Vol. 34, No. 3INFECTION AND IMMUNITY, Dec. 1981, p. 930-9370019-9567/81/120930-08$02.00/0

Effects of Metabolic Inhibitors on ExtracellularFructosyltransferase Production in Actinomyces viscosus

WINNIE CHAK AND H. K. KURAMITSU*

Department ofMicrobiology-Immunology, Northwestern University Medical-Dental Schools,Chicago, Illinois 60611

Received 7 May 1981/Accepted 18 August 1981

Extracellular fructosyltransferase (levansucrase; EC 2.4.1.10) production inActinomyces viscosus T14AV was demonstrated to occur concomitantly withcellular growth. The inhibition of both cellular ribonucleic acid and proteinsynthesis resulted in no further accumulation of enzyme activity. The antibioticsodium clofibrate differentially inhibited the production of fructosyltransferaseby strain T14AV. Furthermore, the antibiotic preferentially inhibited [14C]acetateincorporation into cellular lipid, but did not affect protein synthesis. In addition,no inhibition of fructosyltransferase production was observed upon the additionof the fatty acid acid synthesis inhibitor cerulenin. On the other hand, extracellularfructosyltransferase production was apparently stimulated in the presence of thecell wall synthesis inhibitors penicillin, amphomycin, and tunicamycin. Theseresults are discussed in terms ofthe mechanism of extracellular protein productionin A. viscosus.

Actinomyces viscosus is an oral microorgan-ism which has been implicated in the etiology ofthe human oral tissue diseases gingivitis (18)and periodontitis (19). Studies with germfreeanimal model systems have demonstrated thatmonoinfection of oral cavities with A. viscosusresults in extensive plaque formation, root sur-face caries, and alveolar bone loss typical ofperiodontal disease (17, 19). Based on recentinvestigations, immunological injury has beenproposed as a major factor in the pathogenesisofA. viscosus infection (2, 26). Both specific andnonspecific activation of lymphocytes by A. vis-cosus have also been demonstrated (8, 10, 26). Itis possible that some cellular product of theorganism may serve as a direct irritant of theadjacent tissue or as a mitogen for local lymph-oid elements. However, these putative patho-genic factors have yet to be conclusively identi-fied.

Extracellular levan (polyfructose) has beenreported to be synthesized by A. viscosus (24).Since bacterial polysaccharides appear to act asB-lymphocyte mitogens (15), it is possible thatgingival inflammation after A. viscosus infectionmay represent the result of the blastogenic ac-tivity of levan molecules produced by the organ-ism. In addition, the release of extracellularlevan may play an important role in other as-pects of the pathogenicity of A. viscosus. Thepresence of levan may also promote plaque for-mation by inducing aggregation between A. vis-cosus and other bacteria (17, 19). Furthermore,

extracellular levan may serve as a carbon sourcefor plaque microorganisms capable of hydrolyz-ing levan molecules (9, 23).

Fructosyltransferase (FTF; levansucrase; EC2.4.1.10) catalyzes the formation of levan by thefollowing reaction: sucrose + acceptor -- levan+ glucose. The presence of FTF activity hasbeen demonstrated both in the growth mediumand on the cell surface of A. viscosus (24). Onthe other hand, no large pool of intracellular(compartment within the cell wall) FTF hasbeen reported (24). Recently, FTF from thehuman oral isolate A. viscosus T14V has beenextensively purified and characterized by Pabst(24). However, the regulation of extracellularFTF production has not been investigated indetail.The present investigation was undertaken to

examine the mechanism of FTF secretion in A.viscosus in light of recent studies on procaryoticextracellular protein secretion (14, 27, 29, 31, 33).Because of the possible role of fatty acid synthe-sis in this process (7, 20), the effects of ceruleninand another recently described inhibitor of bac-terial lipid synthesis, sodium clofibrate (the so-dium salt of chlorophenoxyisobutyrate [Na-CPIB] [22]), on FTF synthesis were investi-gated. A recent study has demonstrated that theaddition of penicillin to growing cultures ofStreptococcus mutans results in the apparentstimulation of glucosyltransferase production,due to the increased secretion of putative am-phipathic activators (16). It was therefore of

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EXTRACELLULAR FRUCTOSYLTRANSFERASE 931

interest to investigate the possible analogousrelease of FTF activators in A. viscosus as aresult of the inhibition of cell wall synthesis. Theeffects of cell wall synthesis inhibitors on extra-cellular FTF production in A. viscosus wereassessed by the addition of penicillin, ampho-mycin, and tunicamycin to growing cell cultures.These results are discussed relative to the mech-anism of FTF production (synthesis plus secre-tion) in A. viscosus.

