Streptococcal Hyaluronic Acid: Proposed Mechanisms of Degradation and Loss of Synthesis During Stationary Phase - Journal of Bacteriology
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JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1059-1065 Vol. 156, No. 3
0021-9193/83/121059-07$02.00/0
Copyright 0 1983, American Society for Microbiology
Streptococcal Hyaluronic Acid: Proposed Mechanisms of
Degradation and Loss of Synthesis During Stationary Phase
I. VAN DE RIJN
Department of Microbiology and Immunology, Bowman Gray School of Medicine, Winston-Salem, North
Carolina 27103
Received 8 July 1983/Accepted 13 September 1983
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Streptococcal hyaluronic acid was found to distribute into two discrete sizes.
Cellular hyaluronic acid from strain D181 had an average molecular weight of 10 x
106, whereas the average molecular weight of extracellular hyaluronic acid from
the same strain was 2 x 106. Cellular streptococcal hyaluronic acid was purified to
homogeneity. Proteases were unable to cleave the purified cellular polymer,
indicating that a peptide was not involved in cross-linking five extracellular
hyaluronate polymers to form a cell-bound complex. Lipids apparently are not
part of the cellular hyaluronic acid because phosphorus and glycerol were not
detected by radioisotopic techniques, and denaturing conditions did not change
the size of the polymer. Membranes obtained from various strains of group A and
C streptococci cleaved the cellular form of the hyaluronate polymer demonstrat-
ing the presence of a membrane-bound hyaluronidase-like activity. By contrast,
this activity was not found in the extracellular products of the strains studied.
Furthermore, membranes derived from streptococci at the stationary phase of
growth no longer had the capacity to synthesize hyaluronic acid. The loss of this
property appeared to be due to changes in the structure of the membrane.
Hyaluronic acid is a linear polysaccharide phocytes and alveolar macrophages (15, 17, 18,
composed of repeating subunits of P-1,4-linked 24). Furthermore, abnormalities in the regula-
disaccharides of glucuronic acid ,B-1,3-N-ace- tion of the synthesis of this molecule are be-
tylglucosamine. This polymer is synthesized on lieved to be the basis for Marfan syndrome (1).
the streptococcal membrane with UDP-gluc- Streptococci are ideally suited for studying
uronic acid and UDP-N-acetylglucosamine serv- the biosynthesis of hyaluronic acid due to the
ing as precursors for the molecule (19). Lipid abundant availability of hyaluronate and since in
intermediates in the biosynthetic pathway of hy- this organism the hyaluronate is the only poly-
alutonic acid have not been detected to date mer into which glucuronic acid is incorporated.
(20). In this report, it is demonstrated that group C
Group A and C streptococci produce a cap- streptococcal strain D181 releases hyaluronic
sule that is composed of hyaluronic acid poly- acid polymers in defined sizes that a,'e about
mers identical to that found in mammalian con- 20% of the size of the cellular form. In addition,
nective tissues (14). In Streptococcus spp., the a hyaluronidase-like activity was found associat-
hyaluronic acid capsule has been demonstrated ed with the membrane of streptococci. It is
to be a major virulence factor in addition to the thought that this activity may result in the partial
cell wall M protein (10). The capsule also inhib- degradation of the cellular hyaluronate polymer,
its the binding of the organism to human epitheli- releasing the smaller hyaluronate polymer found
al cells (3) and murine peritoneal macrophages extracellularly. Furthermore, it was found that
(25). the streptococcal membrane, in the stationary
In mammals, hyaluronic acid has been shown phase, lacked the capacity to synthesize hyal-
to affect a number of biological processes such uronic acid polymers from precursors, which
as inhibition of lymphocytes and macrophage correlates with the absence of capsules.
proliferation (2), suppression of the graft-versus-
host reaction (2), stimulation of aggregation of MATERIALS AND METHODS
lymphoma cells to influence gene expression
(18, 24), assembly of epithelial layers during Bacteria and media. Streptococcal strains S43/192/3
development (8), and reduction of the chemotac- (group A) and D181 (group C) were obtained from the
tic movement of leukocytes (6). There are also culture collection of The Rockefeller University.
