Vol. 156, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1059-10650021-9193/83/121059-07$02.00/0Copyright 0 1983, American Society for Microbiology

Streptococcal Hyaluronic Acid: Proposed Mechanisms ofDegradation and Loss of Synthesis During Stationary Phase

I. VAN DE RIJNDepartment of Microbiology and Immunology, Bowman Gray School of Medicine, Winston-Salem, North

Carolina 27103

Received 8 July 1983/Accepted 13 September 1983

Streptococcal hyaluronic acid was found to distribute into two discrete sizes.Cellular hyaluronic acid from strain D181 had an average molecular weight of 10 x106, whereas the average molecular weight of extracellular hyaluronic acid fromthe same strain was 2 x 106. Cellular streptococcal hyaluronic acid was purified tohomogeneity. Proteases were unable to cleave the purified cellular polymer,indicating that a peptide was not involved in cross-linking five extracellularhyaluronate polymers to form a cell-bound complex. Lipids apparently are notpart of the cellular hyaluronic acid because phosphorus and glycerol were notdetected by radioisotopic techniques, and denaturing conditions did not changethe size of the polymer. Membranes obtained from various strains of group A andC 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 ofgrowth no longer had the capacity to synthesize hyaluronic acid. The loss of thisproperty appeared to be due to changes in the structure of the membrane.

Hyaluronic acid is a linear polysaccharidecomposed of repeating subunits of P-1,4-linkeddisaccharides of glucuronic acid ,B-1,3-N-ace-tylglucosamine. This polymer is synthesized onthe streptococcal membrane with UDP-gluc-uronic acid and UDP-N-acetylglucosamine serv-ing as precursors for the molecule (19). Lipidintermediates in the biosynthetic pathway of hy-alutonic acid have not been detected to date(20).Group A and C streptococci produce a cap-

sule that is composed of hyaluronic acid poly-mers identical to that found in mammalian con-nective tissues (14). In Streptococcus spp., thehyaluronic acid capsule has been demonstratedto be a major virulence factor in addition to thecell wall M protein (10). The capsule also inhib-its the binding of the organism to human epitheli-al cells (3) and murine peritoneal macrophages(25).

In mammals, hyaluronic acid has been shownto affect a number of biological processes suchas inhibition of lymphocytes and macrophageproliferation (2), suppression of the graft-versus-host reaction (2), stimulation of aggregation oflymphoma cells to influence gene expression(18, 24), assembly of epithelial layers duringdevelopment (8), and reduction of the chemotac-tic movement of leukocytes (6). There are alsohyaluronic acid receptors on transformed lym-

phocytes and alveolar macrophages (15, 17, 18,24). Furthermore, abnormalities in the regula-tion of the synthesis of this molecule are be-lieved to be the basis for Marfan syndrome (1).

Streptococci are ideally suited for studyingthe biosynthesis of hyaluronic acid due to theabundant availability of hyaluronate and since inthis organism the hyaluronate is the only poly-mer into which glucuronic acid is incorporated.In this report, it is demonstrated that group Cstreptococcal strain D181 releases hyaluronicacid polymers in defined sizes that a,'e about20% of the size of the cellular form. In addition,a hyaluronidase-like activity was found associat-ed with the membrane of streptococci. It isthought that this activity may result in the partialdegradation of the cellular hyaluronate polymer,releasing the smaller hyaluronate polymer foundextracellularly. Furthermore, it was found thatthe streptococcal membrane, in the stationaryphase, lacked the capacity to synthesize hyal-uronic acid polymers from precursors, whichcorrelates with the absence of capsules.

