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J Biol Chem, Vol. 273, Issue 37, 23668-23673, September 11, 1998


An Ectoprotein Kinase of Group C Streptococci Binds Hyaluronan and Regulates Capsule Formation*

Volker Nickel, Sabine Prehm, Manfred Lansing, Andreas Mausolf, Andreas PodbielskiDagger , Josef Deutscher§, and Peter Prehm

From the Institut für Physiologische Chemie und Pathobiochemie, Waldeyerstr. 15, D-48129 Münster, Germany, Dagger  Institut für Medizinische Mikrobiologie und Immunologie, Robert-Koch Str. 8, D-89081 Ulm, Germany, and § Institut de Biologie et Chime des Protéines-CNRS, 7-Passage Du Vercors, F-9367 Lyon, France

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDS-polyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [32P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [32P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [32P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Group A and C streptococci are pathogens capable of causing a variety of infections. Group A streptococci are known to initiate postinfectious sequelae in humans such as acute rheumatic fever and glomerulonephritis. Group C streptococci are primarily animal pathogens. Many strains of both groups A and C streptococci are able to surround themselves with a hyaluronan capsule that has been implicated as a major virulence factor (1-5). Early attempts to clone the hyaluronan synthase from group C streptococci led to the erroneous identification of a 56-kDa protein (6, 7). The identification was based on indirect evidence, since synthase activity could not be reconstituted. This protein was affinity-labeled with the periodate-oxidized nucleotide sugars UDP-GlcNac and UDP-glucuronic acid, and it bound to nascent hyaluronan. Binding of protoplast membrane proteins to nascent hyaluronan was used to develop a new method that extracted the enzyme activity together with two proteins of 42 and 56 kDa (8). Final purification of the synthase yielded the 42-kDa protein in active and electrophoretically homogeneous form. DeAngelis et al. (9-11) and Dougherty and van de Rijn (12) proved by genetic deletion analysis that the 42-kDa protein was the streptococcal hyaluronan synthase. Therefore the function of the 56-kDa proteins was reexamined. Its amino acid sequence contained an ATP binding domain and indicated homology to bacterial transport proteins and several hyaluronan binding sites (7). It had an N-terminal signal sequence that could integrate it into protoplast membranes. The 56-kDa protein was processed to a 54-kDa protein by endogenous proteases and shed into the culture medium (13). This protein also elicited antibodies in patients with rheumatic fever and cross-reacted with surface protein form eukaryotic cells (14). The cross-reacting eukaryotic protein had a molecular mass of 52 kDa and formed a complex with the hyaluronan receptor RHAMM (15). In this publication we showed that the 56-kDa protein from group C streptococci is an extracellular hyaluronan-binding protein that has a threonine kinase activity and regulates hyaluronan capsule formation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Group C Streptococcus equisimilis (strain D181) was obtained from the Rockefeller University collection; other streptococcal strains were received from the culture collection of Dr. Wagner, University of Jena, Germany. Radiochemicals were purchased from Amersham Pharmacia Biotech, and other reagents were from Sigma.

General Methods-- The S. equisimilis strain D181 was grown in Hewitt-Todd medium, and protoplast membranes were prepared as described (7). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide under reducing conditions. Phosphoamino acids were analyzed by the method of Cooper et al. (16). The relative molecular weight of the kinase was determined on polyacrylamide gels by the method of Geahlen et al. (17). Phosphorylation of the phosphate carrier protein Hpr was performed as described previously (18).

