Purification and characterization of hyaluronidase from Streptococcus agalactiae

Zbl. Bakt. 280, 497-506 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Purification and Characterization of Hyaluronidase from Streptococcus agalactiae * JORG-HERMANN OZEGOWSKI, ELISABETH GUNTHER, and WERNER REICHARDT University of Jena, Institute of Experimental Microbiology, Winzerlaer Str. 10, D-07745 Jena, Germany With 7 Figures· Received August 17, 1993 . Revision received October 21, 1993 . Accepted January 12, 1994

Summary Hyaluronidase from two different strains of Streptococcus agalactiae was purified and characterized. The purification was performed successively by chromatography and rechromatography on phenylsepharose, gel filtration with FPLC on Superdex G 200 and isoelectric focusing. The purified hyaluronidase had an isoelectric point of 8.75 and a molecular weight of approximately 116000 D. It showed maximal enzyme activity at pH 6.30 and 40°C. The Michaelis constant was estimated to be 8.17 x 10-2 mg/ml. Hyaluronidasewas stimulated only by Mg++ and inhibited by Zn++, AI+++, Cu++ and Fe++ at a final concentration of 10 mmoVl, respectively. The enzyme splitted hyaluronic acid and in low amounts dermatan sulphate and chondroitin sulphate A. Additionally, synthetic polyanions (like polymers of gentisic acid with formaldehyde and hydroxy sulphonic acid with formaldehyde) turned out to be also potent inhibitors of the enzyme. Introduction Bacterial hyaluronidases are produced by streptococci (7, 10, 12, 21, 28, 31), staphylococci (1, 2), clostridia and streptomycetes (24). Among streptococci, several serological groups possess the ability to produce hyaluronidases. Streptococcal hyaluronidases behave differently concerning their enzymatic activity and molecular weights (8, 12,20,21,28,31), but they share the ability to split mucopolysaccharide hyaluronic acid (HA) into unsaturated disaccharides (16). Whereas hyaluronidases isolated from streptococcal strains are described to be unable to cleave the substrates chondroitin sulphate and dermatan sulphate, the reaction with these substrates occurs when using hyaluronidases of animal origin (18). The chondroitin sulphate-splitting activity in oral streptococci is not an effect of hyaluronidases but it is caused by the * Dedicated to Professor Dr. Dr. Dr.hc. Werner Kohler on the occasion of his 65th birthday. 32 Zbl. Bakt. 280/4

498

J.-H.Ozegowski, E. Gunther, and W. Reichardt

depolymerizing activity of chondroitin sulphate depolymerase (13). HA represents an essential part of tissue structure; its cleavage is considered to be an important step in the process of invasion into the human body by pathogenic microorganisms. Thus, hyaluronidases represent one of the pathogenicity factors in streptococci (9). Because streptococci of serological group B (GBS, Streptococcus agalactiae) exhibit much higher titers of hyaluronidase than do streptococci of other groups, the pathogenicity factor might play an essential role in GBS infections. GBS strains are found mainly in the urogenital tract of humans (4, 5, 25, 27). They are able to cause severe diseases in newborns and young children (3, 6, 11,30). Until now, the hyaluronidase of GBS has been examined only in culture filtrates.