MATERIALS AND METHODS

Bacteria and media. Streptococcal strains S43/192/3(group A) and D181 (group C) were obtained from theculture collection of The Rockefeller University.Strain CS44 (group A) was obtained from P. Cleary,

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University of Minnesota, whereas strain Cl was afresh isolate (group C). All four strains producedcapsules of hyaluronic acid to various degrees.The bacteria were grown in a chemically defined

medium previously described by van de Rin andKessler (23). Stock cultures were kept frozen at-80°C. To maintain optimal capsule production by theorganism, an original vial was opened for each experi-ment.Growth and monitoring of capsule. Growth of cul-

tures in 18-mm tubes was measured in a Spectronic 20spectrophotometer (Bausch & Lomb, Inc., Rochester,N.Y.) at a wavelength of 650 nm. The net observedoptical density readings of the culture were multipliedby 1,000 and then converted to adjusted optical densi-ty units (21).

In the studies presented, the bacteria were grown at37°C in 5-gallon (ca. 19-liter) carboys to the mid-exponential phase or until after the release of thecapsule was observed (early or late stationary phase,depending on the organism).The presence of bound capsule was monitored by

India ink preparations. Briefly, a drop of India ink wasadded and mixed with two drops of bacteria, a coverslip was added, and then the slide was monitored byphase microscopy. The size of the capsule varied fromstrain to strain as well as during the growth cycle. Thelargest capsules appeared at the mid-exponentialphase.

Isolation of extracelular and bacteria-associated hya-luronate polymers. Bacteria were grown to the appro-priate optical density, rapidly chilled with ice, andsedimented with a Sorvall RC SB centrifuge equippedwith a GS-3 rotor at 13,680 x g for 15 min. Thesupematant served as the source of extracellular hyal-uronic acid. The loose pellet then was washed fivetimes with saline at 4°C to remove the majority ofassociated extracellular hyaluronic acid. Both the en-capsulated bacteria and the extracellular supernatantswere treated in a similar manner throughout the re-maining steps.The bacteria were suspended in saline, sodium

dodecyl sulfate was added to a final concentration of0.01%, and the culture was incubated at room tem-perature until the capsule was released as monitoredby India ink preparations. Bacteria were then pelletedby centrifugation at 13,680 x g for 15 min, and thesupernatants were filtered through a 0.22-,um mem-brane filter (Millipore Corp., Bedford, Mass.). Next,titers were determined on samples of the filteredsupernatants containing hyaluronic acid with hexade-cyltrimethylammonium bromide (cetavalon) to deter-mine the optimal concentration for precipitation of thehyaluronic acid (usually 0.3%). Then the cetavalonsolution was added to the supernatants; the precipitatewas permitted to form at room temperature and thencollected by centrifugation or on a molecular sieve(150-pm openings). The precipitates were then washedextensively with distilled water, followed by solubili-zation with 2 M calcium chloride at 4°C with stirring.Next, the solubiized material was centrifuged at20,000 x g to remove particulates and subsequentlywas treated with 2 volumes of ethanol to precipitatethe hyaluronic acid. The precipitates were washedwith cold ethanol-saline (2:1) and finally solubilizedwith distilled water. Sodium chloride was added to afinal concentration of 0.9% to the solubilized hyal-

J. BACTERIOL.

uronic acid, and any insoluble material was removedby centrifugation. The ethanol precipitation, solubili-zation, and centrifugation steps usually were repeatedfive times to obtain high-purity preparations. All hyal-uronic acid samples were dried by solvent dehydrationwith ethanol, acetone, and then ether. Traces of etherwere removed by desiccation in vacuo.

Sizing of hyaluronic acid polymers. Hyaluronic acidwas solubilized in phosphate-buffered saline (0.01 M,pH 7.4) and treated with RNase and DNase at 10 p.g/mlfor 2 h at 37°C to digest any high-molecular-weightnucleic acid. Samples of hyaluronic acid were sizedand further purified on columns (1.5 by 28 cm) ofSepharose 2B (Pharmacia Fine Chemicals, Pis-cataway, N.J.) equilibrated with the appropriate bufferand run at 8 ml/h. Samples of hyaluronic acid weresuspended in buffer (0.01 M Tris-hydrochloride [pH7.5], 6 M guanidine-hydrochloride, or 1% sodiumdodecyl sulfate-0.01 M Tris-chloride [pH 7.5]). Frac-tions were monitored with a flow-through refractome-ter (Waters Associates, Milford, Mass.) and tested forglucuronic acid by the Bittner and Muir (4) assay.