hyaluronic acid receptors on transformed lym- Strain CS44 (group A) was obtained from P. Cleary,
10591060 VAN DE RIJN J. BACTERIOL.
University of Minnesota, whereas strain Cl was a uronic acid, and any insoluble material was removed
fresh isolate (group C). All four strains produced by centrifugation. The ethanol precipitation, solubili-
capsules of hyaluronic acid to various degrees. zation, and centrifugation steps usually were repeated
The bacteria were grown in a chemically defined five times to obtain high-purity preparations. All hyal-
medium previously described by van de Rin and uronic acid samples were dried by solvent dehydration
Kessler (23). Stock cultures were kept frozen at with ethanol, acetone, and then ether. Traces of ether
-80°C. To maintain optimal capsule production by the were removed by desiccation in vacuo.
organism, an original vial was opened for each experi- Sizing of hyaluronic acid polymers. Hyaluronic acid
ment. was solubilized in phosphate-buffered saline (0.01 M,
Growth and monitoring of capsule. Growth of cul- pH 7.4) and treated with RNase and DNase at 10 p.g/ml
tures in 18-mm tubes was measured in a Spectronic 20 for 2 h at 37°C to digest any high-molecular-weight
spectrophotometer (Bausch & Lomb, Inc., Rochester, nucleic acid. Samples of hyaluronic acid were sized
N.Y.) at a wavelength of 650 nm. The net observed and further purified on columns (1.5 by 28 cm) of
optical density readings of the culture were multiplied Sepharose 2B (Pharmacia Fine Chemicals, Pis-
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by 1,000 and then converted to adjusted optical densi- cataway, N.J.) equilibrated with the appropriate buffer
ty units (21). and run at 8 ml/h. Samples of hyaluronic acid were
In the studies presented, the bacteria were grown at suspended in buffer (0.01 M Tris-hydrochloride [pH
37°C in 5-gallon (ca. 19-liter) carboys to the mid- 7.5], 6 M guanidine-hydrochloride, or 1% sodium
exponential phase or until after the release of the dodecyl sulfate-0.01 M Tris-chloride [pH 7.5]). Frac-
capsule was observed (early or late stationary phase, tions were monitored with a flow-through refractome-
depending on the organism). ter (Waters Associates, Milford, Mass.) and tested for
The presence of bound capsule was monitored by glucuronic acid by the Bittner and Muir (4) assay.
India ink preparations. Briefly, a drop of India ink was Ekectrophoresis and staining. Bound and extracellu-
added and mixed with two drops of bacteria, a cover lar hyaluronic acid were differentiated by electropho-
slip was added, and then the slide was monitored by resis in agarose. Hyaluronic acid was suspended to a
phase microscopy. The size of the capsule varied from final concentration of 1 mg/ml in electrophoresis buffer
strain to strain as well as during the growth cycle. The (barbital hydrochloride buffer; ionic strength, 0.02; pH
largest capsules appeared at the mid-exponential 8.6) and 10-p.l samples were applied to a 2- by 2-in. (ca.
phase. 5- by 5-cm) microscope slide covered with 5 ml of
Isolation of extracelular and bacteria-associated hya- 0.8% agarose in the appropriate buffer. Slides were
luronate polymers. Bacteria were grown to the appro- electrophoresed for 45 min at 150 V.
priate optical density, rapidly chilled with ice, and Hyaluronic acid was visualized by placing the slide
sedimented with a Sorvall RC SB centrifuge equipped into a solution containing 1% bovine serum albumin in
with a GS-3 rotor at 13,680 x g for 15 min. The 2 M acetic acid for 15 min. After the incubation period,
supematant served as the source of extracellular hyal- the slides were washed with water, and a white
uronic acid. The loose pellet then was washed five precipitate formed at the site of hyaluronic acid local-
times with saline at 4°C to remove the majority of ization. The slides were then pressed, dried, and
associated extracellular hyaluronic acid. Both the en- stained as previously described by Kessler and van de
capsulated bacteria and the extracellular supernatants Rijn (11). The visualized precipitates were then quanti-
were treated in a similar manner throughout the re- tated with a soft laser scanning densitometer (Biomed-
maining steps. ical Instruments Inc., Fullerton, Calif.).
