An endodextranase inhibitor from batch cultures of Streptococcus ,mutans

BIOCHEMICAL

Vol. 106, No. 3, 1982 June

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 875-880

J5, 1982

AN ENDODEXTRANASE INHIBITOR

FROM BATCH CULTURES OF

STREPTOCOCCUS MLJTANS Ronald Laboratory

Received

April

M. Hamelik

Department of Microbiology Microbiology, of Miami, School of Medicine Miami, Florida 33101

of Oral University

26,

and Mead M. McCabe

1982

An inhibitor of Streptococcus mutans endodextranase was detected in -~ proteins prepared from batch culttires of S . mutans strains representing serotypes a through g. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of -S. mutans known to produce this enzyme.

Introduction Endodextranases depress

the

(EC 3.2.1.11)

synthesis

inhibit

of

many 2.

mutans

strains

glucan,

altering

the

of

glucan

nase

studies began declined

amount

synthesis.

of -~S. mutans

obtain

(3)

early

under

pH control,

indicated in

rapidly

the

(2). would

also

and the

structure

of --in vitro

conditions relied

preparations that

to zero

of

endodextranase

exponential unless

the

phase

of

culture

(4) upon

the

studies free

of

endodextracultures

batch

D-glucosyltransferase.

growth

but

cultures to Recent

in batch that

of

the mechanism

in many laboratories

production

by

inhibiting of

and from

and

synthesis

continuous

pH was controlled

875

(1,2) produced

product,

glucose-limited

a practice

by the mixed

--mutans

influence

preparations

from

controlled

sucrose

markedly

The endodextranase

presumably

been obtained

grown

from

origin

Streptococcus

and complicating

endodextranase-free (5)

pathogen

D-Glucosyltransferase

have

without

or bacterial

glucans

adherence

adherence

activity

grown

the oral

cell-to-surface

cell-to-surface

fungal

of water-insoluble

D-glucosyltransferases thus

of

cultures

enzyme to

activity

prevent

its

Vol. 106, No. 3, 1982 decrease

to pH 5 and lower.

was growth its

dependent

absence It

is our

dextranase is masked

which

that,

that

endodextranase at pH 5,

production

thus

explaining

pH control.

contrary

to the above

from

without

Apparently,

RESEARCH COMMUNICATIONS

enzyme was labile

recovered

by an endogenous purification,

was concluded

without

are grown

activity.

enzyme during Materials

experience

AND BIOPHYSICAL

the

grown

is consistently

cultures

It

and that

in cultures

dextranase batch

BIOCHEMICAL

the fluids

pH control

endodextranase

inhibitor,

which

as described

observations of

stationary

and lack activity

is readily

(5), phase

detectable in such

separated

endo-

endocultures

from

the

here.

and Methods

Streptococcus mutans strains obtained from the Dental Research Unit, Veterans Administration Hospital, Miami, Fla. were grown in batch cultures, Strain 6715-49 was used for most of without pH control, as described (6)* the studies presented below. Cultures were centrifuged after 18 hrs of growth at 37OC to obtain a cell-free culture fluid which was neutralized with 1ON NaOH and adjusted to 50% (NHI,)~SOI, saturation, at 2oC. The resulting precipitate was collected by centrifugation, redissolved, at 10% of original volume, in 0.02% NaNI and subjected to affinity chromatography (7) on Sephadex G50 (Pharmacia Fine Chemicals, Piscataway, N.J.) equilibrated with 0.02% NaNs- Proteins eluting in peak 2 from this column (Figure 1) were further fractionated by gel filtration on Bio Gel PlO (1.6 X 90 cm bed; Bio Rad Laboratories, Richmond, Calif.) equilibrated with O*Ol M pH 5-5 sodium acetate buffer (not shown). A V. peak and a Vt peak were obtained from the PlO column. The V. peak was further fractionated by ion exchange chromatography on DEAE Bio Gel A (2.6 X 8 cm bed; Bio Rad Laboratories) equilibrated with the above buffer" The charged and washed DEAE Bio Gel A column was eluted with a gradient of NaCl (0-0.25M) in the same buffer and 1 ml fractions were collected. Proteins eluting in peak 1 from the G50 column (Figure 1) included endodextranase as well as a mixture of small quantities of the proteins and glucan-synthesizing enzymes characteristically found in peak 2. The glucao synthesizing activity of peak 1 material was partially separated from endodextranase activity by gel filtration on a 2.6 X 90 cm column of Sepharose CL4B (Pharmacia) in 0.02% NaNj (not shown)The specific activity of the dextranase so obtained was 9.9 I.U./mg protein, while contaminating glucan-synthesizing activity was present at 1.6 I.U./mg protein. The presence of endodextranase was confirmed by the identity of products from its action on isomalto-oligosaccharides, usirLg techniques described by Walker (8). Dextranase activity was detected by incubating aliquots (100~1) of protein preparation with dextran T2000 (100~1, 3.5mg, Pharmacia) and 0.5M, pH 5.0 sodium acetate buffer (50~1) at 37OC for 1 or 2 hrs. Dextranase activity was indicated by increased reducing sugar levels, assayed in 200~1 aliquots of reaction mixture by a modification of the Nelson method (9). If activity was not detected using 1 or 2 hr incubation periods, overnight Assays incubations were used to assure the absence of dextranase activity. (25~1, of inhibitor activity comprised partially Pure S. mutans endodextranase 0.01 I.U.), protein preparation (lOOul), dextraFTmlOOn1, 3.5mg) and 0.5M, pH 5.0 sodium acetate buffer (lOOn1). Following incubation for 1 hr at 37o~, 200~1 aliquots were removed for the assay of reducing sugars by the modified Nelson method. Protein contents of preparations were estimated with Coomassie Brilliant Glucan synthesis was assayed as described previously (11). Blue G250 (10). 876