Ekectrophoresis and staining. Bound and extracellu-lar hyaluronic acid were differentiated by electropho-resis in agarose. Hyaluronic acid was suspended to afinal concentration of 1 mg/ml in electrophoresis buffer(barbital hydrochloride buffer; ionic strength, 0.02; pH8.6) and 10-p.l samples were applied to a 2- by 2-in. (ca.5- by 5-cm) microscope slide covered with 5 ml of0.8% agarose in the appropriate buffer. Slides wereelectrophoresed for 45 min at 150 V.

Hyaluronic acid was visualized by placing the slideinto a solution containing 1% bovine serum albumin in2 M acetic acid for 15 min. After the incubation period,the slides were washed with water, and a whiteprecipitate formed at the site of hyaluronic acid local-ization. The slides were then pressed, dried, andstained as previously described by Kessler and van deRijn (11). The visualized precipitates were then quanti-tated with a soft laser scanning densitometer (Biomed-ical Instruments Inc., Fullerton, Calif.).Chemical analysis. Glucuronic acid was quantitated

by Bitter and Muir's carbazole assay for uronic acids(4). N-Acetylglucosamine was analyzed by the proce-dure of Boas (5) after hydrolysis of the samples with 3N HCI for 16 h at 100°C. The resin treatment wasomitted.

All other carbohydrates were analyzed as theiralditol acetates by gas chromatography on a column ofGP 3% SP-2340 on 100/120 Supelcoport (3 ft [ca. 91cm] by 2 mm). In these analyses samples were hydro-lyzed in sealed tubes with Teflon-lined screw caps in 1N sulfuric acid for 8 h at 100°C (9, 13). A Varian 3700gas chromatograph with a flame ionization detectorinterfaced to a Varian CDS 111 computer (VarianInstruments Division, Palo Alto, Calif.) was used toanalyze the alditol acetates. After a temperature of180°C for 6 min, a temperature gradient of 2°C/min wasrun for 30 min to elute the alditol acetate derivatives.Other sugars were analyzed by using a column of GP3% SP-2330 on 100/120 Supelcoport (6 ft [ca. 182 cm]by 2 mm) at a constant temperature of 220°C.For amino acid analysis, samples were hydrolyzed

with 6 N hydrochloric acid at 110°C for 22 h innitrogen-flushed and evacuated ampoules. Amino ac-ids were analyzed by high-pressure liquid chromatog-raphy with a column of Licrosorb RP-18 (25 cm by 4.5

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STREPTOCOCCAL HYALURONIC ACID 1061

mm) to separate the Dabsyl-CI derivatives as recentlydescribed 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 streptococcalstrains used in this study fermented glycerol, nomodifications had to be made to the medium to incor-porate glycerol into hyaluronic acid. However forstudies on incorporation of phosphorus, the mediumhad to be modified as follows. Chemically definedmedium was prepared without potassium or sodiumphosphates. The medium was buffered with N-2-hy-droxyethylpiperazine-N-2-ethanesulfonic acid (0.1 N,pH 7.0). In addition, sufficient sodium phosphate wasadded for the strain to reach the same optical densityas compared with when the strain is grown in completechemically defined medium. This value varied for eachstrain tested. Subsequently, potassium chloride wasadded to the medium to a final concentration of 9.4mM.

Preparation of streptococcal membranes. Mem-branes were prepared from both group A and Cstreptococcal strains by using phage-associated lysinas described by van de Rijn and Kessler (22). Mem-branes purified by this method contained less than0.1% cell wall and cytoplasmic components.

Degradation of hyaluronic acid. Cellular hyaluronicacid (1 mg/ml) in phosphate-buffered saline (0.05 M,pH 7.4) was treated with either enzyme (pepsin,trypsin, chymotrypsin, thermolysin, group A prote-ase, or hyaluronidase; 100 U/ml) or membrane prepa-rations (5 mg/ml) for 2 h at 37°C. The reaction mixtureswere centrifuged at 175,000 x g for 30 min in a

Beckman Airfuge (Beckman Instruments, Inc., Fuller-ton, Calif.) to remove particulates. The supematantwas then tested for intact hyaluronic acid by electro-phoresis (see above).

