivapreviously shown to inactivate salivary lactoper-oxidase (10) retained the ability of saliva toreduce the inhibitory activity ofF on whole cells.Therefore, we used heated saliva to examinefurther the role of DTT in the saliva-dependentreduction of S. mutans sensitivity to F. Cellswere incubated separately, but successively, inDTT and saliva rather than simultaneously asin the previous experiments. Thus, S. mutanswas pretreated with 1 mM DTT, washed inphosphate buffer to reduce residual DTT toineffective levels (less than 10 ,M), and thensuspended in either 50% heated saliva or inphosphate buffer, each supplemented with 0 to6 mM F, for glucose uptake (Fig. 7). Cells sus-pended in phosphate buffer were essentiallycompletely inhibited by 2 mM F (Fig. 7b). Cellssuspended in heated saliva, however, exhibitedalmost complete resistance to 2 mM F and failed

minutes

FIG. 6. Glucose uptake by S. mitis. Cells were preincubated in (a) 50% saliva in 50 mM phosphate buffersupplemented with 1 mM DTT. (b) 50% saliva in buffer, or (c) buffer alone. Additions to preincubationmixtures were: none (0), 4 mMNaF (0), 6 mM NaF (A), 8 mM NaF (H).

I I

'o F

0

1

VOL. 34, 1981

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

876 GERMAINE AND TELLEFSON

to be completely inhibited even by 6mM F (Fig.7a). Note also that the inclusion ofDTT with 6mM F further enhanced the F resistance ofsaliva-suspended cells but had no effect onbuffer-suspended cells. Thus, the simultaneouspresence of DTT and saliva with the organismis not required to induce reduced F sensitivity.

Finally, we investigated the effect of salivapretreatment of S. mutans on the subsequent Fsensitivity of the cells in phosphate buffer (Fig.8). Cells pretreated with 100% saliva or phos-phate buffer, after several washes, were sus-pended in buffer alone and in buffer containing(i) DTT or (ii) 4 mM F in the presence andabsence of DTT. Note the typical sensitivity toF of control cells (i.e., pretreated in buffer alone)and the failure of DTT to influence the F inhi-bition. In marked contrast, saliva-pretreatedcells exhibited a striking resistance to 4 mM F.Note also thatDTT had no additional enhancingeffect on the degree of F resistance exhibited bysaliva-pretreated cells. Thus, pretreatment ofcells with 100% (undiluted, unaltered) saliva ren-dered the cells F resistant and furthermore re-

I I I(a) (b)

50 -

E

40-Ec

3030

0

10

0

0 2 40 2 4 6minutes

FIG. 7. Effect of saliva with and without DTT onthe F sensitivity ofglucose uptake by DTT-pretreatedS. mutans. Washed cells were incubated with I mMDTT in 50 mMphosphate buffer for 20 min at 37°C.The cells were collected by centrifugation and washedtwice with 40 ml of buffer per wash. Cells were thensuspended in buffer to give a stock suspension (ab-sorbance at 540 nm of 10). Preincubation mixtureswere then established with cells at an absorbance of1.0. Cells werepreincubated in (a) heat-treated (800C,10 min) saliva at 50% in 50 mMphosphate buffer, or(b) buffer alone. Other additions to preincubationmixtures were: none (A), 2mM NaF (A), 4 mM NaF(-), 6mM NaF (U), and 6mM NaFplus 1 mMDTT(Ol).

t%.XE

.! 400E

30 F 4Drr

:200

10

0 2 4 0 2 4 6minutes

FIG. 8. Effect of pretreatment of S. mutans withsaliva on the subsequent F sensitivity ofglucose up-take. Washed cells were collected by centrifugation,and the pellets were suspended to an absorbance of1.0 in (a) 100% whole salivary supernatant or (b) 50mMphosphate buffer. After incubation for 20 min at37°C, the cells were collected by centrifugation andthepellets were washed twice with 40 ml of bufferperwash. Cells were then suspended in buffer to anabsorbance of 4.0. Preincubation mixtures were sub-sequently set up containing cells (absorbance of 1.0)in 50 mM phosphate buffer and the following: noadditions (0), I mM DTT (0), 4mM NaF (A), 1 mMDTT plus 4 mM NaF (A). Note that saliva was notpresent duringpreincubation or glucose uptake.

moved any requirement for DTT to observemaximum reduction in F sensitivity.