BIOCHEMICAL

Vol. 106, No. 3, 1982

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

lU/fractlon

2i

fraction Figure 1. Affinity chromatography of a crude protein preparation (4001~11) from ~~-. S mutans 6715-49 on Sephadex C50. Fractions 20 through 120 contained lox: levels of wadsorbed proteins (as shown), as well as considerable levels of culture medium peptides and carbohydratz?s, which are not shown. tXution with 6M guanidine-HCl began at fraction 160. 5ml fractions were collected. Solid dots: LIP protein/fraction; open dots: I .u . glucosyltransferase/frac~ion. The region of the elution profile containing endodextranase activity is indicated by the open rectangle,

Results Batch mately

cultures,

4.5.

activity, precipitation

removed

from

with

G50 (Figure

that protein

the

dextranase

1, peak

was not

had passed

without

binding uninoculated

did

l),

suggesting

not culture

the

pH values lacked

from

protein

Assays

these

preparations

affinity

chromatography

column.

such

present Penicillium

by these

preparations.

The effluent

Sephadex

G50 affinity

chromatography

contain medium.

inhibitor,

nor

could

by

invariably on

chromatography

was completely by proteins

fluids

chromatography

to detect

endodextranase

of approxi-

endodextranase

to affinity that

and 90% inhibited

inhibited through

crude

when subjected

purified

G50 affinity

fluids obtained

endodextranase.

preparations

Sephadex

However,

had final

culture

preparations

activity

partially

which

from

protein

of

pH control,

cell-free

(NH,+)zSOb.

an inhibitor

revealed crude

crude

endodextranase

Sephadex

without

The neutralized as did

yielded

grown

inhibitor

an inhibitor inhibited

by

in peak 9.

2 endo-

proteins, column be recovered

BIOCHEMICAL

Vol. 106, No. 3, 1982

lU/fraction

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

.

0

pg/fractlon - 300

!OO

00

0

60

100 fraction

3

140

Figure 2, Ion exchange chromatography of Sephadex G50 peak 2 proteins (Figure 1) on DEAE-Bio Gel A. The column and applied proteins were equilibrated with 0.01 M, pH 5.5 sodium acetate. 5ml fractions were collected. Solid dots: ng protein/fraction; open dots: 1.u. glLlcosyltransferase/ fraction; triangles: percent inhibition (%I) of endodextranase activity (see Materials and Methods); X: NaCl gradient, molar concentration of NaCl in fractions.

Endodextranase Bio

Gel PlD column

activity

eluted,

major

dextran-binding

of

the Bio

at V. but

transferase

inhibitor activity

Treatment

of

However,

treatment

co.19

Pronase

E (Type

Sigma

Chemical

Co.)

24 hrs

with

partially

eluting

all

in the

Vt protein

peak,

which

of -~ S. mutans

(7).

Ion

pool

three

closely-spaced

(Fig.

