Institute of Dental Research, United Dental Hospital, Surry Hills, NSW 2010, Australia1
Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans, LA, USA2
Author for correspondence: Derek W. S. Harty. Tel: +61 2 9293 3348. Fax: +61 2 9293 3368. e-mail: derekh{at}dentistry.usyd.edu.au
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ABSTRACT |
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Keywords: Infective endocarditis, Streptococcus gordonii, pH, enzymes
Abbreviations: 7-AMC, 7-amido-4-methylcoumarin; Ca, activated protein C; CDM, chemically defined medium; FBS, foetal bovine serum; IE, infective endocarditis; 4-MU, 4-methyl-umbelliferyl; PAS, periodic acid/Schiff; Xa, activated protein X
a Present address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
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INTRODUCTION |
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Several studies have examined the enzymes produced by streptococci that enable them to metabolize complex molecules, mainly concentrating on the model glycoprotein, pig gastric mucin. Homer et al. (1996) examined the ability of Streptococcus oralis to synthesize sialidase and N-acetylglucosaminidase, enzymes important in the metabolism of glycoproteins, and found that both were induced when grown in the presence of pig gastric mucin (Rafay et al., 1996
). It has also been observed that growth in pig gastric mucin leads to higher levels of bacterial glycosidase activity, whilst some protease activities are repressed. Conversely, the presence of glucose represses glycosidase activity (Rafay et al., 1996
; Beighton et al., 1995
). Mayo et al. (1995)
showed that S. gordonii FSS2 grown in a complex medium produced a variety of glycosidase and peptidase enzymes which were modulated by both growth rate and environmental pH. Enzyme activities were generally higher at pH 6·5 than at 5·5 or 7·5. The exception was thrombin-like activity which was found to be fivefold higher at more neutral pH than under more acidic conditions. Another activity, Hageman factor, was apparently subject to catabolite repression by glucose, as activity was threefold higher in cultures grown on galactose compared with those grown on glucose. Enzymes important for catabolism in S. gordonii FSS2 therefore seem to be regulated by environmental conditions.
The shift from a low oral pH to blood pH has been shown in S. gordonii to induce or up-regulate the expression of several genes including one, msrA, which is involved in protection against oxidative stress and enhanced bacterial growth (Vriesema et al., 2000 ). In this work we investigate the effects of environmental pH and carbon source on the expression of cell-associated and secreted enzymes of S. gordonii grown in a fully defined medium.
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METHODS |
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Culture media.
S. gordonii FSS2 was stored as brain-heart infusion broth (Oxoid)/20% (v/v) glycerol stocks at -70 °C and was routinely grown on Columbia blood agar plates (Oxoid) with 5% (v/v) horse blood. Batch cultures were grown on a filter-sterilized (0·2 µm pore; Sartorius) chemically defined medium (CDM) modified from that described by Terleckyj et al. (1975) and Wittenberger et al. (1978)
, containing (mg l-1 unless stated otherwise): trisodium citrate, 250; NH4SO4, 600; CaCl2 . 2H2O, 20; MnSO4 . 4H2O, 11; MgSO4 . 7H2O, 700; FeSO4 . 7H2O, 18; Na2MoO4 . 2H2O, 0·15; NaCl, 2000; NaHCO3, 500; adenine, 20; guanine, 20; uracil, 20; thiamin, 8; pyridoxamine, 2; calcium pantothenate, 8; riboflavin, 8; nicotinamide, 4; p-aminobenzoic acid, 0·4; biotin, 0·2; folic acid, 0·2; inositol, 2; thiamin, 2; L-glutamic acid, L-glycine and L-cysteine, 3 mM; L-arginine, L-lysine, L-isoleucine, L-leucine, L-methionine, L-tryptophan, L-tyrosine, L-histidine, L-proline, DL-alanine, L-aspartic acid, L-phenylalanine, L-asparagine, L-serine, L-threonine, L-valine and L-glutamine all 1 mM; 30 mM potassium phosphate buffer pH 7·5; 0·5% (w/v) glucose (27·8 mM). The medium was brought to pH 7·5 by the addition of 5 M KOH. All chemicals were of analytical grade and were purchased from either Sigma or Merck. In one series of experiments, 50% (v/v) foetal bovine serum (FBS; Commonwealth Serum Laboratories), heated at 80 °C for 30 min to inactivate serum-enzyme activities, was added to CDM devoid of glucose.
Growth conditions.