DISCUSSIONThe results of this investigation demonstrate

that saliva may promote a significant reductionin the F sensitivity of S. mutans and S. mitis asjudged by the effects on the early rate of glucoseuptake by whole cells. The capacity of saliva topromote F resistance was markedly enhancedby DTT and several other thiols when salivawas present during glucose uptake. The exactnature of the role of DTT (or other thiols) inthe phenomenon described here is not known.In our studies it was considered possible thatsaliva promoted access of thiols to cytoplasmicenzymes in whole cells; more specifically weconsidered it possible that DTT directly reducedthe F sensitivity of enolase. The results obtainedwith decryptified S. mutans, however, failed toprovide any evidence that DTT reduced thesensitivity of enolase to a wide range of F con-centrations. The observation that the DTT pre-treatment of cells, followed by extensive wash-

INFECT. IMMUN.

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALIVA, FLUORIDE, AND S. MUTANS 877

ing, rendered the organism more receptive tothe subsequent effects of saliva indicates thatDTT imparts its effect by direct action on themicroorganism and not via some fluid phaseinteraction with salivary constituents. DTT pre-treatment of cells, however, is not a sufficientcondition to observe a maximum decrease in Fsensitivity of cells suspended in 50% saliva dur-ing glucose uptake. Supplementation of saliva-containing uptake mixtures with DTT was nec-essary to attain the maximum decrease in Fsensitivity. In contrast, cells pretreated with100% saliva, followed by extensive washing andsuspension in buffer (no saliva present) for esti-mation of glucose uptake, exhibited the maxi-mum reduction in F sensitivity without the re-quirement for inclusion of DTT in the uptakemixture. Cells pretreated with either 90% saliva(diluted with water) or 90% saliva containing afinal concentration of 50 mM phosphate buffer(data not shown) also yielded maximum reduc-tion in F sensitivity when tested in buffer with-out saliva or DTT. Taken together, the experi-ments employing pretreatment of cells suggestthat thiols enhance the efficiency of the inter-action of diluted (i.e., 50%, vol/vol) saliva withthe microorganisms but are unnecessary whenbacteria are treated with undiluted saliva speci-mens.

It seems especially unlikely that saliva servesas a source of substances such as calcium, phos-phate, or other salts that sequester F. This no-tion is consistent with the failure to observereductions in the inhibitory effect of F in thephosphate or tris(hydroxymethyl)aminometh-ane-hydrochloride buffer systems supplementedwith combinations of calcium, phosphate, F, andDTT. Furthermore, the retention of the F re-sistance-promoting ability of saliva after dialysisand the demonstration that saliva pretreatmentof cells resulted in decreased F sensitivity areboth inconsistent with models in which inor-ganic constituents of saliva are the active prin-cipals. The results from experiments that em-ployed DDT or saliva pretreatment of cells orboth strongly suggest that the principal actionof saliva (and DTT) when present during glucoseuptake was on the cell directly and not based onsaliva-DTT-F interactions.Accumulation ofF by bacteria. Fluoride is

an inhibitor of numerous enzymes via a varietyof mechanisms (27) and is widely known toinhibit the production of phosphoenolpyruvatein bacteria. Fluoride inhibition of glucose uptakevia phosphoenolpyruvate-dependent phospho-transferase transport systems is thus a directconsequence of the F sensitivity of enolase (5,12). To inhibit bacterial enolase and other intra-cellular enzymes, F must gain entry into the cell.