Material and Methods Streptococci. Two streptococcal strains of serological group B were used. Strain B 3402 Typ II: clinical isolate (diabetic gangrene of the left heel) Strain B 4755: non typablelR: clinical isolate (pyelonephritis) Fermentation. The fermentation was performed in a stirred (200 rpm) glas fermentor with a working volume of 21. The culture medium contained 25 gil standard I nutrient broth (Merck). Glucose solution (1 g in 10 ml distilled water) was sterilized separately. The fermentor was fitted with devices for automatic regulation of the pH value by using pH 7.05 with sodium hydroxide solution (20%), coupled with feeding of a glucose solution (50%) at a ratio of 1: 1 (v/v) to sodium hydroxide. The main culture was inoculated with a batch culture (18 h at 37"C). The medium of the batch culture was preparated according to Ozegowski et a1. (22). Streptococci were incubated for 16 h at 35°C. Estimation of fermentation parameters. The cell density was estimated optically at 600 nm according to Maller et a1. (19). An extinction of 1.0 was found to be equal to 0.288 g biomass dry weightll. Hyaluronic acid. Streptococcal hyaluronic acid was isolated and purified from S. zooepidemicus according to Ozegowski et a1. (patent submitted). Hyaluronidase activity. Hyaluronidase activity was determined quantitatively by photometry (21). A sample to be tested (0.5 ml) was added to 0.5 ml acetate buffer (0.1 M, pH 6.0) and diluted geometrically. To each tube, 0.5 ml of substrate solution (0.2 mg hyaluronic acidlml of acetate buffer, 10 mM/I, pH 6.0) were added. After 30 min at 37°C, the enzyme reactions were terminated by addition of 3 ml of a cetyltrimethylammoniumbromide solution (2.5% cetyltrimethylammoniumbromide in 0.5 N NaOH). The samples were incubated at room temperature for 20 min and the turbidity was estimated at 600 nm. One unit of hyaluronidase was able to split 0.05 mg hyaluronic acid within 30 minutes. Purification of Hyaluronidase. After fermentation of GBS streptococci, the cells were removed by centrifugation (1 h, 5000 rpm) and subsequent filtration (average pore diameter 0.2 !lm, Sartorius Gottingen). The culture supernatant was saturated by addtion of ammonium sulphate at a final concentration of 40%. This hyaluronidase solution was then applied to a phenyl-sepharose column (2.2 x 10 cm, Pharmacia, Uppsala, Sweden) equilibrated with acetate-buffered ammonium sulphate solution (40%, pH 6.5). The column was subsequently washed with equilibration solution and the hyaluronidase was then eluted with a step gradient of decreasing ammonium sulphate concentrations. The hyaluronidase was eluated at an ammonium sulphate concentration of 30%. Fractions with the highest hyaluronidase activity were pooled and saturated once more with ammonium sulphate solution up to a final concentration of 40%. This hyaluronidase solution was rechromatographed on a phenyl-sepharose column (1.6 x 15 cm) equilibrated also with acetate-buffered ammonium sulphate solution (40%), pH 6.0. The hyaluronidase was eluted by a continuous

Hyaluronidase from GBS Strains

499

gradient of decreasing ammonium sulphate concentrations. The peak with highest activity of hyaluronidase was collected, dialyzed and lyophilized. Two mg of lyophilized protein were dissolved in 0.2 ml distilled water and then applied to a column packed with Superdex G 200 (Pharmacia, Uppsala) which was equilibrated with 20 mM Trisl150 mM NaCl, pH 7.5. The pooled active hyaluronidase peak was dialyzed and applied to preparative isoelectric focusing in Sephadex gel G 100 according to (23). The carrier ampholyte (Ampholine, Pharmacia, Uppsala) ranged from pH 3.5 to 10. After focusing, the hyaluronidase peak was determined, collected and eluted from Sephadex gel with distilled water. The protein was precipitated by addition of ammonium sulphate to a final concentration of 80%. Molecular weight determination. Molecular weight was estimated by sodium dodecylsulph ate (SDS)-polyacrylamide electrophoresis (12.5% slab gel). Samples containing approximately 5 Ilg protein were subjected to SDS-polyacrylamide gel electrophoresis for 3 h at 120 rnA as described by Laemmli et al. (14). The gel was stained with Coomassie blue. The molecular weight was estimated by comparing the relative migration of purified hyaluronidase to the migration of standard molecular weight markers (myosin 212000, (lrmacroglobulin 170000, ~-galactosidase 116000, transferrin 76000 and glutamic dehydrogenase 53000; Pharmacia, Uppsala) according to Weber and Osborn (32). Protein determination. The soluble protein was precipitated by addition of 10% (w/v) trichloroacetic acid (TCA) at a ratio of 1 : 9. After 20 minutes, precipitates were centrifuged and washed 3 times in 10% TCA. The protein content was measured by the method of Lowry et al. (17) with bovine serum albumin and ribonuclease as standards. Enzyme kinetics. The Michaelis constant (Km) was determined using 5 III of purified hyaluronidase and varying concentrations (0.03-0.1 mg/ml) of HA in the hyaluronidase assay described above. The velocity of enzyme reaction was expressed as mg HA splitted after 30 min. at 37°C. Double-reciprocal plots of velocity versus HA concentrations according to Lineweaver and Burk (15) served to determine Km-values. Enzyme characterization. Hyaluronidase activity was measured in phosphate buffer (10mmoVI phosphate with 10 mmoVi NaCl) at pH values from 4.0 to 9.5. Subsequently, hyaluronidase activity at pH 6.0 was determined after 30 min at various temperatures between 30 and 60°C. Hyaluronidase activity was evaluated also after addition of various cations (10 mmoVl) in comparison to unsupplemented controls. Inhibition of hyaluronidase activity was attempted with hydroquinone sulphonic acid formaldehyde polymer and gentisic acid formaldehyde polymer according to (26). The substrate specifity of hyaluronidase was investigated with dermatan sulphate (Sigma) and chondroitin sulphate A (Sigma) at a substrate concentration of 0.2 mg/ml.