Biosynthesis of hyaluronic acid. The assay for the hy-aluronic acid synthesis system used in these experi-ments was previously described by Sugahara et al.(20).

RESULTS

Molecular species of streptococcal hyaluronicacid. Previous investigators have demonstratedthat hyaluronic acid isolated from streptococcihas an average molecular weight in excess of 1 x

106 (20). The initial observations with strainD181 appeared to indicate that total streptococ-cal hyaluronic acid varied in molecular weightfrom 1 x 106 to 10 x 106 as determined by gelfiltration chromatography. To determine the na-ture of the size heterogeneity of this polymer,isolated cell-bound or extracellular hyaluronicacid was chromatographed on Sepharose 2Bcolumns (Fig. 1). The elution profile of cellularhyaluronic acid with 1% sodium dodecyl sulfateas the buffer is given in Fig. la and indicates thatthe majority of these polymers have a molecularweight of 10 x 106. By contrast, most of theextracellular hyaluronate polymers eluted at a

molecular weight of 2 x 106 (Fig. ld). The

25

15

- 5

25

1z-, 15

n) 5

25

15

5

10 30 50 10 30 50

VOLUME (ml)FIG. 1. Gel permeation chromatography of cellular

and extracellular streptococcal hyaluronic acid. Cellu-lar (a, b, c) or extracellular (d, e, f) hyaluronic acid (2mg/ml) was loaded onto columns (1.5 by 28 cm) ofSepharose 2B equilibrated with either 1% sodiumdodecyl sulfate in 0.01 M Tris-hydrochloride, pH 7.5(a, d), 6 M guanidine-hydrochloride (b, e), or 0.01 MTris-chloride, pH 7.5 (c, f). Sufficient sodium dodecylsulfate or guanidine-hydrochloride was added to theappropriate samples to simulate running buffer condi-tions. The hyaluronic acid samples were separated at aflow rate of 8 ml/h. Fractions were analyzed forglucuronic acid (see the text). Void and total volumeswere calculated by using latex spheres and tritiatedwater. Sized dextran fractions served as molecularweight standards for calibration of the columns.

shoulder in the elution profile of the cellularform of the polymer (Fig. la) represents somecontaminating extracellular hyaluronate poly-mers. This was removed by chromatography ofthe cellular hyaluronic acid fractions. Both mo-lecular species were also separated by eitherdenaturing conditions such as 4 M guanidine-hydrochloride or native conditions such as 0.02M Tris-hydrochloride, pH 7.4 (Fig. lb and e orFig. lc and f, respectively). This set of experi-ments indicated that the cellular form of hyal-uronic acid was not a micelle or noncovalentlybound aggregate of extracellular forms.

In addition to using gel filtration chromatogra-phy to demonstrate the difference in the size ofthe cellular and extracellular species, the sizevariation was also demonstrated by agaroseelectrophoresis (Fig. 2). As would be expected,the cellular hyaluronic acid (top) migrated slow-er than the extracellular form (middle).

a VO Vt d. VO VTI i I i_l .1 . I . I ...

b. e. I

c. f

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FIG. 2. Electrophoresis of cellular and extracellu-lar streptococcal hyaluronic acid. Ten microliters ofcellular (top), extracellular (middle), and membrane-treated cellular hyaluronic acid were loaded into theappropriate well. After electrophoresis at 150 V for 45min, the hyaluronic acid was visualized and stained(see the text).