Fluoride is the salt of a weak acid (hydrogenfluoride [HF], pK - 3.45), and since the F sen-sitivity of bacteria is dramatically pH dependent(12) (F sensitivity increases as the pH decreases),it has been generally accepted that accumulationof F inside the cell depends upon (i) simplediffusion of HF across the cell membrane, and(ii) subsequent dissociation of HF inside the cell(28). In this model it is assumed that the cellularmembrane is essentially impermeable to F an-ion. Microorganisms usually maintain an intra-cellular pH (pHi) that is alkaline relative to theextracellular pH (pHe) when pHe ' 7.5 (15, 16).If HF is in equilibrium across the membrane, anet accumulation of F in the more alkaline cellcytoplasm will occur (8, 28). Fluoride accumu-lation is dependent upon the magnitude of thepH difference (ApH) between the cellular cyto-plasm and the extracellular medium (28). As thePHe is lowered, the ApH will increase and theratio of intracellular F to extracellular F willincrease, hence the increased F sensitivity ofbacteria as pHe is decreased. It should also benoted that accumulations of cell-associated Fmay exceed that predicted solely on the basis ofApH considerations. The excess F may be dueto Donnan effects within the cells or to essen-tially "irreversible" F binding to cellular com-ponents (14, 17, 18, 29).Possible mechanisms of action of saliva.

The mechanism underlying the saliva-depend-ent reduction in the F sensitivity of S. mutansand S. mitis is unknown. Several possible mech-anisms exist, however. (i) The ApH may beaffected by changes in pHi and pHe separatelyor in combination. In all cases in our experi-ments, 50 mM buffer was present during theshort (10 min) preincubation of cells before ad-dition of glucose and also during estimation ofglucose uptake. This concentration of buffer waschosen because it was demonstrated in prelimi-nary studies that 50 mM buffer maintained aconstant pHe under our conditions. Determina-tion of pHe immediately after a glucose uptakestudy demonstrated that pHe changed less than0.05 units over the time course of the uptakeexperiment with either buffer-pretreated or sa-liva-pretreated cells. The saliva-dependent re-duction in the F sensitivity of S. mutans cannot,therefore, be explained on the basis of differen-tial effects on pHie of buffer- versus saliva-pre-treated cells. Another possibility is a saliva-de-pendent decrease in pHi such that the ApH isreduced. A salivary effect on pHi could resultfrom (a) some metabolic change induced in thecells that is involved in maintenance of a pHgradient across the membrane, or (b) a directeffect on the cell membrane by salivary constit-uents that increase the proton permeability (i.e.,

VOL. 34, 1981

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

878 GERMAINE AND TELLEFSON

ionophore-like substances) of the membrane. Toexplain the magnitude ofchanges in F sensitivityof S. mutans reported here purely in terms ofdifferences in ApH (at constant pHe), the pHi ofsaliva-pretreated or saliva-suspended cell sys-tems would need to be at least 0.5 units moreacidic than the pHi of buffer-pretreated orbuffer-suspended cells.

(ii) Reports have appeared in which it hasbeen demonstrated that bacterial membranepermeability to ionic solutes was altered by cat-ionic proteins (including muramidase-inactivelysozyme) (22, 24, 30). Since human salivarysecretions contain lysozyme plus several othercationic proteins (1, 20, 23), it thus seems possi-ble that saliva could induce alterations in mem-brane permselectivity such that otherwise trans-ported ionic solutes now may gain entry intocells via diffusion. If this were the case, anionicF might now be able to enter and exit from cellsby diffusion. This would restrict net accumula-tions of F in the cells to that level dependentupon Donnan equilibria. This series of eventswould be expressed as a reduction in the Fsensitivity of the microorganism.