Measurement of Hyaluronan Synthesis and Release-- Protoplast membranes were prepared as described by Prehm et al. (8) and suspended in 50 mM Tris-malonate, pH 7.0, at a concentration of 2 mg/ml. In some experiments the 100 µg of membranes were dephosphorylated with 2 units of alkaline phosphatase (Boehringer Mannheim) in 50 µl of 50 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, for 1 h at 37 °C and recovered by ultracentrifugation in an Airfuge (Beckman) at 100,000 × g for 30 min. Controls were incubated similarly without alkaline phosphatase. Aliquots of 200 µl were mixed at 37 °C with 200 µl of a solution of 160 µM UDP-GlcNac and 8 µM UDP-[14C]glucuronic acid (specific activity, 320 mCi/mmol), 1 mM dithiothreitol, 10 mM MgCl2 and incubated at 37 °C. At various time points, aliquots of 50 µl were withdrawn, and 5 µl of a solution of 0.1 M HgCl2 was added to stop hyaluronan synthesis. The solution was chilled in ice water and subjected to ultracentrifugation in an Airfuge (Beckman) for 30 min at 100,000 × g at 0 °C. The sediment was dissolved in 50 µl of 1% SDS. A solution of 10% SDS was added to the supernatant to inactivate any synthase activity. The dissolved sediment and the supernatant were applied to descending paper chromatography on Whatman MM paper for 18 h in 1 M ammonium acetate, pH 5.5, ethanol (13:7). The origin was cut out, and the radioactivity was determined.

For determining the effect of antibodies against the 56-kDa protein, 1 ml of protoplast membranes in 50 mM Tris-malonate, pH 7.0, at a concentration of 2 mg/ml were incubated with 50 µl of polyclonal rabbit antiserum against the 56-kDa protein or with preimmune serum for 30 min at 4 °C and centrifuged at 100,000 × g for 30 min. The sediment was suspended in 1 ml of 50 mM Tris-malonate, pH 7.0, and the kinetics of hyaluronan synthesis were measured as described above.

Determination of Hyaluronan Concentration on Bacteria and in the Medium-- Streptococci were grown in 10 ml of Hewitt-Todd medium in the presence or absence of ATP, alkaline phosphatase (1 unit/ml), the cysteine protease inhibitor e64 (0.1 mg/ml) or proteinase E (Pronase) (0.1 mg/ml) to A600 = 0.6 and harvested by centrifugation at 10,000 × g for 15 min. To dissociate the hyaluronan capsule, the pellet was resuspended in 10 ml of phosphate-buffered saline, 0.1% Triton X-100 and shaken for 1 h at room temperature. Insoluble material was removed by centrifugation at 10,000 × g for 15 min. A solution of 10% cetylpyridinium chloride was added to the supernatant to give a final concentration of 0.5% and incubated at 37 °C for 1 h. The precipitate was sedimented at 3,000 × g for 30 min and dissolved in 3 ml of 0.5 M NaCl by ultra sonication. Hyaluronan was again precipitated from the solution by the addition of 9 ml of 95% ethanol containing 1.3% potassium acetate. The precipitate was pelleted by centrifugation at 3,000 × g for 30 min and dissolved in 1 ml of water. Aliquots were used for glucuronic acid determination (19), and the amount of hyaluronan was calculated. The culture supernatant of the bacteria was diluted with 5 ml of water, and hyaluronan was precipitated by the addition of cetylpyridinium chloride and determined as described above.

Phosphorylation of Streptococcal Membranes-- Protoplast membranes (100 µg) were incubated in 50 mM Tris-malonate, pH 7.0, with 10 µCi [32P]ATP (specific activity 1000 Ci/mmol), 10 mM MgCl2 for 10 min at room temperature. The proteins were separated on a 10% polyacrylamide gel, and radioactive bands were visualized by autoradiography.

Phosphorylation of Escherichia coli Lysates-- Exponentially growing cultures of E. coli Y1090 were infected with lambda gt 11 or lambda gt 11/2LK that contained the recombinant 56-kDa gene. After cell lysis, the debris were sedimented at 3,000 × g for 10 min, and the supernatants were centrifuged at 100,000 × g for 30 min. The pellets were resuspended in 50 mM Tris-malonate buffer, pH 7.0, at a protein concentration of 5 mg/ml. These suspensions (100 µg) were incubated with [32P]ATP for 15 min and analyzed as described above for phosphorylation of streptococcal membranes.