S. gordonii FSS2 was grown at 37 °C as a 100 ml stirred, pH-controlled batch culture gassed with 5% (v/v) CO2/95% (v/v) N2 at a rate of 50 ml min-1. The pH was controlled by automatic addition of a mixture of 2 M NaOH and 2 M KOH (to avoid changes in the sodium:potassium ratio of the medium during growth; Pitty & Jacques, 1989 ). Batch cultures were inoculated at 15% (v/v) from a 20 ml static batch culture grown for 18 h in CDM. In some experiments, CDM was modified to provide carbon-excess/nitrogen-limited growth conditions by removing the ammonium sulphate and adding one-third the concentration of amino acids (1 mM glutamic acid, glycine and cysteine; 0·33 mM all other amino acids). This resulted in residual glucose [0·93 mg ml-1 (5·2 mM)] being detected in the medium at stationary phase.
Bacteria were harvested by centrifugation (27000 g, 4 °C, 5 min), washed three times in 50 mM potassium phosphate buffer pH 7·5 containing 0·02% (w/v) sodium azide and resuspended to an OD600 1·00±0·05. All determinations of cell-associated protein concentration were made at OD600. Supernatants were filtered through 0·2 µm pore membranes (Sartorius) prior to assaying for enzyme activity. In some experiments, bacteria were resuspended in 50 mM citrate/phosphate buffer at pH 4·0, pH 5·0 or pH 6·0 and incubated at 37 °C for 2 h before being recentrifuged (27000 g, 4 °C, 5 min) and resuspended in 50 mM potassium phosphate buffer pH 7·5 prior to assaying for enzyme activities.
The ability of S. gordonii FSS2 to utilize FBS as a source of nutrients was determined in pH-controlled stirred cultures by the addition of an equal volume of heat-inactivated FBS to the defined medium devoid of glucose. After treatment, the only residual enzyme activities in FBS were thrombin [5·7 (4·17·4) nmol h-1 (100 ml culture)-1] and Hageman factor [9·8 (8·111·4) nmol h-1 (100 ml culture)-1] representing 0·2% and 0·02%, respectively, of the initial activities prior to heating at 80 °C. Bacterial growth was monitored spectrophotometrically at OD600 and by the number of viable cells in 1 ml culture samples after being sonicated (10 °C, 5 min, 130 W) in a cup horn (Branson model 450 sonifier, Branson Ultrasonics) to break up aggregates of bacteria before being diluted and plated on Columbia 5% (v/v) horse-blood agar.
A partial separation of the fibrous precipitate from streptococcal cells grown on FBS was achieved by a slow-speed centrifugation (500 g, 22 °C, 5 min). Examination by light microscopy of the pellet after centrifugation indicated that few bacterial cells were present.
Enzyme assays.
Four glycosidase and eight peptidase activities were measured using a range of synthetic substrates attached to the fluorogens, 4-methylumbelliferone or 7-amido-4-methylcoumarin (7-AMC), that were purchased from Sigma. The substrates (with the activity they detected shown in parentheses) were: 4-methylumbelliferyl (4-MU) -ß-D-glucoside (ß-D-glucosidase); 4-MU-ß-D-galactoside (ß-D-galactosidase); 4-MU-N-acetyl-ß-D-glucosaminide (N-acetyl-ß-D-glucosaminidase); 4-MU-N-acetyl-ß-D-galactosaminide (N-acetyl-ß-D-galactosaminidase); N-tert-butoxycarbonyl-Val-Pro-Arg-7-AMC (thrombin); N-tert-butoxycarbonyl-Leu-Ser-Thr-Arg-7-AMC [activated protein C (Ca)]; N-tert-carbobenzyloxy-Phe-Arg-7-AMC (kallikrein); N-tert-carbobenzyloxy-Lys-7-AMC (Hageman factor); N-tert-butoxycarbonyl-Ile-Glu-Gly-Arg-7-AMC [activated protein X (Xa)]; N-tert-butoxycarbonyl-Glu-Lys-Lys-7-AMC (plasmin); N-succinyl-Leu-Leu-Val-Tyr-7-AMC (chymotrypsin); and N-succinyl-Gly-Pro-Leu-Gly-Pro-7-AMC (collagenase) (Mayo et al., 1995 ; Oakey et al., 1995
). All substrates were dissolved in a minimal amount of dimethylsulphoxide and then diluted to 100 µg ml-1 in 50 mM potassium phosphate buffer pH 7·5 containing 0·02% (w/v) sodium azide. Enzyme assays were performed in duplicate at room temperature (1820 °C) using a Perkin-Elmer Luminescence spectrometer LS50-B with a microtitre plate assembly using white 96-well microtitre plates (Perkin-Elmer). The assay volume of 200 µl consisted of 50 µl substrate, 50 µl bacterial cells or 10 µl culture supernatant diluted to 150 µl with 50 mM potassium phosphate buffer pH 7·5. The controls contained 50 µl substrate and 150 µl 50 mM potassium phosphate buffer pH 7·5. Fluorescence readings were taken at 0, 10, 20, 30, 40, 50, 60 and 120 min and at 24 h. The maximum rate of release of fluorogen was determined and calculated from a standard curve of authentic 4-MU or 7-AMC and expressed as specific activity for cell-associated enzymes [nmol h-1 (mg protein)-1] or total activity for culture-supernatant activities [nmol-1 h-1 (100 ml culture)-1].