The bacteria were suspended in saline, sodium Chemical analysis. Glucuronic acid was quantitated
dodecyl sulfate was added to a final concentration of by Bitter and Muir's carbazole assay for uronic acids
0.01%, and the culture was incubated at room tem- (4). N-Acetylglucosamine was analyzed by the proce-
perature until the capsule was released as monitored dure of Boas (5) after hydrolysis of the samples with 3
by India ink preparations. Bacteria were then pelleted N HCI for 16 h at 100°C. The resin treatment was
by centrifugation at 13,680 x g for 15 min, and the omitted.
supernatants were filtered through a 0.22-,um mem- All other carbohydrates were analyzed as their
brane filter (Millipore Corp., Bedford, Mass.). Next, alditol acetates by gas chromatography on a column of
titers were determined on samples of the filtered GP 3% SP-2340 on 100/120 Supelcoport (3 ft [ca. 91
supernatants containing hyaluronic acid with hexade- cm] by 2 mm). In these analyses samples were hydro-
cyltrimethylammonium bromide (cetavalon) to deter- lyzed in sealed tubes with Teflon-lined screw caps in 1
mine the optimal concentration for precipitation of the N sulfuric acid for 8 h at 100°C (9, 13). A Varian 3700
hyaluronic acid (usually 0.3%). Then the cetavalon gas chromatograph with a flame ionization detector
solution was added to the supernatants; the precipitate interfaced to a Varian CDS 111 computer (Varian
was permitted to form at room temperature and then Instruments Division, Palo Alto, Calif.) was used to
collected by centrifugation or on a molecular sieve analyze the alditol acetates. After a temperature of
(150-pm openings). The precipitates were then washed 180°C for 6 min, a temperature gradient of 2°C/min was
extensively with distilled water, followed by solubili- run for 30 min to elute the alditol acetate derivatives.
zation with 2 M calcium chloride at 4°C with stirring. Other sugars were analyzed by using a column of GP
Next, the solubiized material was centrifuged at 3% SP-2330 on 100/120 Supelcoport (6 ft [ca. 182 cm]
20,000 x g to remove particulates and subsequently by 2 mm) at a constant temperature of 220°C.
was treated with 2 volumes of ethanol to precipitate For amino acid analysis, samples were hydrolyzed
the hyaluronic acid. The precipitates were washed with 6 N hydrochloric acid at 110°C for 22 h in
with cold ethanol-saline (2:1) and finally solubilized nitrogen-flushed and evacuated ampoules. Amino ac-
with distilled water. Sodium chloride was added to a ids were analyzed by high-pressure liquid chromatog-
final concentration of 0.9% to the solubilized hyal- raphy with a column of Licrosorb RP-18 (25 cm by 4.5VOL. 156, 1983
mm) to separate the Dabsyl-CI derivatives as recently
described by Chang et al. (7).
Phosphorus was detected as previously described
(16).
Radiolabeling of bacteria. For all isotope incorpo-
ration studies, the streptococci were grown in chemi-
cally defined medium. Since none of the streptococcal
strains used in this study fermented glycerol, no
modifications had to be made to the medium to incor-
porate glycerol into hyaluronic acid. However for
studies on incorporation of phosphorus, the medium
had to be modified as follows. Chemically defined
medium was prepared without potassium or sodium
-
25
1
25
1
5
5
a VO
b.
I
_l
STREPTOCOCCAL HYALURONIC ACID
Vt
i
.1
d. VO
e.
I
I
. I
VT
.
i
I
1061
...
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phosphates. The medium was buffered with N-2-hy- z-, 15
droxyethylpiperazine-N-2-ethanesulfonic acid (0.1 N,
pH 7.0). In addition, sufficient sodium phosphate was n) 5
added for the strain to reach the same optical density
as compared with when the strain is grown in complete c. f
chemically defined medium. This value varied for each 25
strain tested. Subsequently, potassium chloride was
added to the medium to a final concentration of 9.4 15
mM.
Preparation of streptococcal membranes. Mem-
branes were prepared from both group A and C 5
streptococcal strains by using phage-associated lysin
as described by van de Rijn and Kessler (22). Mem- 10 30 50 10 30 50
branes purified by this method contained less than VOLUME (ml)
0.1% cell wall and cytoplasmic components.