For immunoprecipitation, the suspension of membranes (100 µl) was supplemented with 10 µl of 10% Triton X-100 and incubated for 1 h at 0 °C. The suspension was centrifuged in an Airfuge at 100,000 × g for 30 min. The supernatant (20 µl) was incubated with 20 µl of polyclonal rabbit antiserum against the 56-kDa protein or preimmune serum and 20 µl of a 1:1 suspension of protein-A-Sepharose for 1 h. The protein-A-Sepharose beads were sedimented by centrifugation for 1 min at 10,000 × g and washed with 1 ml each of phosphate-buffered saline, 1% Triton X-100, and 1 M NaCl. The sediment was resuspended and incubated in 100 µl of 50 mM Tris-malonate, pH 7.0, with 10 µCi of [32P]ATP, 10 mM MgCl2 for 10 min at room temperature. The proteins were separated on a 10% polyacrylamide gel, and radioactive bands were visualized by autoradiography.

Identification of the 56-kDa Protein in Bacterial Colonies-- Streptococci were grown on Todd-Hewitt agar plates that contained 1 mg/ml of testis hyaluronidase (specific activity, 440 units/mg), and replicas on nitrocellulose filters were prepared. After blocking with a solution of 3% bovine serum albumin in phosphate-buffered saline, the filters were incubated with a 1:1000 dilution of antiserum against the 56-kDa protein in blocking solution for 1 h. The filters were washed with phosphate-buffered saline and developed with anti-rabbit-IgG alkaline phosphatase conjugate as described (20).

Extracellular Phosphorylation of Streptococci-- An overnight culture of group C streptococci (strain D181) in Todd-Hewitt medium (400 ml) was diluted with prewarmed Todd-Hewitt medium to a final A600 = 0.5 and incubated with 10 mg of testis hyaluronidase (specific activity, 440 units/mg) for 30 min at 37 °C. Bacteria were sedimented by centrifugation for 15 min at 10,000 × g and resuspended in 40 ml of prewarmed Todd-Hewitt medium containing 4 mM MgCl2. Aliquots of 4 ml were incubated with 200 µl of [32P]phosphate (specific activity 10 mCi/ml) or with 200 µl of [gamma -32P]ATP (specific activity, 10 mCi/ml) in the absence and presence of 160 units of the extracellular adenosine 5'-triphosphatase apyrase. Aliquots of 1 ml were withdrawn after 5 and 15 min and centrifuged at 10,000 × g for 1 min. The bacterial sediments were frozen until further analysis. The samples were resuspended in sample buffer and subjected to gel electrophoresis on 10% SDS-polyacrylamide. Radioactivity was visualized by autoradiography.

Inhibition of phosphorylation by extracellular trypsin was determined by incubation of 3 ml of the above bacterial suspension with 30 µCi of [gamma -32P]ATP in the presence of 0.005% trypsin for 15 min at 37 °C. Phosphorylation was visualized as described above.

Phosphorylation of extracellular casein by growing streptococci was determined by incubating 3 ml of the above bacterial suspension with 30 µCi of [gamma -32P]ATP in the presence of 0, 1, 10, and 100 µg of casein for 15 min at 37 °C. Bacteria were sedimented by centrifugation for 5 min at 10,000 × g, proteins in the culture media were precipitated by the method of Wessel and Flügge (21) and separated by gel electrophoresis on 10% SDS-polyacrylamide, and radioactivity was visualized by autoradiography.

Adsorption of Radioactive Hyaluronan to the Recombinant 56-kDa Protein-- Radioactive hyaluronan was prepared by incubating 150 µg of streptococcal membranes at 37 °C with 500 µl of a solution of 160 µM UDP-GlcNac and 8 µM UDP-[14C]glucuronic acid (specific activity 320 mCi/mmol), 1 mM dithiothreitol, 10 mM MgCl2 for 2 h. Proteins were denatured by heating for 5 min to 100 °C, and the solution was clarified by centrifugation for 5 min at 14,000 × g. The supernatant was dialyzed against water and used for adsorption with E. coli lysates.