Since the above two procedures might notdemonstrate the presence of low-molecular-weight oligomers, the extracellular fluid of expo-nential- and stationary-phase cultures werelyophilized and suspended at a 25-fold concen-tration. A portion of this material was loadedonto a column of Sephadex G-50 and eluted withdistilled water. Only a small portion of theglucuronic acid in the sample (less than 5%)eluted after the void volume of the column, withthe remainder eluting in the void volume. Theincluded glucuronic acid-containing materialeluted at the position of a dodecasaccharide.Chemical composition of cellular hyaluronic

acid. Initially the cellular hyaluronic acid frac-tions from the Sepharose 213 column werepooled, precipitated with ethanol, treated withRNase and DNase, suspended in 0.5% sodiumdodecyl sulfate, and chromatographed. Frac-tions from the chromatographed hyaluronic acidwere pooled, precipitated with 2 volumes ofethanol, and then dried with absolute ethanol,acetone, and ether, respectively. Chemical anal-ysis of this material from strain D181 demon-strated that it was composed of glucurbnic acid(50.2%) and N-acetylglucosamine (50.6%),whereas strain S43/192/3 gave values of 49.5 and50.6%, respectively (Table 1). The only other

k components quantifiable were amino acids at0.03 and 0.04%. Glycerol, sugars, and phospho-rus were not detected.

Since hyaluronic acid polymers are very largemolecules, the possibility remained that glycerolor phosphorus (or both) may have gone -unde-tected and yet formed part of the structureinvolved in the binding of the molecule to themembrane as a lipid or phospholipid. To deter-mine whether this was possible, streptococcalstrain D181 was grown in mediu-m containingeither (32P]phosphorus or [ 4CJglycerol. Whenthe cellular and extracellular hyaluronic acidwas isolated and chromatographed on Sepha-rose 2B, neither of the isotope labels cochroma-

tographed with the cellular or extracellular hyal-uronate polymers, indicating that glycerol andphosphorus indeed are not present in the mole-cule (Fig. 3). By comparison with nondenaturingconditions, lipoteichoic acid and RNA, twophosphorus-containing polymers, were shiftedto near the total volume of the column (data notshown).

Relative susceptibility of cellular hyaluronatepolymer to degradation by enzymes and purifiedsreptococcal membranes. Purified cellular hyal-uronate was exposed to various enzymes todetermine whether a peptide may be involved inthe covalent linkage of five extracellular hyal-uronate polymeric subunits. Neither pepsin,trypsin, chymotrypsin, nor thermolysin cleavedthe hyaluronate polymer, indicating that this isnot a likely possibility. Although the group Astreptococcal protease cleaved the cellular hya-luronate polymer to the extracellular size, dith-ioerythritol (DTE) at a concentration of lo- Mwas present in the reaction mixture because theetizyme requires DTE for activity. Because re-ducing agents like DTE are known to cleavehigh-molecular-weight polysaccharides such ashyaluronic acid in the presence of oxygen (26), itseems likely that cleavage of cellular hyaluron-ate by group A streptococcal protease is due toDTE. DTE at a concentration of 3 x 10-4 M inthe absence of the protease was capable ofcleaving the hyaluronate polymer in the pres-ence of oxygen, but lost this capability whennitrogen was substituted for air. Furthermore,the group A protease was inactive when thereaction was attempted in the presence of nitro-gen. Furthermore, solutions of DTE and groupA streptococcal protease plus DTE were unableto cleave cellular hyaluronate under nitrogen.These data would appear to verify the abovehypothesis.

Isolated streptococcal membranes from strainD181 effectively cleaved the cellular hyaluronate

TABLE 1. Chemical composition of cellularhyaluronic acid'

Cellular hyaluronic acid

Component D181 S43/192/3FLmol F81gtlOO Fmol "/100

Glucuronic acid 0.232 50.2 0.229 49.5(sodium salt)

N-Acetylglucosamine 0.229 50.6 0.229 50.6Amino acids 0.03 0.04Phosphorus NDb NDGlycerol ND NDSugars ND ND

All samples were done in triplicate.b ND, Not detectable to the 0.1% level.