MATERIALS AND METHODSOrganisms. A. viscosus T14AV and T14V, human

oral isolates, were kindly supplied by S. Brecher (For-syth Dental Center, Boston, Mass.) and F. McIntire(University of Colorado, Denver, Col.), respectively.Growth medium. The organism was routinely

grown in a static culture at 37°C in actinomyces broth(BBL Microbiology Systems, Cockeysville, Md.)which contained the following: brain heart infusion,trypticase (BBL), peptone, yeast extract, cysteine, so-dium phosphate, ammonium sulfate, magnesium sul-fate, and calcium chloride. The broth was also supple-mented with 1% glucose.Production of FTF. For each experiment, 10 to 20

ml of actinomyces broth was inoculated with a 1%(vol/vol) inoculum ofA. viscosus T14AV from culturesgrown for 12 h. After growth for an additional 12 h,the cells were harvested by centrifugation at 1,000 xg for 15 min at 22°C. The cells were washed twice withsaline (0.9% NaCl) and resuspended in the actino-myces broth so that the initial turbidity yielded aKlett reading of approximately 75 in a Klett-Summer-son colorimeter (no. 54 filter). At 0 time and at varioustime intervals during logarithmic-phase and early sta-tionary-phase growth, turbidity readings were re-corded, and 1.5-ml samples of the cultures were with-drawn and placed in centrifuge tubes. The sampleswere then centrifuged immediately at 10,000 x g for10 min (Sorvall RC-2B centrifuge) at 4°C, and theculture supematants were removed with Pasteur pi-pettes. The supematant samples were dialyzed against2.0 liters of 0.005 M potassium phosphate buffer (pH6.0). The dialyzed samples were then assayed for FTFactivity as described below.FTF assay. The enzyme activities were determined

by a modification of the isotope procedure previouslydescribed (24) involving measurement of the incorpo-ration of [3H]fructose from [3H]fructose-sucrose intomethanol-insoluble polysaccharide. The standard in-cubation mixture contained 0.02 M potassium phos-phate buffer (pH 6.0), 7.3 mM [3H]fructose-sucrose(0.75 ,LCi/ml), water, and enzyme in a total volume of0.5 ml. After incubation for 1 h at 37°C, the reactionswere terminated by the addition of 8.0 ml of methanol.The samples were then filtered through glass fiberfilters (Whatman GF/A) and washed twice with 8.0 mlof methanol. The filters were dried in an oven andsuspended in 8.0 ml of toluene-based Liquiflor (NewEngland Nuclear Corp., Boston, Mass.) scintillationfluid. The samples were then counted in a Packardliquid scintillation spectrometer (model 3385). Oneunit of enzyme activity was defined as the amount ofenzyme required to incorporate 1.0 Lmol of fructose

from sucrose into methanol-insoluble fructan per min-ute under standard assay conditions.Macromolecular synthesis. Cells were grown in

15 ml of actinomyces broth containing ['4C]acetate(0.33 ,uCi/ml) for the measurement of lipid synthesis,['4C]uracil (0.656 t,Ci/ml) for the determination ofribonucleic acid synthesis, or 3H-amino acids mixture(1.3 ,uCi/ml) for the measurement of protein synthesis.At the indicated time intervals, 1.0-ml portions wereremoved and added to an equal volume of cold 10%trichloroacetic acid. The suspensions were then fil-tered through glass fiber filters (Whatman GF/A), andthe filters were washed twice with 2.0 ml of coldtrichloroacetic acid. After drying, the filters werecounted as described previously.