(iii) Another possible basis for our findingsmight involve the mechanism of entry of glucoseinto the test microorganisms. In addition tophosphoenolpyruvate-phosphotransferase-me-diated glucose transport, it was recently sug-gested (13) and subsequently demonstrated thatunder certain conditions a glucose permeasetransport system energized by a transmembraneelectrochemical potential exists in S. mutans (I.R. Hamilton and E. J. St. Martin, J. Dent. Res.60A, abstract 698, 1981). In general, the F sen-sitivity of cells in a chemostat decreased underthose conditions which resulted in decreasedphosphoenolpyruvate-phosphotransferase ac-tivity and increased glucose permease activity.It is therefore possible that saliva-treated cellsrecruit the glucose permease system such thatthe proportion of glucose transported by thephosphoenolpyruvate-phosphotransferase sys-tem is reduced, hence the reduced F sensitivityof glucose uptake by saliva-pretreated cells.

Finally, regardless of the mechanism(s) in-volved, the saliva-induced reduction in the Fsensitivity of S. mutans reported here may havesignificant in vivo implications. We demon-strated that essentially complete insensitivity to2 to 2.5 mM F and significant resistance to 4 to6 mM F resulted when cells were exposed to100% saliva. These levels of F exceed thosewhich are usually reported in plaque, exceptperhaps in some cases immediately after topicalF applications (14). Recent studies have failedto note differences in the composition of theplaque flora of individuals exposed to high and

low F levels (19, 26). These results suggest a lackof an effect of F ingested via the water supply orsupplements on the ecology of the microflora.Our results may indicate that effects of F on themetabolic activities of the plaque flora are alsounlikely except perhaps at uncommonly high Fconcentrations.

ACKNOWLEDGMENTThis study was supported in part by a grant from the

University of Minnesota Graduate School, and Public HealthService grants RR 05322 and DE 05652 from the NationalInstitutes of Health.

LITERATURE CITED1. Armstrong, W. G. 1971. Characterization studies on the

specific human salivary proteins adsorbed in vitro byhydroxyapatite. Caries Res. 5:215-227.

2. Birkeland, J. M. 1971. Fluoride ion activity in vitro andin vivo of two sodium fluoride dentrifrices. Caries Res.5:193-201.

3. Birkeland, J. M. 1973. The effect ofpH on the interactionof fluoride and salivary ions. Caries Res. 7:11-18.

4. Birkeland, J. M., and G. Rolla. 1972. In vitro affinity offluoride to proteins, dextrans, bacteria and salivarycomponents. Arch. Oral Biol. 17:455-463.

5. Cimasoni, G. 1972. The inhibition of enolase by fluoridein vitro. Caries Res. 6:93-102.

6. Coombs, T. L., Felber, J.-P., and B. L. Vallee. 1962.Metallocarboxypeptidases: mechanisms of inhibition bychelating agents, mercaptans, and metal ions. Biochem-istry 1:899-905.

7. Cornell, N. W., and K. E. Crivaro. 1972. Stability con-stant for the zinc-dithiothreitol complex. Anal. Bio-chem. 47:203-208.

8. Eisenberg, A. D., and R. E. Marquis. 1980. Uptake offluoride by cells of Streptococcus mutans in dense sus-pensions. J. Dent. Res. 59:1187-1191.

9. Germaine, G. R., and L. M. Tellefson. 1981. Simplefilter paper procedure for the estimation of glucoseuptake via group translocation by whole-cell suspen-sions of bacteria. Appl. Environ. Microbiol. 41:837-839.

10. Germaine, G. R., and L. M. Tellefson. 1981. Effect ofhuman saliva on glucose uptake by Streptococcus mu-tans and other oral microorganisms. Infect. Immun. 31:598-607.

11. Gracy, R. W., and E. A. Noltmann. 1968. Studies onphosphomannose isomerase. II. Characterization as azinc metalloenzyme. J. Biol. Chem. 243: 4109-4116.

12. Hamilton, I. R. 1977. Effects of fluoride on enzymaticregulation of bacterial carbohydrate metabolism. CariesRes. 11 (Suppl. 1):262-291.