Degradation of hyaluronic acid. Cellular hyaluronic FIG. 1. Gel permeation chromatography of cellular
acid (1 mg/ml) in phosphate-buffered saline (0.05 M, and extracellular streptococcal hyaluronic acid. Cellu-
pH 7.4) was treated with either enzyme (pepsin, lar (a, b, c) or extracellular (d, e, f) hyaluronic acid (2
trypsin, chymotrypsin, thermolysin, group A prote- mg/ml) was loaded onto columns (1.5 by 28 cm) of
ase, or hyaluronidase; 100 U/ml) or membrane prepa- Sepharose 2B equilibrated with either 1% sodium
rations (5 mg/ml) for 2 h at 37°C. The reaction mixtures dodecyl sulfate in 0.01 M Tris-hydrochloride, pH 7.5
were centrifuged at 175,000 x g for 30 min in a (a, d), 6 M guanidine-hydrochloride (b, e), or 0.01 M
Beckman Airfuge (Beckman Instruments, Inc., Fuller- Tris-chloride, pH 7.5 (c, f). Sufficient sodium dodecyl
ton, Calif.) to remove particulates. The supematant sulfate or guanidine-hydrochloride was added to the
was then tested for intact hyaluronic acid by electro- appropriate samples to simulate running buffer condi-
phoresis (see above). tions. The hyaluronic acid samples were separated at a
Biosynthesis of hyaluronic acid. The assay for the hy- flow rate of 8 ml/h. Fractions were analyzed for
aluronic acid synthesis system used in these experi- glucuronic acid (see the text). Void and total volumes
ments was previously described by Sugahara et al. were calculated by using latex spheres and tritiated
(20). water. Sized dextran fractions served as molecular
weight standards for calibration of the columns.
RESULTS shoulder in the elution profile of the cellular
Molecular species of streptococcal hyaluronic form of the polymer (Fig. la) represents some
acid. Previous investigators have demonstrated contaminating extracellular hyaluronate poly-
that hyaluronic acid isolated from streptococci mers. This was removed by chromatography of
has an average molecular weight in excess of 1 x the cellular hyaluronic acid fractions. Both mo-
106 (20). The initial observations with strain lecular species were also separated by either
D181 appeared to indicate that total streptococ- denaturing conditions such as 4 M guanidine-
cal hyaluronic acid varied in molecular weight hydrochloride or native conditions such as 0.02
from 1 x 106 to 10 x 106 as determined by gel M Tris-hydrochloride, pH 7.4 (Fig. lb and e or
filtration chromatography. To determine the na- Fig. lc and f, respectively). This set of experi-
ture of the size heterogeneity of this polymer, ments indicated that the cellular form of hyal-
isolated cell-bound or extracellular hyaluronic uronic acid was not a micelle or noncovalently
acid was chromatographed on Sepharose 2B bound aggregate of extracellular forms.
columns (Fig. 1). The elution profile of cellular In addition to using gel filtration chromatogra-
hyaluronic acid with 1% sodium dodecyl sulfate phy to demonstrate the difference in the size of
as the buffer is given in Fig. la and indicates that the cellular and extracellular species, the size
the majority of these polymers have a molecular variation was also demonstrated by agarose
weight of 10 x 106. By contrast, most of the electrophoresis (Fig. 2). As would be expected,
extracellular hyaluronate polymers eluted at a the cellular hyaluronic acid (top) migrated slow-
molecular weight of 2 x 106 (Fig. ld). The er than the extracellular form (middle).1062 VAN DE RIJN J. BACTERIOL.
tographed with the cellular or extracellular hyal-
uronate polymers, indicating that glycerol and
phosphorus indeed are not present in the mole-
cule (Fig. 3). By comparison with nondenaturing
conditions, lipoteichoic acid and RNA, two
phosphorus-containing polymers, were shifted
to near the total volume of the column (data not
shown).