Materials. [3H]fructose-sucrose (10 Ci/mmol),['4C]uracil (58 mCi/nmmol), and 3H-amino acids mix-ture (0.35 to 60 Ci/mmol) were obtained from NewEngland Nuclear Corp. ['4C]acetate (56.1 mCi/mmol)was obtained from ICN Pharmaceuticals, Inc., Irvine,Calif. Cerulenin, penicillin G, chloramphenicol, andrifamycin were obtained from Sigma Chemical Co., St.Louis, Mo. Sodium clofibrate was generously providedby Ayerst Laboratories, Montreal, Canada. Tunica-mycin was kindly supplied by G. Tamura (TokyoUniversity, Japan). Amphomycin was a gift from W.F. Minor (Bristol Laboratories, Syracuse, N.Y.).

RESULTSGrowth and production of extracellular

FTF. The rate of extracellular FTF productionconstituted the major concern in the selection oforganisms for the investigation of FTF secretionby A. viscosus. Compared with the virulentstrain T14V, the avirulent strain T14AV (6)accumulated significantly higher levels of FTFactivity (data not shown). Therefore, T14AVwas selected as the organism to be examined inthe present study.Growth resumed immediately when T14AV

cells grown as a preinoculum for 12 h werewashed and suspended in fresh actinomycesbroth (Fig. 1). The production of extracellularFTF was concomitant with the growth of thecells. FTF activity appeared in the medium dur-ing early logarithmic-phase growth, and furtheraccumulation ceased when the cells reached thestationary phase of growth. In some, but not allexperiments, a small decline in FTF activity wasapparent early in the stationary phase.A significant proportion of FTF activity pro-

duced in A. viscosus T14AV is associated withthe cell surface (24). In addition, cell-associatedenzyme has been suggested to be identical to theextracellular enzyme (25). Under the presentconditions, cell-associated FTF activity repre-sented approximately 70% of the total cultureactivity. However, as shown in Table 1, changesin extracellular FTF activity in the presence ofinhibitors were paralleled by alterations in cell-associated activity.

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932 CHAK AND KURAMITSU

200 5

1- -n~~~~~~~~~~~1

100 C0 2 3

TIME (hr)FIG. 1. Cellular growth and production of FTF.

Cells grown for 12 h were washed and suspended infresh medium. The growth of the cells was then mon-itored by measuring cell turbidity. Samples were re-moved at the indicated intervals and assayed forFTF activity. Symbols: 0, cellular growth; A, enzymeactivity.

Effects of chloramphenicol on extracel-lular FTF production. For investigating thesecretion mechanism ofextracellular FTF, it wasimportant to determine whether the productionof FTF is tightly coupled to cellular proteinsynthesis or whether intracellular pools of FTFare involved in this process. T14AV cells activelysecreting FFT and had been growing for 2 hwere divided into two portions, and chloram-phenicol (100 ,ug/ml) was added to one culture.Cellular protein synthesis was immediately in-hibited upon the addition of chloramphenicol(data not shown). At the same time, the produc-tion of extracellular FTF was completely in-hibited in the chloramphenicol-treated sample,whereas secretion continued in the control me-dium (data not shown). These results are com-patible with the absence of significant intracel-lular pools of preformed FTF molecules.Effects of rifamycin on extracellular FTF

production. Previous reports (5, 11) have sug-gested that certain microorganisms continue tosecrete extracellular proteins after ribonucleicacid synthesis is inhibited. This has been ration-alized by postulating the existence of stable mes-senger ribonucleic acid for extracellular proteinsor the synthesis of an overabundance of messen-ger ribonucleic acid which is able to sustainprotein synthesis for a period of time after thetermination of transcription. To determinewhether the production of extracellular FTFrequires concomitant ribonucleic acid synthesis,we examined the effects of rifamycin on extra-cellular FTF synthesis. T14AV cells were di-vided into two portions after 2 h of growth, andrifamycin (10 jig/ml) was added to one culture.

["4C]uracil incorporation into trichloroaceticacid-insoluble material was monitored duringgrowth in parallel cultures, and rapid inhibitionof uracil incorporation took place after the ad-dition of rifamycin (Fig. 2A). Under these con-ditions, the antibiotic also inhibited furthergrowth and protein synthesis. Moreover, thetreated sample yielded no further accumulationof extracellular FTF activity (Fig. 2B), and adecrease in activity, which may represent pro-teolysis, was observed.