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I

0

U)

UD

VO VtIp

---

N

3001

100 X

-5000 Ld

08

- 1000 R

Volume (ml)FIG. 3. Analysis of streptococcal hyaluronic acid

for isotope phosphorus or glycerol. Hyaluronic acidisolated from strain D181 grown in the presence of 32por [3H]glycerol was loaded onto a column (1.5 by 28cm) of Sepharose 2B equilibrated with 1% sodiumdodecyl sulfate in 0.01 M Tris-chloride (pH 7.5). Thehyaluronic acid samples were separated at a flow rateof 8 ml/h. Fractions were analyzed for glucuronic acid,32p, and 3H.

polymer to the extracellular size; this capacitywas heat labile, but insensitive to inactivation bynitrogen. Hyaluronidase treatment of the hyal-uronate polymer cleaved the polymer to oligo-mers that were too small for our electrophoresisassay to detect.To determine whether the capacity to cleave

the cellular hyaluronic acid was a general phe-nomenon with group A and C streptococci,membranes were prepared from representativestrains. In addition, membrane preparationswere isolated from both exponential- and sta-tionary-phase cells (Table 2). In all of the strainsexamined, the membranes from stationary-phase cells were able to degrade the hyaluronicacid irrespective of their ability to produce acapsule (Table 2). Membranes from exponential-phase bacteria, however, were markedly lessefficient in this process. Extracellular productsof organisms that produce capsules did notcleave the cellular hyaluronic acid (data notshown).

Biosynthesis of hyaluronic acid. To establishthe temporal relationship between loss of cap-sule formation and the capacity to synthesizehyaluronate, membranes were purified fromseveral strains of streptococci in the exponentialand stationary phases of growth. These mem-brane preparations met the criteria for purity aspreviousIdescribed by van de Rijn and Kessler(22). When membranes from the mid-exponen-tial phase were examined for their capacity tosynthesize hyaluronic acid, they were capable oftransferring glucuronic acid from UDP-gluc-uronic acid at a rate of 430 to 954 nmollh per mgof protein, indicating a good capacity to synthe-size hyaluronic acid (Table 3). By contrast,membranes isolated from streptococci that had

just lost their capsule during the stationaryphase had an appreciably diminished capacity tosynthesize the hyaluronate, as evidenced by thelow rate of glucuronic acid transfer from UDP-glucuronic acid (12 to 17 nmol/h per mg ofprotein).To determine whether an inhibitor of biosyn-

thesis was formed during the stationary phase,membrane preparations from both the exponen-tial and stationary phases were mixed and mildlysonicated to insure proper mixing, and a samplewas used in the assay. The specific activity ofthe mixed preparations was approximately one-half that of the exponential phase membranesalone (Table 3). These results would be expectedif membranes from streptococci in the stationarygrowth phase did not have an inhibitor of hyalur-onate biosynthesis.

DISCUSSION

Hyaluronic acid was isolated and purifiedfrom cultures of streptococcal strains D181 andS43/192/3 to apparent homogeneity (100.8 and100.1%, respectively). Besides glucuronic acidand N-acetylglucosamine, the only other com-ponents that were detectable were amino acidsat 0.03 to 0.04%. However, there were probablydue to contamination since the amino acids wererestricted to those usually found in buffer con-trols.Minor contaminants have been known to pro-

duce problems in interpreting results. It becameevident from our studies that small amounts oflipoteichoic acid and RNA were present in ourpreparations unless denaturing conditions (i.e.,1% sodium dodecyl sulfate, 4 M guanidine) wereused during the column chromatography step.This method of purification of hyaluronic acid

TABLE 2. Cleavage of cellular hyaluronic acid tothe extracellular form by streptococcal membranes'

Membrane prepn Phase % Con-version

Encapsulated strainsS43/192/3 Exponential 50S43/192/3 Stationary 90D181 Exponential 100D181 Stationary 100

Unencapsulated strainsD420 Exponential 0D420 Stationary 90GL318 Stationary 70F301 Stationary 90A928 Exponential 60

D181 cellular hyaluronic acid 0without membrane (control)

a All strains were obtained from The RockefellerUniversity collection. Membrane preparations met thestandard criteria for purity.