Relative susceptibility of cellular hyaluronate
FIG. 2. Electrophoresis of cellular and extracellu- polymer to degradation by enzymes and purified
lar streptococcal hyaluronic acid. Ten microliters of sreptococcal membranes. Purified cellular hyal-
cellular (top), extracellular (middle), and membrane- uronate was exposed to various enzymes to
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treated cellular hyaluronic acid were loaded into the determine whether a peptide may be involved in
appropriate well. After electrophoresis at 150 V for 45 the covalent linkage of five extracellular hyal-
min, the hyaluronic acid was visualized and stained uronate polymeric subunits. Neither pepsin,
(see the text).
trypsin, chymotrypsin, nor thermolysin cleaved
the hyaluronate polymer, indicating that this is
Since the above two procedures might not not a likely possibility. Although the group A
demonstrate the presence of low-molecular- streptococcal protease cleaved the cellular hya-
weight oligomers, the extracellular fluid of expo- luronate polymer to the extracellular size, dith-
nential- and stationary-phase cultures were ioerythritol (DTE) at a concentration of lo- M
lyophilized and suspended at a 25-fold concen- was present in the reaction mixture because the
tration. A portion of this material was loaded etizyme requires DTE for activity. Because re-
onto a column of Sephadex G-50 and eluted with ducing agents like DTE are known to cleave
distilled water. Only a small portion of the high-molecular-weight polysaccharides such as
glucuronic acid in the sample (less than 5%) hyaluronic acid in the presence of oxygen (26), it
eluted after the void volume of the column, with seems likely that cleavage of cellular hyaluron-
the remainder eluting in the void volume. The ate by group A streptococcal protease is due to
included glucuronic acid-containing material DTE. DTE at a concentration of 3 x 10-4 M in
eluted at the position of a dodecasaccharide. the absence of the protease was capable of
Chemical composition of cellular hyaluronic cleaving the hyaluronate polymer in the pres-
acid. Initially the cellular hyaluronic acid frac- ence of oxygen, but lost this capability when
tions from the Sepharose 213 column were nitrogen was substituted for air. Furthermore,
pooled, precipitated with ethanol, treated with the group A protease was inactive when the
RNase and DNase, suspended in 0.5% sodium reaction was attempted in the presence of nitro-
dodecyl sulfate, and chromatographed. Frac- gen. Furthermore, solutions of DTE and group
tions from the chromatographed hyaluronic acid A streptococcal protease plus DTE were unable
were pooled, precipitated with 2 volumes of to cleave cellular hyaluronate under nitrogen.
ethanol, and then dried with absolute ethanol, These data would appear to verify the above
acetone, and ether, respectively. Chemical anal- hypothesis.
ysis of this material from strain D181 demon- Isolated streptococcal membranes from strain
strated that it was composed of glucurbnic acid D181 effectively cleaved the cellular hyaluronate
(50.2%) and N-acetylglucosamine (50.6%),
whereas strain S43/192/3 gave values of 49.5 and
50.6%, respectively (Table 1). The only other TABLE 1. Chemical composition of cellular
k components quantifiable were amino acids at hyaluronic acid'
0.03 and 0.04%. Glycerol, sugars, and phospho- Cellular hyaluronic acid
rus were not detected.
Since hyaluronic acid polymers are very large Component D181 S43/192/3
molecules, the possibility remained that glycerol FLmol F81gtlOO Fmol "/100
or phosphorus (or both) may have gone -unde-
tected and yet formed part of the structure Glucuronic acid 0.232 50.2 0.229 49.5
involved in the binding of the molecule to the (sodium salt)
membrane as a lipid or phospholipid. To deter- N-Acetylglucosamine 0.229 50.6 0.229 50.6
mine whether this was possible, streptococcal Amino acids 0.03 0.04
strain D181 was grown in mediu-m containing Phosphorus NDb ND
either (32P]phosphorus or [ 4CJglycerol. When Glycerol ND ND
the cellular and extracellular hyaluronic acid Sugars ND ND
was isolated and chromatographed on Sepha- All samples were done in triplicate.
b ND, Not detectable to the 0.1% level.
rose 2B, neither of the isotope labels cochroma-VOL. 156, 1983 STREPTOCOCCAL HYALURONIC ACID 1063
VO Vt just lost their capsule during the stationary
I
Ip phase had an appreciably diminished capacity to
--- -5000
Ld
synthesize the hyaluronate, as evidenced by the
N
low rate of glucuronic acid transfer from UDP-
0 glucuronic acid (12 to 17 nmol/h per mg of
3001 08 protein).