2).

G50 peak subsequent

these

proteins

XIY protease,

purified

The elution

profiles

upon

resulted

yielded

in one instance,

elution

Sephadex

of

the proteins

where

coincided

had no effect

in

(65% inhibition),

protein

enzyme and inhibitor

minutes

not

was present

Gel PlO V. protein

endodextranase

of

inhibitor

in

but

was not

the loss

contained exchange

trypsin Chemical of

inhibitor

glycosidases

only

the

chromatography peaks

inhibitor

of

and D-glucosyl-

a consistent

correlation

observed.

effectiveness with

the

D-glucosyltransferase

1) proteins

2 (Fig.

Sigma

mixed

of

from

at 100°C

of

the

(Type

IX,

Co.),

15

inhibitor. Sigma

or papain

action. (Charonia

for

Chemical (Type

Treatment lampus,

Miles

III, for

BIOCHEMICAL

Vol. 106, No. 3, 1982 Laboratories, Sigma

Inc,,

Chemical

Elkhart,

Co.)

produced

the

AHT, which produce

mutans

or with

effect

strains

endodextranase

RESEARCH COMMUNICATIONS

fungal

endodextranase

upon subsequent

representing

inhibitor,

had been reported

endodextranase

BIOPHYSICAL

Indiana)

was without

Streptococcus

AND

to lack

inhibitor

serotypes

as well

(Grade action.

a through

g each One strain,

as endodextranase.

endodextranase

I,

activity

(3,5)

did

not

inhihitor.

Discussion The observations dextranase

activity

the lability

of

from the

"dextranase-free" It

m&y.

Se mutans -~-

which

insoluble

glucans

nase

relied

have

pattern

not

upon (8)

glucan

cells

surfaces. suggests

modulation

of

since

synthesis

that

chromato-

of

endodextra-

Preparations

of

an inhibited

form

upon

studies

of

from

the

the

other

effects

endodextranases: of

possesses

however, would

its

have

depression

the adherence

of a reversible

of water-

of endodextra-

species.

for

other

synthesis

of S. mutans

inhibitor

a mechanism

for

of

of the

extra-

activity.

Acknowledgements National

These studies Institute

were supported by U.S.P.H.S. of Dental Research.

grant

DE 04321

from

References 1. 2. 3.

of

activity.

and prevention

endodextranase

to

from

affinity

inhibitor

endodextranase

S . mutans

endo-

be attributed

of

S. mutans

The production

of

obtained

contain

endodextranase

observed

easily

procedure

therefore,

regaining

that

cannot

a reversible

the use of enzymes

to those

to smooth

of

known,

indicates

water-insoluble

endodextranase

capable

is

absence

of enzyme activity-

can,

of -~ S. mutans is

similar

cellular

the absence

is

enzyme

of

the

S. mutans

the simple

the presence

for

The influence

effects

by

that

of

active

D-glucosyltransferases

endodextranase

action

fluids

instead,

indicate

cultures

since

enzyme,

accounts

here

batch

culture is,

nase which

described

Walker, G.J. (1972) J. Dent. Res., 15, 409-414. Schachtele, C.F., Staat, R.J. and Harlander, S.K. (1975) Infect. Immun., 12, 309-317. Staat, R.J. and Schachtele, C.F. (1974) Infect. Immun., 9, 467-469.

the

Vol. 106, No. 3, 1982 4. 5. 6. 7.

8. 9. 10. 11.

BIOCHEMICAL

AND BIOPHYSICAL

Walker, G.J., Murray, V.L. and Morrey-Jones, 115, 206-208. Walker, G.J., Pulkownik, A. and Morrey-Jones, Microbial., 127, 201-208. McCabe, M.M. and Smith, E-E. (1973) Infect. McCabe, M.M., Hamelik, R.M. and Smith, E.E. Res. Comm., 78, 273-278. Pulkownik, A., Thoma, J.A. and Walker, G.J. 61, 493-497. Robyt, J.F. and Whelan, W.J., in J*A. Radley, its Derivaties, pp 432-433, Chapman and Hall, Sedmak, J.J. and Grossberg, S.E. (1977) Anal. McCabe, M.M. and Smith, E.E. (1977) Infect.

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(1980) (1981)

FEBS Letter, J. Gen.

Immun., 7, 829-838. (1977) Biochem. Biophys (1978)

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