13. Hamilton, I. R., and D. C. Ellwood. 1978. Effects offluoride on carbohydrate metabolism by washed cells ofStreptococcus mutans grown at various pH values in achemostat. Infect. Immun. 19:434-442.

14. Jenkins, G. N., and W. M. Edgar. 1977. Distributionand forms of F in saliva and plaque. Caries Res. 11(Suppl. 1):226-242.

15. Kashket, E. R., and S. L. Barker. 1977. Effects ofpotassium ions on the electrical and pH gradients acrossthe membrane of Streptococcus lactis cells. J. Bacteriol.130:1017-1023.

16. Kashket, E. R., A. G. Blanchard, and W. C. Metzger.1980. Proton motive force during growth of Streptococ-cus lactis cells. J. Bacteriol. 143:128-134.

17. Kashket, S., and F. J. Bunick. 1978. Binding of fluoridein oral streptococci. Arch. Oral Biol. 23:993-996.

18. Kashket, S., and V. M. Rodriguez. 1976. Fluoride ac-cumulation by a strain of human oral Streptococcus

INFECT. IMMUN.

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

VOL. 34, 1981

sanguis. Arch. Oral Biol. 21:459-464.

19. Kilian, M., A. Thylstrup, and 0. Fejerskov. 1979.Predominant plaque flora of Tanzanian children ex-

posed to high and low water fluoride concentration.Caries Res. 13:330-343.

20. Levine, M., and P. J. Keller. 1977. The isolation ofsomebasic proline-rich proteins from human parotid saliva.Arch. Oral Biol. 22:3741.

21. Michelson, M. N. 1977. Glucose transport in Streptococ-cus agalactiae and its inhibition by lactoperoxidase-thiocyanate-H202. J. Bacteriol. 132:541-548.

22. Odeberg, H., and I. Olsson. 1976. Mechanisms for themicrobicidal activity of cationic proteins ofhuman gran-

ulocytes. Infect. Immun. 14:1269-1275.23. Peters, E. H., and E. A. Azen. 1977. Isolation and partial

characterization of human parotid basic proteins. Bio-chem. Genet. 15:925-946.

24. Pollock, J. J., G. G. Bicker, L. I. Katona, M. I. Cho,and V. J. Iacono. 1979. Lysozyme bacteriolysis, p. 429-447. In I. Kleinberg, S. A. Ellison, and I. W. Mandel(ed.), Saliva and dental caries, Sp. Suppl. Microbiol.Abstr. Information Retrieval Inc., New York.

SALIVA, FLUORIDE, AND S. MUTANS 879

25. Sullen, L G., and A. E. Martell. 1964. Stability constantsof metal-ion complexes. Special Publication No. 17.Chemical Society, London.

26. van Houte, J., R. Aasenden, and T. C. Peebles. 1978.Oral colonization of Streptococcus mutans in humansubjects with low caries experience given fluoride sup-plements from birth. Arch. Oral Biol. 23:361-366.

27. Wiseman, A. 1970. Effect of inorganic fluoride on en-zymes, p. 48-97. In F. A. Smith (ed.), Pharmacology offluorides (part 2). Handbook of experimental pharam-cology, vol. 20. Springer-Verlag, New York.

28. Whitford, G. M., G. S. Schuster, D. H. Pashley, andP. Venkateswarlu. 1977. Fluoride uptake by Strepto-coccus mutans 6715. Infect. Immun. 18:680-687.

29. Yotis, W. W., S. Mante, P. C. Brennan, F. R. Kirchner,and L. E. Glendenin. 1979. Binding of fluorine-18 bythe oral bacterium, Streptococcus mutans. Arch. OralBiol. 24:853-860.

30. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteinsof polymorphonuclear leukocyte lysosomes. II. Com-position, properties, and mechanism of antibacterialaction. J. Bacteriol. 91:755-762.

on October 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from