U)
To determine whether an inhibitor of biosyn-
thesis was formed during the stationary phase,
UD 100 X - 1000 R membrane preparations from both the exponen-
tial and stationary phases were mixed and mildly
sonicated to insure proper mixing, and a sample
was used in the assay. The specific activity of
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Volume (ml)
FIG. 3. Analysis of streptococcal hyaluronic acid the mixed preparations was approximately one-
for isotope phosphorus or glycerol. Hyaluronic acid half that of the exponential phase membranes
isolated from strain D181 grown in the presence of 32p alone (Table 3). These results would be expected
or [3H]glycerol was loaded onto a column (1.5 by 28 if membranes from streptococci in the stationary
cm) of Sepharose 2B equilibrated with 1% sodium growth phase did not have an inhibitor of hyalur-
dodecyl sulfate in 0.01 M Tris-chloride (pH 7.5). The onate biosynthesis.
hyaluronic acid samples were separated at a flow rate
of 8 ml/h. Fractions were analyzed for glucuronic acid, DISCUSSION
32p, and 3H.
Hyaluronic acid was isolated and purified
from cultures of streptococcal strains D181 and
polymer to the extracellular size; this capacity S43/192/3 to apparent homogeneity (100.8 and
was heat labile, but insensitive to inactivation by 100.1%, respectively). Besides glucuronic acid
nitrogen. Hyaluronidase treatment of the hyal- and N-acetylglucosamine, the only other com-
uronate polymer cleaved the polymer to oligo- ponents that were detectable were amino acids
mers that were too small for our electrophoresis at 0.03 to 0.04%. However, there were probably
assay to detect. due to contamination since the amino acids were
To determine whether the capacity to cleave restricted to those usually found in buffer con-
the cellular hyaluronic acid was a general phe- trols.
nomenon with group A and C streptococci, Minor contaminants have been known to pro-
membranes were prepared from representative duce problems in interpreting results. It became
strains. In addition, membrane preparations evident from our studies that small amounts of
were isolated from both exponential- and sta- lipoteichoic acid and RNA were present in our
tionary-phase cells (Table 2). In all of the strains preparations unless denaturing conditions (i.e.,
examined, the membranes from stationary- 1% sodium dodecyl sulfate, 4 M guanidine) were
phase cells were able to degrade the hyaluronic used during the column chromatography step.
acid irrespective of their ability to produce a This method of purification of hyaluronic acid
capsule (Table 2). Membranes from exponential-
phase bacteria, however, were markedly less
efficient in this process. Extracellular products TABLE 2. Cleavage of cellular hyaluronic acid to
of organisms that produce capsules did not the extracellular form by streptococcal membranes'
cleave the cellular hyaluronic acid (data not
shown). Membrane prepn Phase % Con-
version
Biosynthesis of hyaluronic acid. To establish
the temporal relationship between loss of cap- Encapsulated strains
sule formation and the capacity to synthesize S43/192/3 Exponential 50
hyaluronate, membranes were purified from S43/192/3 Stationary 90
several strains of streptococci in the exponential D181 Exponential 100
and stationary phases of growth. These mem- D181 Stationary 100
Unencapsulated strains
brane preparations met the criteria for purity as D420 Exponential 0
previousIdescribed by van de Rijn and Kessler D420 Stationary 90
(22). When membranes from the mid-exponen- GL318 Stationary 70
tial phase were examined for their capacity to F301 Stationary 90
synthesize hyaluronic acid, they were capable of A928 Exponential 60
transferring glucuronic acid from UDP-gluc- D181 cellular hyaluronic acid 0
uronic acid at a rate of 430 to 954 nmollh per mg without membrane (control)
of protein, indicating a good capacity to synthe- a All strains were obtained from The Rockefeller
size hyaluronic acid (Table 3). By contrast, University collection. Membrane preparations met the
membranes isolated from streptococci that had standard criteria for purity.1064 VAN DE RIJN J. BACTERIOL.
TABLE 3. Biosynthesis of hyaluronic acid by ficity for cleavage of the molecule at specific
streptococcal membranes sites along a molecule containing repeating di-
Hyaluronic acid saccharide subunits. In addition, the question
Strepto- Exponential Stationary Mixeda arises of how membrane-bound activity could
coccal cleave a molecule that extended far beyond the
membrane nmolb Slp nmol Sp nmol Sp cell wall.
actc act act The experiments in this report also contribute
Group A some knowledge to the mechanism of capsule
S43/192/3 15.9 430 0.45 12 17.9 240 loss during the stationary phase of growth.