Effects of cerulenin on extracellular FTFproduction. One approach toward determiningwhether de novo lipid biosynthesis is involvedin extracellular protein secretion has been to usethe antibiotic cerulenin. This antibiotic specifi-cally inhibits the condensation reaction betweenacyl and malonyl thioesters by acting as a com-petitive inhibitor of the enzyme catalyzing fattyacid chain elongation (32). Several reports havesuggested that the secretion of some extracellu-lar proteins involves a cerulenin-sensitive step(1, 7, 20).When T14AV cells were incubated with cer-

ulenin (20 ,ug/ml), growth was inhibited approx-imately 40%. That such inhibition resulted froman inhibition of fatty acid synthesis is indicatedby the reduction of the differential rate of[I4C]acetate incorporation in the presence of theantibiotic (Fig. 3B). However, during the timeinterval when differential fatty acid synthesiswas inhibited approximately 42%, the differen-tial rate of appearance of FTF activity was vir-tually the same in the presence or absence ofcerulenin (Fig. 3A). These results suggest thatde novo fatty acid synthesis is not required forthe synthesis or secretion of FTF or both.

Effects of NaCPIB on extracellular FTF

TABLE 1. Effects ofmetabolic inhibitors on cell-associated activity'

Metabolic inhibitor Increase in FTFactivityb

None ... 9.38 (100)NaCPIB (4 mg/ml).3.25 (35)PeniciUin (0.5 Ag/ml) .. 15.0 (160)Cerulenin (20 ug/ml) 12.5 (134)

a T14AV cells were incubated with the indicatedconcentrations of inhibitors as described in the text.At various intervals, samples were removed, and thecells were suspended after centrifugation. The super-natant fluids were treated as described in the figurelegends. The washed cells were assayed for FTF activ-ity in the presence of 0.3% NaF.

b Data represent the change in cell-associated FTFactivity (in milliunits per milliliter) per 100 Klett unitsafter 1 h of incubation at 37°C. The numbers inparentheses represent the percent increase in FTFactivity per 100 Klett units relative to the control.

INFECT. IMMUN.

EXTRACELLULAR FRUCTOSYLTRANSFERASE 933

0

0

x

E9

0 2 4 5 0 2 3TIME (hr) TIME- (hr)

FIG. 2. (A) Effects of rifamycin on uracil incorporation. ['4C]uracil incorporation was measured asdescribed in the text. Rifamycin (10 pg/mi) was added after 1.5 h ofgrowth. Symbols: 0, uracil incorporationin the absence of rifamycin; 0, uracil incorporation in the presence of rifamycin. (B) Effects of rifamycin onextracellular FTF production. Cells grown for 12 h were washed and suspended in fresh medium. Sampleswere removed at the indicated intervals and assayed for FTF activity. Rifamycin (10 pg/ml) was added after2 h ofgrowth. Symbols: 0, enzyme activity in the absence of rifamycin; 0, enzyme activity in the presence ofrifamycin.

=- l

I- 5

-50 100 00

KLETT UNTS

lb o.so2 O0.a

025-

20 -50 00 s0KLETT UNITS

FIG. 3. (A) Effects ofcerulenin on extracellular FTFproduction. Preinoculum cells were suspended in freshmedium without or with cerulenin (20 pg/ml). Samples were removed at the indicated intervals and assayedfor FTF activity. Symbols: 0, enzyme activity in the absence of cerulenin; 0, enzyme activity in the presenceof cerulenin. (B) Effects of cerulenin on lipid synthesis. Cells were grown in the presence and absence ofcerulenin. ['4Clacetate incorporation was measured as described in the text. Symbols: 0, acetate incorporationin the absence of cerulenin; 0, acetate incorporation in the presence of cerulenin.