After cell lysis of E. coli Y1090 lysed by growth of lambda gt 11 or lambda gt 11/2LK, the debris were sedimented at 3,000 × g for 10 min, and the supernatants were centrifuged at 100,000 × g for 30 min. The pellets were resuspended in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.0, at a concentration of 3 mg/ml, and 100 µl were mixed with 5 µl of radioactive hyaluronan and 10 µl of 10 mM ATP or 10 µl of water and incubated at 37 °C for 10 min. The particular fractions were sedimented by ultracentrifugation in an Airfuge (Beckman) for 1 h at 100,000 × g. The supernatants were removed, the pellets were dissolved in 100 µl of 1% SDS, and the radioactive counts were determined.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the Kinase-- Streptococcal membranes were separated on a SDS-polyacrylamide gel that contained casein as kinase substrate and tested for a kinase activity. After electrophoresis, proteins were reconstituted, and the gel was incubated with gamma -(32P)ATP. The radioactive substrate was washed out, and the gel was autoradiographed. One band was visualized with a molecular mass of 56 kDa (Fig. 1).


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  		Fig. 1.   Detection of kinase activity in streptococcal membranes. Streptococcal membrane proteins were separated on a 10% SDS-polyacrylamide gel containing casein as substrate for phosphorylation. After renaturation of proteins, the gel was incubated with [gamma -32P]ATP. The gel was washed to remove nonincorporated radioactivity and subjected to autoradiography as described under "Experimental Procedures."

The recombinant 56-kDa protein was expressed also in E. coli and analyzed for kinase activity. Membranes isolated from E. coli lambda gt-11 and E. coli lambda gt-11/2LK lysates were incubated with [gamma -32P]ATP, and the amount of radioactivity incorpororated into proteins was determined to be 3916 cpm/µg for normal and 5781 cpm/µg for recombinant cells. After SDS-polyacrylamide gel electrophoresis, the pattern of phosphorylated proteins was compared. Fig. 2 shows that both lysates contained phosphorylated proteins. However, the E. coli lambda gt-11/2LK lysate containing the cloned 56-kDa protein showed at least two additional phosphorylated proteins with 56 and 30 kDa. Antibodies against the 56-kDa protein were used for immunoprecipitation of the 56-kDa protein from a detergent extract of the particular fraction from an E. coli lambda gt-11/2LK lysate. The antiserum has been shown to react specifically with the 56-kDa protein in Western blots from membranes of the streptococcal strain D181 and recombinant E. coli lambda gt-11/2LK lysates (7). The immunoprecipitate was incubated with [gamma -32P]ATP, and the phosphorylated proteins were analyzed. Fig. 2 (lane C) shows that the recombinant 56-kDa protein was phosphorylated in the immunoprecipitate. This protein was also immunoprecipitated from a detergent extract of 32P-labeled membranes (data not shown). An immunoprecipitate from a control lysate of E. coli lacking the recombinant 56-kDa protein with antiserum against the 56-kDa protein or an immunoprecipitate from the recombinant E. coli lysate with preimmune serum did not yield any phosphorylated proteins (data not shown). These results indicated that the 56-kDa protein could be autophosphorylated.


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  		Fig. 2.   Kinase activity of the recombinant 56-kDa protein. Particular fractions from E. coli lysed with lambda gt11 (A) or lambda gt11/2LK (B) were incubated with [gamma -32P]ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described under "Experimental Procedures." An aliquot of the particular fraction from E. coli lysed with lambda gt11/2LK was immunoprecipitated with polyclonal rabbit antiserum against the 56-kDa protein (C), incubated with [gamma -32P]ATP, and analyzed as above.

Previously, a membrane-bound kinase of the Hpr system has been described in streptococci that was involved in sugar transport (18). Therefore, we tested whether the 56-kDa protein was able to phosphorylate the phosphate carrier protein Hpr. An E. coli lysate containing the active recombinant 56-kDa kinase did not phosphorylate the streptococcal Hpr protein (data not shown).