Enzyme synthesis.
To confirm that de novo enzyme synthesis was occurring, cells from a 20 ml CDM static batch culture containing 30 mM potassium phosphate buffer pH 7·5 and grown for 18 h were harvested as above and resuspended in 5 ml phosphate buffered saline. A portion of the resuspended cells was reserved to determine enzyme activities. Of the remaining cells, 1 ml was inoculated into duplicate pre-warmed CDM cultures containing 100 mM potassium phosphate buffer pH 7·5, or duplicate CDM cultures supplemented with 100 mM potassium phosphate buffer pH 7·5 and 50% (v/v) heat-inactivated FBS. Increasing the concentration of phosphate buffer raised the terminal pH of the culture. Chloramphenicol was added to a final concentration of 100 µg ml-1 to one of each of the duplicate cultures to inhibit protein synthesis. The cultures were incubated for 6 h in an anaerobic jar as described above before being harvested by centrifugation (27000 g, 4 °C, 5 min) and washed three times in 50 mM potassium phosphate buffer pH 7·5 containing 0·02% (w/v) sodium azide. The cells were finally resuspended to an OD600 1·00±0·05 for determination of enzyme activities. To confirm that chloramphenicol was inhibiting protein synthesis, static batch cultures of S. gordonii were grown in CDM supplemented with 185 kBq [3H]leucine (Amersham Life Sciences). After harvesting and washing, the level of incorporation of [3H]leucine into the cells was determined using a Packard 1500 Tricarb Liquid scintillation analyser (Canberra Packard).
Analytical procedures.
The concentration of bacterial cell-associated proteins (at OD600 1·00±0·05) and the protein concentration in the culture supernatant were determined by the Pierce Coomassie Protein Plus Assay Reagent (Pierce) using bovine serum albumin as the standard. The concentration of residual glucose in CDM and in serum was determined using a glucose oxidase test kit (Sigma). Statistical analysis, Friedman non-parametric repeated-measures test, two-tailed, was carried out using the statistical program Instat v2·02 (Graphpad Inc.). SDS-PAGE analysis of FBS culture components was carried out on duplicate 10% gels (Laemmli, 1970 ). One gel was stained for protein with Gelcode Blue Coomassie blue (Pierce) and the other stained for carbohydrate [periodic acid/Schiff (PAS) stain] using the method described by Pitty et al. (1989)
. Protein molecular masses were estimated using Bio-Rad broad range molecular mass markers.
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RESULTS |
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Effect of glucose-excess/nitrogen-limited conditions on enzyme activities
At stationary phase, in cultures grown under glucose excess/nitrogen limitation at pH 7·5, the cell-associated protein concentration (50·1±0·3 µg ml-1) was approximately 50% of the value for the other culture conditions. Culture-supernatant protein concentration (19·9±0·1 µg ml-1) was similar to the pH 7·5 culture grown on CDM but nearly double the concentration of cultures grown with no pH control or at pH 6·5 on CDM. Cell-associated and supernatant glycosidase activities were undetectable, whilst peptidase activities were not significantly different from the other culture conditions (Tables 1 and 2
).
Effect of incubating cells in various pH buffers
Washed cell suspensions of S. gordonii were incubated at 37 °C in 50 mM citrate/phosphate buffer at pH 4·0, 5·0 or 6·0 for 2 h. Incubation at pH 4·0 resulted in the total loss of measurable activity for all enzymes except N-acetyl-ß-D-glucosaminidase, ß-D-glucosidase and Hageman factor. These enzymes retained 50%, 30% and 1% of their pre-incubation activities, respectively. Incubation at pH 5·0 or above had little effect on any of the enzyme activities (data not shown).
Effect of growth on 50% (v/v) FBS on the expression of enzyme activities
Cultures were grown in CDM medium without glucose and supplemented with 50% (v/v) FBS; 50% (v/v) FBS contained 0·52 mg glucose ml-1. The inoculum for the cultures was centrifuged to pellet the cells and resuspended in a small volume of 50 mM potassium phosphate buffer pH 7·5 prior to inoculation into the batch culture vessel. Culturing in FBS was repeated three times and a representative growth curve is shown in Fig. 2. Two hours after inoculation, the culture OD600 had reached 0·57 and no glucose could be detected in the culture supernatant. The culture optical density then fell to an OD600 of 0·25 after 24 h. The viable count, which had increased from 3·0x108 c.f.u. ml-1 at inoculation to 1·4x109 c.f.u. ml-1 at 2·5 h, also declined until at 24 h it was 3·0x108 c.f.u. ml-1. However, over the next 92 h the culture OD600 increased to 0·95 while the viable count declined slightly to 7·2x107 c.f.u. ml-1 (Fig. 2
). Centrifugation of culture samples and examination by Gram staining and light microscopy revealed that a fibrous precipitate was being formed in the culture. The precipitate had little solubility in 50 mM potassium phosphate buffer pH 7·5 or deionized water.