production. NaCPIB is a water-soluble deriv-ative of the mammalian phosphoglyceride inhib-itor chlorophenoxyisobutyrate ethyl ester (22).A previous report has suggested that NaCPIBinhibits lipid synthesis in S. mutans but doesnot affect the secretion of extracellular glucosyl-transferase (20). Therefore, it was of interest toinvestigate the effects of NaCPIB on lipid bio-synthesis and FTF secretion in T14AV cells.Washed preinoculum cells were initially sus-

pended in fresh medium in the presence andabsence of NaCPIB (4 mg/ml). NaCPIB appar-

ently inhibited the growth of T14AV cells (ap-proximately 25% inhibition under these condi-tions) by preferentially inhibiting [14C]acetateincorporation into cellular lipids (Fig. 4A). Inaddition, extracellular FTF production was dif-ferentially inhibited by NaCPIB (Fig. 4B). Nev-ertheless, this inhibition of enzyme productioncould reflect a concomitant inhibition of cellularprotein synthesis by the antibiotic. This possi-bility was ruled out by the observation thatdifferential 3H-amino acid incorporation into cel-lular protein was unaffected by NaCPIB (Fig.

aA

0

200

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934 CHAK AND KURAMITSU

KLETT UNITS

KLETT UNITS

] KLETT UNITS

300 FIG. 4. (A) Effects of NaCPIB on lipid synthesis.Cells were grown in the absence and presence ofNaCPIB (4 mg/ml). ['4C]acetate incorporation wasmeasured as described in the text. Symbols: 0, ace-tate incorporation in the absence of NaCPIB; 0,acetate incorporation in the presence ofNaCPIB. (B)Effects ofNaCPIB on extracellular FTFproduction.Cells were grown in the absence and presence ofNaCPIB (4 mg/ml). Samples were removed at theindicated intervals and assayed for FTF activity.Symbols: 0, enzyme activity in the absence of Na-CPIB; 0, enzyme activity in the presence ofNaCPIB.(C) Effects ofNaCPIB on protein synthesis. Cellularprotein synthesis was measured in the absence andpresence of NaCPIB (4 mg/ml) as described in thetext. Symbols: *, 3H-amino acid incorporation in the

300 absence ofNaCPIB; 0, 3H-amino acid incorporationin the presence of NaCPIB.

4C). Moreover, the inhibition of FTF secretionwas directly related to the inhibition of ['4C]-acetate incorporation. No differential inhibitionof extracellular FTF production was achievedwith lower concentrations of NaCPIB, whichalso had little effect on lipid synthesis.

Effects of penicillin on extracellular FTFproduction. A previous report has suggestedthat extracellular glucosyltransferase productionin S. mutans is enhanced by penicillin treatment(16). The addition of penicillin (0.5 ,ug/ml) toT14AV cells during the logarithmic phase ofgrowth resulted in a 50% inhibition of the growthrate (data not shown). However, cells grown inpenicillin-containing medium produced extracel-lular FTF at a significantly faster rate than cellsgrown in the absence of the antibiotic (Fig. 5).Moreover, the differential rate of cellular proteinsynthesis did not appear to be increased by theaddition of penicillin (data not shown). Directaddition of penicillin to FTF preparations didnot result in any detectable stimulation of theenzyme activity. Therefore, the apparent stim-ulation of extracellular FTF production by pen-

icillin may also be related to the effects of peni-cillin on cell wall synthesis.

Effects of tunicamycin and amphomycinon extracellular FTF production. Tunica-mycin is an antibiotic which has been suggested

I0

E"I.D 5

0 100 200

KLETT UNITS

FIG. 5. Effects ofpenicillin on extracellular FTFproduction. Cells were grown in the absence andpresence of penicillin (0.5 jig/ml). Samples were re-

moved at the indicated intervals and assayed forFTF activity. Symbols: 0, enzyme activity in theabsence ofpenicillin; 0, enzyme activity in the pres-ence ofpenicillin.

to inhibit the incorporation of glucosamine intomacromolecules (3). In Bacillus subtilis, tuni-camycin inhibits cell wall synthesis, subse-quently inducing the formation of spherical or

tadpole-shaped cells and subsequent cell lysis(28). Therefore, it was of interest to study theeffects of tunicamycin on extracellular FTF pro-

duction in A. viscosus T14AV. Cells grown infresh medium in the presence and absence of

0x

E.