Hyaluronan Binding Activity in Recombinant 56-kDa Protein-- The 56-kDa protein contains several hyaluronan binding motifs (7) that are characterized by BX7B ,where B is a basic amino acid, and X is any other amino acid (22). It was also extracted together with the hyaluronan synthase from streptococcal membranes by a recently developed procedure that enriched only hyaluronan-binding membrane proteins (8). We therefore examined whether the hyaluronan binding activity was retained by the recombinant protein expressed in E. coli. E. coli lysates were prepared from lambda gt-11/2LK and lambda gt-11-control phages. Membranes were prepared by sedimentation in the ultracentrifuge. The membranes were incubated in the absence and presence of 1 mM ATP with radioactive hyaluronan to analyze the possibility that hyaluronan binding was influenced by phosphorylation. The membranes were again sedimented by ultracentrifugation, and the radioactivity in the membrane pellet was determined. Fig. 3 shows that the lysate containing recombinant 56-kDa protein bound hyaluronan only in the absence of ATP.


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  		Fig. 3.   Binding of hyaluronan by recombinant 56-kDa protein. Particular fractions from E. coli lysed with lambda gt11 (C and D) or lambda gt11/2LK (A and B) were incubated with [14C]hyaluronan in the absence (B and D) and presence (A and C) of 1 mM ATP for 10 min at 37 °C and sedimented by ultracentrifugation as described under "Experimental Procedures." The supernatant was removed, and the radioactivity in the sediments were determined. The error bars indicate the S.D. of triplicate samples (p value, 0.013).

Kinetics of Hyaluronan Synthesis and Release-- The kinetics of hyaluronan synthesis and release was measured in the presence and absence of ATP. Fig. 4 shows that hyaluronan is synthesized at membranes and then released into the soluble fraction. Hyaluronan release ceased after 60 min in the absence of ATP. In the presence of ATP, membranes continued to release hyaluronan into the soluble fraction.


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  		Fig. 4.   Stimulation of hyaluronan synthesis and release by ATP. Streptococcal membranes were incubated in the absence (, black-square) and presence (bullet , open circle ) of 1 mM ATP with radioactive substrates for hyaluronan synthesis. At the time points indicated, aliquots were withdrawn and subjected to ultracentrifugation to separate membrane-bound hyaluronan (black-square, bullet ) from released hyaluronan (, open circle ), and the amount of labeled hyaluronan was determined as described under "Experimental Procedures." The error bars indicate the S.D. of triplicate samples (p value for the difference of the supernatants at 120 and 240 min, 0.013).

Attempts to delete the 56-kDa kinase by homologous recombination were not successful, suggesting that it plays a critical, nonredundant role in the cell. Therefore specific antibodies were utilized to verify its influence on hyaluronan synthesis. The kinetics of hyaluronan synthesis and release was measured before and after binding of antibodies to streptococcal membranes. Fig. 5 shows that antibodies to the 56-kDa protein increased the rate of hyaluronan release from membranes into the supernatant.


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  		Fig. 5.   Influence of anti-56-kDa protein antibodies on the kinetics of hyaluronan synthesis and release. Streptococcal protoplast membranes (5 mg in 5 ml of phosphate-buffered saline) were incubated with 0.5 ml of preimmune serum or with antiserum against the 56-kDa protein for 24 h at 4 °C and reisolated by ultracentrifugation. The kinetics of hyaluronan synthesis and release were determined as described under "Experimental Procedures." open circle , preimmune serum, bullet , anti-56 protein, ---, membrane-bound, - - -supernatant. The error bars indicate the S.D. of triplicate samples (p value for the difference at 24 h, 0.05).

Extracellular Localization and Strain Specificity of the 56-kDa Protein-- The following experiments should clarify the cellular localization and strain specificity of the 56-kDa protein. Bacterial colonies on agar plates containing hyaluronidase were overlaid with nitrocellulose filters to produce replicates. The filters were developed with polyclonal rabbit antibodies against the 56-kDa protein and anti-rabbit peroxidase. Group C streptococci (15 strains) and group A streptococci (18 strains) were tested. Positive signals were obtained from all group C streptococci but not from group A streptococci (data not shown). When protoplast membranes were prepared from different strains and analyzed by Western blotting with antiserum against the 56-kDa protein, the 56-kDa protein could again be detected only in membranes from group C streptococci but not in protoplast membranes form group A streptococci. Furthermore it was impossible with polymerase chain reaction and southern hybridizations at low stringency to identify the homologous gene in group A streptococci.