Table 1. Purification of hyaluronidase from GBS type strain B 4755 Step of purification Culture supernatant

Specific activity (u1mg) * 46

Purification factor 1

5230

113.6

Rechromatography on phenyl sepharose

12400

269.5

FPLC chromatography on Superdex G 200

39600

860.9

Isoelectric focusing

81400

1769.6

Hydrophobic adsorption and chromatography on phenyl sepharose

* Units of hyaluronidase per mg protein.

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J.-H.Ozegowski, E. Gunther, and W. Reichardt

Results S. agalactiae strains 3402 (type II) and 4755 (n.t.lR) revealed the highest hyaluroniase activity and were therefore selected for isolation of the enzyme. A highly purified hyaluronidase could be isolated from GBS strains by stepwise purification (Tab. 1). In the first step for isolation of enzyme from cell-free culture supernatant, the hyaluronidase was absorbed to phenylsepharose at an ammonium sulphate concentration of 40%.95-98% of absorbed hyaluronidase was bound to phenyl sepharose under these conditions and eluted by an ammonium sulphate concentration of 30%. The specific activities of this enzyme were 5230-6100 u/mg. Further purification was achieved by rechromatography on phenyl sepharose followed by gel filtration on Superdex G 200. On this way, the specific activities increased to 40000 u/m!. Finally, isoelectric focusing yielded homogeneous preparations of highly purified hyaluronidase with a specific activity of 81400-83 000 u/mg, an isoelectric point of pH 8.75 (Fig. 1) and a molecular weight of 116000 D (Fig. 2). The purified enzyme exhibited maximal activity at pH 6.3

A

B

Fig. 1. Isoelectric focusing of purified hyaluronidase from GBS type strains B 3402 and B4755 on polyacrylamide gel with pH gradient from pH 4.0 to 9.0 (A,A': purified hyaluronidase from GBS strain B 3402, 0.5 mg/ml; 0.1 mg/ml B,B': purified hyaluronidase from GBS strain B 4755, 0.2 mg/ml; 1 mg/ml).

Hyaluronidase from GBS Strains

501

Fig. 2. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis of GBS hyaluronidase. As standards (ST) served myosin, 212 000; az-macroglobulin, 170 000; ~-galac­ tosidase, 116000; transferrin, 76000; glutamic-dehydrogenase, 53000 (A,A': purified hyaluronidase from GBS strain B 3402 B, B': purified hyaluronidase from GBS strain B 4755).

(Fig. 3) and 40°C (Fig.4). The Michaelis constant was estimated to be 8.19 X 10-2 mg! m!. Hyaluronidase activity increased in the presence of Mg + +, was not influenced by K+, Ca++ and Co++ and decreased in the presence of Cu++, Mn++, Zn++, AI+++ while the strongest inhibition was found with Fe++ (Fig. 5). The activity was also inhibited by synthetic polyanionic inhibitors from gentisic acid and hydroquinone sulphonic acid with formaldehyde (Fig. 6). The investigation of substrate specificity showed that the hyaluronidase from GBS type strains also splitted dermatan sulphate and chondroitin sulphate A, however, to a low extent (Fig. 7). Purified hyaluronidases from GBS-strains B 3402 (type II) and B 4755 (non-typablel R) were identical in isoelectric points, molecular weights and other properties.

502

J.-H.Ozegowski, E. Gunther, and W. Reichardt 1,2

relative activity

0,8

0,6

0,4 0,2

oL-~~~~-L~~~~~L-~~~~-L~-L~~~L-~

3,5

4

4,5

5

5,5

6

6,5 7 pH

7,5

8

8,5

9

9,5

10

Fig. 3. pH dependence of hyaluronidase activity from GBS type strains.

relative activity

I 0,6

r

/

\

0,4 0,2

o~~----.J~

25

30

35

40 45 50 temperature ("C)

55

60

Fig. 4. Influence of temperature on the hyaluronidase activity from GBS strains.

65

Hyaluronidase from GBS Strains

503

relative activity

4

3

2

o

Orig.