0.C-

1.0

0.5

C

100 200c

INFECT. IMMUN.

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EXTRACELLULAR FRUCTOSYLTRANSFERASE 935

tunicamycm (4 ,tg/ml) were monitored forgrowth and FTF production. The addition oftunicamycin resulted in a very slight inhibitionof cell proliferation (data not shown). Extracel-lular FTF production did appear to be stimu-lated in the presence of tunicamycin (Fig. 6)although not to the same degree as in the pres-ence of penicillin. Amphomycin, another poly-peptide antibiotic which inhibits peptidoglycansynthesis in Bacillus cereus (30), also produceda similar stimulatory effect on the differentialproduction of extracellular FTF (data notshown) under conditions in which growth waspartially inhibited.Effects ofmetabolic inhibitors on cell-as-

sociated FTF activity. When cell-associatedFTF activity was measured at different timeintervals in the presence of inhibitors, altera-tions similar to those for extracellular activitywere observed (Table 1). Penicillin produced anapparent increase in cell-associated FTF activ-ity, whereas cell-associated activity in the pres-ence of NaCPIB was severely depressed. On theother hand, cerulenin did not inhibit the increasein cell-associated FTF activity within the initial1-h incubation period and actually producedsome stimulatory effects. Indeed, as was ob-served with extracellular FTF activity (Fig. 3),cell-associated FTF activity accumulated underconditions in which fatty acid synthesis wasstrongly inhibited.

DISCUSSIONThe appearance of extracellular FTF activity

in A. viscosus T14AV appeared to be closelyrelated to cellular growth. No obvious lag wasobserved between the appearance of extracellu-lar FTF and the growth of strain T14AV (Fig.1). In addition, further accumulation of FTF

150KLETT UNITS

FIG. 6. Effects of tunicamycin on extracellularFTFproduction. Cells weregrown in the absence andpresence of tunicamycin (4 pg/ml). Samples were re-

moved at the indicated intervals and assayed forFTF activity. Symbols: 0, enzyme activity in theabsence of tunicamycin; 0, enzyme activity in thepresence of tunicamycin.

activity ceased when the cells reached the sta-tionary phase of growth (Fig. 1). The apparentdecline in FTF activity noted in some experi-ments in the early stationary phase may repre-sent the induction of fructanase activity (9) orthe release of proteolytic activity. The release ofpotential inhibitors of FTF activity during thestationary phase of growth could not be detectedin mixing experiments. Furthermore, extracel-lular FTF production was immediately inhibitedupon the addition of the protein synthesis inhib-itor chloramphenicol. This latter observation isconsistent with the absence of large pools ofintracellular FTF, although it is possible thatintracellular pools requiring protein synthesisfor secretion are present. However, disruption ofT14AV cells yielded no significant release ofFTF activity (data not shown).The existence of stable messenger ribonucleic

acids for extracellular FTF could not be dem-onstrated in the present investigation (Fig. 2B).In this regard, the production of extracellularFTF was concomitant with ribonucleic acid syn-thesis. However, since the production of FTFwas measured at 30-min intervals, these resultsdo not rule out the possible existence of FTFmessenger ribonucleic acids with half-liveslonger than the 1- to 2-min half-lives of mostbacterial messenger ribonucleic acids (4, 5, 11).

Relative to the results in other bacterial sys-tems secreting extracellular proteins (1, 7, 20),the addition of growth-inhibiting concentrationsof cerulenin did not appear to affect the differ-ential rate of production of extracellular FTF(Fig. 3A). This suggests that de novo fatty acidsynthesis in strain T14AV is not specificallyrequired for the expression of extracellular FTFactivity. Altematively, if fatty acids are in factrequired for such expression, T14AV cells maycontain significant pools of these fatty acidsunder the growth conditions used in the presentinvestigation.