CS44 24.9 623 0.52 14 25.8 335 Whereas membranes from exponential-phase
Group C cultures produce hyaluronic acid at high specific
D181 35.3 954 0.96 27 35.6 490 activity (430 to 954 nmol/h per mg of protein),
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C1 17.5 490 0.64 18 18.8 263
membranes from stationary-phase cells isolated
a Equal amounts of exponential- and stationary- after capsule release did not have this high rate
phase membrane were added to the reaction mixture. of synthesis. Three possibilities for the loss of
b Nanomoles of glucuronic acid incorporated into biosynthetic activity appear reasonable. First,
hyaluronic acid. an inhibitor could be formed. However, when
c Specific activity, nanomoles per hour per milli-
gram of protein.
exponential- and stationary-phase membranes
were mixed in a 1:1 ratio, the rate of hyaluronate
synthesis was not appreciably reduced, dispel-
then permits its use for sensitive immunological ling the possibility of inhibitors avidly associated
analyses. with the membranes from streptococci in the
The initial studies demonstrating that strain stationary phase of growth (Table 3). A second
D181 released its hyaluronic acid polymers in possibility would be that the topography of the
discrete sizes during its various phases of membrane changes and the biosynthetic mecha-
growth prompted us to speculate on how this nism is altered. Previously we reported that
might occur. Three possibilities were tested, membranes from the stationary phase expressed
including that the hyaluronate was attached to only one-half the amount of outer surface pro-
the membrane by a lipid, that hyaluronate sub- tein as that of exponential-phase membranes
units were cross-linked by a peptide, and that (22). Third, the enzyme system could be diluted
hyaluronate was released by a hyaluronidase- out or degraded. Both of the latter two possibili-
like activity. The fact that phosphorus, glycerol, ties remain viable until specific probes for the
and fatty acids (data not shown) were not detect- molecules involved in the biosynthesis of hyal-
ed in the hyaluronate polymers indicated that uronic acid can be developed.
the polymers of hyaluronic acid were not at- ACKNOWLEDGMENTS
tached by lipids to the membrane. In addition, I am indebted to N. Dawson and C. Eastby for invaluable
because glycerol was not found associated with and expert laboratory assistance. I am also indebted to M.
the polymer, a deacylation reaction like that of McCarty and E. Gotschlich for stimulating conversations
lipoteichoic acid (12) does not appear to be regarding this paper.
involved in the release mechanism of extracellu- This work was supported by Public Health Service grant Al-
lar hyaluronate from the bacterium. 19756 from the National Institutes of Health. I.v.d.R. Estab-
lished Investigator with the American Heart Association.
Experiments with various proteases demon-
strated that a peptide bridge did not connect five LITERATURE CITED
polymers to produce a cellular polymer. Fur- 1. Appel, A., A. L. Horwitz, and A. Dorfman. 1979. Cell free
thermore, preparations of streptococcal mem- synthesis of hyaluronic acid in Marfan syndrome. J. Biol.
branes were demonstrated to have the capacity Chem. 254:12199-12203.
2. Balazc, E. A., and Z. Darzynkiewicz. 1973. The effect of
to cleave the cellular form to the extracellular hyaluronic acid on fibroblasts, mononuclear phagocytes
form. The membranes lost this capacity under and lymphocytes, p. 237. In E. Kulonen and J. Pikkar-
conditions that destroy enzymatic activity. ainen (ed.), Biology of fibroblasts. Academic Press, Inc.,
Membranes from stationary-phase cells ap- London.
3. Bartelt, M. A., and J. L. Duncan. 1978. Adherence of
peared to have a greater capacity to cleave the group A streptococci to human epithelial cells. Infect.
cellular hyaluronic acid to the extracellular form Immun. 20:200-208.
(Table 2). The hyaluronidase-like activity asso- 4. Bitter, T., and H. M. Muir. 1%2. A modified uronic acid
ciated with the membrane was not apparent in carbazole reaction. Anal. Biochem. 4:330-334.
5. Boas, N. F. 1953. Method for the determination of hexosa-
the extracellular products of the four strains mines in tissues. J. Biol. Chem. 204:553-563.
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