For extracellular localization of the kinase, intact group C streptococci were incubated with [32P]phosphate or [32P]ATP for various periods, and phosphorylation was followed by polyacrylamide gel electrophoresis and autoradiography (Fig. 6) in the presence and absence of adenosine 5'-triphosphatase. Two proteins with molecular masses of 56 and 75 kDa were phosphorylated only by extracellular ATP after 5 and 15 min in the absence of adenosine 5'-triphosphatase. Longer incubation times did not yield labeled proteins, indicating that phosphorylation was transient and susceptible to endogenous surface phosphatases. Neither extracellular [32P]phosphate that could be taken up by bacteria nor [32P]ATP in the presence of adenosine 5'-triphosphatase led to phosphorylation, excluding the possibility that intracellular [32P]phosphate caused the phosphorylation. Low concentrations of extracellular trypsin eliminated phosphorylation. Extracellular casein was also a target of the kinase (Fig. 6).


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  		Fig. 6.   Extracellular phosphorylation. Whole streptococci (strain D181) were incubated with [32P]phosphate (P) or [32P]ATP for 5 and 15 min in the presence and absence of adenosine 5'-triphosphatase or with increasing concentrations of casein or with trypsin. Phosphorylated proteins were separated by polyacrylamide gel electrophoresis and visualized by autoradiography as described under "Experimental Procedures."

When membranes were labeled for 15 min and chased with unlabeled ATP for 15 min, the 56-kDa protein disappeared and gave rise to a 36-kDa protein (data not shown), suggesting that the 56-kDa protein was rapidly degraded. The radioactive proteins were eluted from the gel and subjected to phosphoamino acid analysis. The phosphorylated amino acid was threonine (Fig. 7).


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  		Fig. 7.   Phosphoamino acid analysis. The 32P-labeled 56-kDa protein was eluted and subjected to phosphoamino acid analysis as described under "Experimental Procedures." P-, phospho-.

Extracellular ATP Decreases Capsule Formation and Increases Hyaluronan Production-- The influence of the ectoprotein kinase and extracellular ATP on the hyaluronan synthesis and shedding was investigated on growing cultures of group C and group A streptococci. Increasing concentrations of ATP were added to group C streptococcal strain D181. The bacteria were separated from the culture medium, and the amount of hyaluronan on bacteria and in the medium was determined (Fig. 8). ATP reduced the amount of hyaluronan on bacteria and increased the concentration in the culture medium. This led to an overall increase in hyaluronan synthesis. Treatment with alkaline phosphatase decreased the amount of hyaluronan in the medium (Table I). In comparison, group A streptococcal strain 36487 released most of its hyaluronan capsule into the medium also in the absence of ATP, and alkaline phosphatase reduced the amount of hyaluronan in the medium only slightly. Treatment of group C streptococci with proteinase E led to an increased shedding of hyaluronan into the culture medium, whereas a cysteine proteinase inhibitor showed only marginal effects. Despite the differences in hyaluronan capsule between group A and group C streptococci, the macroscopic appearance of colonies on agar plates were mucoid with both strains.


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  		Fig. 8.   Stimulation of hyaluronan synthesis and release by extracellular ATP. Streptococci (strain D181) were grown to A600 = 0.6 in the presence of increasing concentrations of ATP. The amount of hyaluronan on the bacterial sediment (bullet ) and in the culture supernatant (open circle ) was determined as described under "Experimental Procedures." , total hyaluronan.