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Effect of FBS on enzyme activities in late-stationary phase
Cell-associated specific enzyme activities in FBS cultures (Table 3) were initially increased (at 2·5 h and 5·5 h) from those detected at inoculation. However, over the extended time period of the culture, enzyme activities were generally reduced, though not significantly (Table 3
). Cell-associated enzyme activities were not significantly different from those activities found in cultures grown at pH 7·5 on CDM when compared with any time point in the FBS culture.
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DISCUSSION |
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Previous chemostat studies showed that glucose, although present at a low level (915 µg ml-1), was still detectable in all steady-state conditions (Mayo et al., 1995 ). In the current batch-culture studies, no glucose was detectable by stationary phase. When grown under glucose-excess/nitrogen-limited conditions, it was noted that glycosidase expression was significantly repressed whereas peptidase expression was virtually unchanged. This suggested that the presence of glucose was repressing the expression of the glycosidase enzymes. Catabolite repression of glycosidases by glucose in S. oralis has been previously reported (Rafay et al., 1996
).
In contrast to the oral environment, in an IE vegetation large fluctuations in carbohydrate concentration and therefore in acidity are unlikely to occur. The pH of the local environment would therefore be expected to be more stable with a near-neutral pH similar to that in the oral cavity in the absence of dietary carbohydrate. Even though it is not known what nutrients are available to streptococci in a platelet fibrin vegetation, serum glucose is present in the blood at 1 mg ml-1. The relative availability of blood glucose to bacteria in a vegetation is not known; however, transmission electron microscopic images of sections through staphylococcal IE vegetations show densely packed bacterial cells embedded in an amorphous fibrous matrix, probably comprising fibrin, platelets and other host and bacterial products, with no bacteria visible on the surface (Marrie et al., 1987 ; Ferguson et al. 1986
). When the surface of a damaged vegetation is examined (Marrie et al., 1987
), the exposed subsurface shows large numbers of bacteria covered by a flocculent material which has been described as being similar to the exopolysaccharide material found by Mills et al. (1984)
. Durack & Beeson (1972)
have also reported that bacteria near the surface of the vegetation are metabolically active whilst those buried deeper are metabolically inactive. These findings would imply that those metabolically active bacteria in the surface layers of the vegetation are exposed to glucose from blood. However, the nutritional status of the bacteria in the bulk of the vegetation is unknown but they may be existing in near-starvation conditions. The increased expression of glycosidases at pH 7·5 and when also cultured in the presence of serum indicate an adaptive response to the available nutrients in the environment. In our studies, serum clearly up-regulates the synthesis and secretion of the glycosidases by S. gordonii that would be required for acquisition of oligosaccharides from host sources.
This study also showed that S. gordonii is able to regulate specific peptidase activities involved in clot formation [i.e. thrombin, Hageman factor (lysine peptidase) and Xa] as well as clot dissolution (i.e. plasmin, kallikrein and Ca). Hageman factor was the most abundant activity found under any condition. It was the least affected by pH and was continuously accumulated in the culture supernatant when S. gordonii was grown on FBS. The ability of S. gordonii FSS2 to synthesize enzymes similar to those that promote the formation of clots would amplify the effect of clot formation already stimulated by the platelet-aggregating ability of this IE strain when it first enters the bloodstream (Manning et al., 1994a , b
). Conversely, activities that promote clot dissolution would clearly assist in the penetration and release of potential nutrients into the clot in close proximity to the bacteria. It is perhaps pertinent to speculate that clot dissolution on a larger scale may also assist in the dissemination of an IE infection to other sites in the body. IE vegetations are known to be friable and to fragment, spreading the infection to other sites where they cause abscesses in organs such as the lungs, liver and kidneys (Ferguson et al., 1986
).
In conclusion, the glycosidases and peptidases of S. gordonii have been shown to be up-regulated in neutral pH environments and in the presence of serum. The increased expression of these enzymes would greatly assist S. gordonii in acquiring nutrients, particularly in the environment of the IE vegetation, but also in healthy dental plaque or between meal times when it relies on intrinsic sources of nutrients for survival.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 January 2000;
revised 14 April 2000;
accepted 16 May 2000.