K+

Ca++

Mg++

Co++

Cu++

Mn++

Fe++

Zn++

AI+++

metal ions

Fig. 5. Influence of metal ions at a concentration of 10 mMlL on the hyaluronidase activity from GBS type strains.

activity

100%·+---~-------------------------------------------

80%

60%

\

\

\

40%

20%

oj

I

1,000E-04

I

!

~

\

I11111

~

I

1,000E-03

I

I

0,01 0,1 polyanionic inhibitor (mg/ml)

10

Fig. 6. Inhibition of GBS hyaluronidase by synthetic polyanionic inhibitors. (+) polymer from gentisic acid with formaldehyde (Q) polymer from hydroxysulphonic acid with formaldehyde.

504

J.-H.Ozegowski, E. Giinther, and W. Reichardt split 100%

80%

60%

40%

20%

O%JL==== hyaluronic acid

dermatan sulphate substrate

chondroitin sulphate

Fig. 7. Depolymerization of mucopolysaccharide (hyaluronic acid, dermatan sulphate and chondroitin sulphate A) by GBS hyaluronidase.

Discussion Concerning GBS, until now only the publications of Gochnauer et al. (10) and Seller (29) exist, dealing with partial characterization of hyaluronidase in culture supernatants. The results must be compared with the hyaluronidases of other streptococcal serological groups. Hyaluronidase could be isolated by applying hydr~phobic phenylsepharose chromatography, gel filtration and isoelectric focusing. In the first purification step, the proteins were bound to phenylsepharose in a hydrophobic mode at a final ammonium sulphate concentration of 40%. By applying this procedure, the highest content of hyaluronidase was obtained. Precipitation with an ammonium sulphate concentration of 60%, which has been used by many authors (7, 12,31) produced only very low yields. Probably, the precipitation of the first step from culture supernatant solution was disturbed by medium components, in further purification steps, concentration with ammonium sulphate precipitation was possible without difficulties. The isoelectric point of purified hyaluronidase was determined to be pH 8.75. Considering this IP, the hyaluronidase of GBS represents an alkaline protein contrary to hyaluronidases from other serological groups (8, 12,21,31), isoelectric points of which were estimated to be in the acidic pH-range. Similar to hyaluronidases from S. pyogenes (8), S. dysgalactiae and S. equi (31), or S. mitis (20), this enzyme has a low temperature stability. At temperatures above 40°C, the activity decreases very quickly. Gochnauer et al. (10) found a somewhat higher stability of hyaluronidase from GBS, possibly as an effect of medium components, whereas the pH optimum was in accordance at pH 6.3. The molecular weight was about 116000 D, which is higher than the formerly described values of hyaluronidases from group A streptococci at 50000 D

Hyaluronidase from GBS Strains

505

(12) or 75000 D (8) and from S. equi, S. zooepidemicus or S. dysgalactiae of group C at 55000 (31), but similar to the hyaluronidase from S. equisimilis (also group C) at 110000 D (21). The Michaelis constant (Km) of hyaluronidase from GBS type strains at 8.19 X 10-2 mglml was similar to the Km-values found in group C streptococci (31) and Streptococcus uberis (28). In contrast to the hyaluronidases of the two-last mentioned streptococcal species, the activity was only stimulated in the presence of Mg++, but reduced in the presence of Zn++, Cu++, Fe++ and Al++. Even polyanionic inhibitors on the basis of polymers between gentisic acid and formaldehyde or hydroxysulphonic acid and formaldehyde inhibited the hyaluronidase activity of GBS type strains. The ability of GBS hyaluronidase, in contrast to hyaluronidases from other streptococcal serological groups (18), to split also dermatan sulphate and chondroitin sulphate A which represent further structural elements of human tissue indicates, that GBS hyaluronidase probably can be considered as a major pathogenic factor of this group. Because of the rather low specificity of GBS hyaluronidase, it enables group B streptococci to destroy different tissue structures and thus facilitates invasion.

Acknowledgements. This work was supported by grants of Deutsche Forschungsgemeinschaft, Oz 511-1 and Bundeszuwendung 1993 ftir das Nationale Referenzzentrum fur Streptokokken des Bundesministeriums ftir Gesundheit. The authors are grateful to Dr. D. Gerlach for assistance in FPLC chromatography and to Mrs. Karin Schohknecht and Mrs. Sabine Urban for excellent technical assistance.

References

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Dr. J.-H. Ozegowski, Institut fur Experimentelle Mikrobiologie, Winzerlaer Str. 10, D-07745 Jena