                              
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Table I
Amount of hyaluronan (µg/ml) in the capsule and in the culture supernatant of 10-ml cultures of selected streptococci at an A600 = 0.6 

Because an ectoprotein kinase was found to be involved in growth regulation of fibroblasts (23), we measured the effect of increasing ATP concentrations on the growth rate of group A and group C streptococci. Extracellular ATP concentrations above 10 mM had only slight growth inhibitory effects.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In previous publications we reported cloning of a 56-kDa protein from protoplast membranes of group C streptococci that was erroneously characterized as the hyaluronan synthase (6, 7). Here we have reexamined the function of this protein that bound hyaluronan (8) and exhibited sequence similarity with ATP-binding transport proteins (7). We show that a protein from streptococcal membranes with a molecular mass of 56 kDa possessed a protein kinase activity that was also retained when the protein was expressed in E. coli. The recombinant 56-kDa protein could be autophosphorylated in an immunoprecipitate. The kinase was not identical to the previously identified streptococcal Hpr kinase of the sugar transport system (18). The 56-kDa protein and its gene was only detected in group C streptococci but not in group A streptococci. In streptococcal membranes, the kinase activity was directed against a 75-kDa and the 56-kDa membrane proteins. The function of the 75-kDa protein phosphorylation remains elusive, but it is conceivable that it mediates transduction of other signals in response to extracellular ATP.

The localization of the 56-kDa kinase was investigated by the kinetics of phosphorylation with exogenous [32P]phosphate and [32P]ATP of intact growing bacteria. Rapid phosphorylation was only observed with [32P]ATP and was abolished by extracellular trypsin. Extracellular casein could also serve as a substrate. To our knowledge this is the first demonstration of an ectoprotein kinase in prokaryotes, although there are several examples of eukaryotic ectoprotein kinases that mediate signal transfer from extracellular ATP into the cell (23-29).

In addition to the kinase activity, a hyaluronan binding activity could be demonstrated in recombinant E. coli lysates that bound exogenous hyaluronan only in the absence of ATP, suggesting that hyaluronan binding could be influenced by autophosphorylation. Thus, extracellular ATP influenced the kinetics of hyaluronan synthesis and release from isolated streptococcal protoplast membranes. Membranes continued to release hyaluronan in the presence of ATP but stopped this process after 60 min in its absence. Antibodies against the 56-kDa protein also increased the shedding rate of hyaluronan from membranes.

Extracellular ATP at low millimolar concentrations stimulated hyaluronan synthesis and shedding from growing group C streptococci but not in group A streptococci. In the infected host, extracellular ATP can be released from attacking neutrophils or degranulated platelets (30). The effective ATP concentrations seem to be physiological, because they were slightly below the intracellular concentration that has been measured to be about 6 mM (31). Extracellular phosphatase reduced and extracellular proteinase increased hyaluronan shedding rate. These processes were only slightly influenced by an inhibitor of the extracellular cysteine proteinase that was shown to contribute to the virulence of group A streptococci (32). Nevertheless, it is possible that other proteases participate in regulation of capsule formation.

Van de Rijn has shown that the hyaluronan capsule is lost during the stationary phase (33). In addition we demonstrated here that group C streptococci, but not group A streptococci, regulate their capsule in response to extracellular ATP during the growth phase. The differences in hyaluronan synthesis between group A and group C streptococci may be a reflection of different host specificity. Retention of a larger hyaluronan capsule by group C streptococci in skin surfaces may be more advantageous for their survival in preventing desiccation. Invasion into necrotic host tissue would expose them to host defense that calls for rapid shedding of surface hyaluronan together with adhered antibodies or host cells. Thus the 56-kDa ectoprotein kinase may contribute to the virulence of group C streptococci.

Our results also showed that binding of nascent hyaluronan by a cell surface receptor did not only inhibit hyaluronan release but also hyaluronan synthase activity in isolated membranes and hyaluronan synthesis in whole cells. This finding may be a reflection of a novel mechanism for the regulation of polymer biosynthesis. It appears that chain initiation or elongation is suppressed if dissociation of nascent hyaluronan from the synthase into the medium is inhibited by cell surface receptors. This mechanism may also apply for hyaluronan synthesis in eukaryotic cells that could be influenced by cell surface receptors such as CD44 or RHAMM.

    ACKNOWLEDGEMENTS

We thank A. Blanke, G. Reinhold, and U. Rasmussen for excellent technical assistance and Dr. Wagner for group A and C streptococcal strains.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB310 and Po391/6-1).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-251-8355579; Fax: 49-251-8355596; E-mail: prehm{at}uni-muenster.de.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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