In contrast to the secretion of glucosyltrans-ferase in S. mutans (20), the production of ex-tracellular FTF was shown to be differentiallyinhibited by the addition of NaCPIB (Fig. 4B).Furthermore, a direct correlation between theinhibition of FTF secretion and [14C]acetate in-corporation was indicated (Fig. 4A and B). Thisobservation is compatible with the role of denovo lipid biosynthesis in extracellular proteinproduction. However, the inhibitory effects ofNaCPIB in bacterial systems have not beenstudied extensively, and further investigation isrequired to depict the specific action of NaCPIBon biochemical processes in T14AV cells. There-fore, the divergent effects of NaCPIB and ceru-

lenin on FTF expression probably reflect differ-ent sites of action of the two antibiotics in lipid

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936 CHAK AND KURAMITSU

biosynthesis. In this regard, it is of interest thatcerulenin, but not NaCPIB, differentially in-hibits extracellular glucosyltransferase expres-sion by S. mutans GS-5 (20). For clarifying therole of lipid synthesis in these systems, it will benecessary to specifically identify the site of ac-tion of NaCPIB in bacterial systems and todetermine the role of the lipid molecules inextracellular protein secretion. Efforts in thisregard are currently in progress in this labora-tory.

Elevated FTF activities were also demon-strated in the presence of several inhibitors ofcell wall synthesis with different sites of action(Fig. 5 and 6). Therefore, it is unlikely that theseelevated activities occur as a result of secondaryeffects at non-cell wall target sites. These effectswere immediate, and no enhancement of activitycould be detected by the direct addition of cellwall synthesis inhibitors to FTF preparations.Since the surface barriers of the cells are alteredby these antibiotics, it is possible that the stim-ulation of extracellular FTF production mayoccur subsequent to the release of some amphi-pathic cellular molecules (phospholipids, tei-choic acid-like molecules) which are able to pro-mote or stabilize enzyme activity by interactingwith the enzyme secreted during growth. A re-cent study has suggested that extracellular glu-cosyltransferase from S. mutans interacts withamphipathic molecules secreted during growth,resulting in the stabilization of enzyme activity(21). Furthermore, the release of lipid materialhas been demonstrated to be enhanced in thepresence of the cell wall inhibitor penicillin in S.mutans (13). Therefore, it is possible that ananalogous release of molecules which are capa-ble of enhancing FTF activity may take place inT14AV cells upon the addition of inhibitors ofcell wall synthesis. Moreover, FTF activity hasbeen demonstrated to be affected by the directaddition of lipid-containing detergents to theenzyme preparations; i.e., FTF activity was stim-ulated roughly twofold in the presence ofTween80. This may indicate that FTF molecules areable to interact with certain lipid-containingsubstances, resulting in a stimulation of enzymeactivity. In addition, direct alteration of the cellsurface after antibiotic treatment may providean alternate explanation for the stimulation ofFTF activity. Since the cell wall represents thefinal barrier to extracellular enzyme secretion, itis conceivable that the distortion of the surfacestructures of the cells may account for the stimn-ulation of extracellular FTF activity. However,no large pools of precursor extracellular proteinmolecules have been demonstrated to exist be-tween the membranes and cell walls of gram-positive bacteria. Therefore, it is unlikely that

the enhancement ofFTF activity is solely causedby changes in the permeability of the cell sur-face.The results of the present study represent an

initial investigation of the regulation of extracel-lular FTF secretion in A. viscosus T14AV. Fur-ther investigation is required for a detailed un-derstanding of the mechanism of FTF secretion.In this regard, it will be of interest to investigatethe possible existence of a membrane-associatedFTF precursor in this organism. Relative to theapparent increase in extracellular enzyme activ-ity in the presence of cell wall synthesis inhibi-tors, the possible release of putative lipid-con-taining activators after antibiotic treatmentshould also be examined. Furthermore, the pres-ent results indicate that strain T14AV produceshigher levels of FTF than does strain T14V.However, it is not clear whether the greaterproduction of slime polysaccharide by the for-mer strain contributes to this difference. It willalso be of interest to extend these studies tostrain T14V as well as rodent strains of A. vis-cosus. These, as well as other, approaches shouldprovide the basis for further investigation intothe mechanism of FTF secretion in A. viscosus.

ACKNOWLEDGMENTSThis project was supported by Public Health Service grant

DE-05141 from the National Institute of Dental Research.We acknowledge the helpful assistance of Lillian Wondrack

in this project.

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