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TABLE 3. Biosynthesis of hyaluronic acid bystreptococcal membranes

Hyaluronic acidStrepto- Exponential Stationary Mixedacoccal

membrane nmolb Slp nmol Sp nmol Spactc act act

Group AS43/192/3 15.9 430 0.45 12 17.9 240CS44 24.9 623 0.52 14 25.8 335

Group CD181 35.3 954 0.96 27 35.6 490C1 17.5 490 0.64 18 18.8 263

a Equal amounts of exponential- and stationary-phase membrane were added to the reaction mixture.

b Nanomoles of glucuronic acid incorporated intohyaluronic acid.

c Specific activity, nanomoles per hour per milli-gram of protein.

then permits its use for sensitive immunologicalanalyses.The initial studies demonstrating that strain

D181 released its hyaluronic acid polymers indiscrete sizes during its various phases ofgrowth prompted us to speculate on how thismight occur. Three possibilities were tested,including that the hyaluronate was attached tothe membrane by a lipid, that hyaluronate sub-units were cross-linked by a peptide, and thathyaluronate was released by a hyaluronidase-like activity. The fact that phosphorus, glycerol,and fatty acids (data not shown) were not detect-ed in the hyaluronate polymers indicated thatthe polymers of hyaluronic acid were not at-tached by lipids to the membrane. In addition,because glycerol was not found associated withthe polymer, a deacylation reaction like that oflipoteichoic acid (12) does not appear to beinvolved in the release mechanism of extracellu-lar hyaluronate from the bacterium.Experiments with various proteases demon-

strated that a peptide bridge did not connect fivepolymers to produce a cellular polymer. Fur-thermore, preparations of streptococcal mem-branes were demonstrated to have the capacityto cleave the cellular form to the extracellularform. The membranes lost this capacity underconditions that destroy enzymatic activity.Membranes from stationary-phase cells ap-peared to have a greater capacity to cleave thecellular hyaluronic acid to the extracellular form(Table 2). The hyaluronidase-like activity asso-ciated with the membrane was not apparent inthe extracellular products of the four strainsstudied. Whether this membrane-bound activityis responsible for the release and cleavage of thehyaluronic acid polymer remains to be estab-lished. It is difficult to conceive how such amembrane-bound activity would have the speci-

J. BACTERIOL.

ficity for cleavage of the molecule at specificsites along a molecule containing repeating di-saccharide subunits. In addition, the questionarises of how membrane-bound activity couldcleave a molecule that extended far beyond thecell wall.The experiments in this report also contribute

some knowledge to the mechanism of capsuleloss during the stationary phase of growth.Whereas membranes from exponential-phasecultures produce hyaluronic acid at high specificactivity (430 to 954 nmol/h per mg of protein),membranes from stationary-phase cells isolatedafter capsule release did not have this high rateof synthesis. Three possibilities for the loss ofbiosynthetic activity appear reasonable. First,an inhibitor could be formed. However, whenexponential- and stationary-phase membraneswere mixed in a 1:1 ratio, the rate of hyaluronatesynthesis was not appreciably reduced, dispel-ling the possibility of inhibitors avidly associatedwith the membranes from streptococci in thestationary phase of growth (Table 3). A secondpossibility would be that the topography of themembrane changes and the biosynthetic mecha-nism is altered. Previously we reported thatmembranes from the stationary phase expressedonly one-half the amount of outer surface pro-tein as that of exponential-phase membranes(22). Third, the enzyme system could be dilutedout or degraded. Both of the latter two possibili-ties remain viable until specific probes for themolecules involved in the biosynthesis of hyal-uronic acid can be developed.

ACKNOWLEDGMENTS

I am indebted to N. Dawson and C. Eastby for invaluableand expert laboratory assistance. I am also indebted to M.McCarty and E. Gotschlich for stimulating conversationsregarding this paper.This work was supported by Public Health Service grant Al-

19756 from the National Institutes of Health. I.v.d.R. Estab-lished Investigator with the American Heart Association.

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