Br. J. exp. Path. (1976) 57, 105
STUDIES ON ANTISTREPTOLYSIN 0 ACTIVITY GENERATED IN SERUM BY MICROORGANISMS K. C. WATSON AND E. J. C. KERR From the Central Mificrobiological Laboratories, Western General Hospital, Edinburgh EH4 2XU Received for publication September 1, 1975
Summary.-An antistreptolysin factor (ASF) was generated in normal human serum by the growth of Staph. aureus and Pseud. aeruginosa. Alpha toxin producing strains of the former were usually positive but activity was not restricted to such strains. Positive strains produce cholesterol esterase which was obtained from DEAE-cellulose column fractions of 18 h broth cultures. Antistreptolysin factor develops slowly in serum, being maximal between the 5th and 10th days and is associated with alterations and disappearance of beta lipoproteins on gel electrophoresis. Activity also appeared in beta lipoproteins precipitated from normal serum with dextran sulphate and redissolved in nutrient broth before inoculation with Staph. aureus. The slow appearance of antistreptolysin activity in serum appears to be due to an esterase inhibitor which is present in high concentration in some sera. Activity is also modified by the production of a staphylococcal fraction capable of binding to the antistreptolysin factor and reducing its activity. It is suggested that antistreptolysin factor which can be demonstrated in small amounts in normal human serum represents a readily available non-specific defence mechanism capable of binding to certain bacterial products and possibly to other foreign protein molecules.
NON-ANTIBODY inhibition of streptolysin 0 has been noted in sera from certain patients with jaundice (Packalen, 1948) and in hypercholesterolaemia (Winblad, 1966; Watson, Rose and Kerr, 1972). In addition, Hewitt and Todd (1939) showed that sera contaminated with bacteria could become inhibitory and we have shown previously that such activity occurs mainly amongst strains of Staphylococcus aureUs and Pseudomonas aeruginosa (Watson and Kerr, 1 974a). Preliminary evidence suggested that the mechanism involves production of cholesterol esterase by the organism, acting in association with a proteolytic enzyme, to produce lipoprotein fragments with molecular weights between 25,000 and 100,000. In these fragments the cholesterol moiety appears to be spatiallv oriented in a manner that exposes the hydroxyl group
at C3 and the hydrophobic chain at C17, required for inactivation of streptolysin 0 (Howard, Wallace and Wright, 1953; Watson and Kerr, 1 974b). The present investigation was undertaken to study some of the complex changes in sera contaminated with bacteria and the light which this may shed on our previously stated hypothesis which attributes a homoeostatic protective function to catabolic breakdown products of lipoprotein metabolism (Watson and Kerr, 1975). MATERIALS AND METHODS Bacteria. Most investigations were carried out with suitable strains of Staph. aureus. In general, these produced higher levels of antistreptolysin activity in serum than did Pseud. aeruginosa. Also, the latter tended to increase serum viscosity after 4-6 days incubation, mnaking filtration difficult. Cultures of Staph.
106
K. C. WATSON AND E. J. C. KERR
aureus included (a) 30 freshly isolated strains from wound swabs and all shown to produce alpha toxin as well as antistreptolysin activity in serum, (b) a non-alpha toxin producing strain isolated from a patient with staphylococcal septicaemia and whose serum had a non-antibody antistreptolysin ) titre of 1280 and (c) the following NCTC strains: No. 5661, producing beta lysin and an abnormal lysin for rabbit cells but not alpha lysin; 5663 and 5664, producing beta and gamma lysin but not alpha; 5662, producing alpha1 and alpha2 lysins and 7121 (Wood 46 strain), producing alpha lysin, and a local strain of Pseud. aeruginosa (strain 3). Strains were maintained on nutrient agar slopes and incubated in nutrient broth for 18 h before inoculation into serum. Sera.-Normal human sera were obtained from volunteers and from specimens submitted for syphilis serology. Pools of 40-50 ml were prepared and tested for antistreptolysin activity. Only those with titres less than 100 were employed. In addition, 15 sera from patients with hyperbetalipoproteinaemia (either Type llA or 1lB) were kindly supplied by Professor T. D. V. Lawrie, Royal Infirmary, Glasgow. Sera were stored at -700 and before use were filtered through Millipore membranes of 0 45 ,Im and 0-22 ,m porosity. Precipitation of beta lipoproteins.-To each ml of serum 0-02 ml of 10% dextran sulphate was added along with 0-1 ml of M CaCl2. After standing at room temperature for 30 min the precipitate was removed by centrifugation. Precipitates were redissolved in an aliquot of sterile buffered saline, pH 7-2. To investigate the effect of organismal growth on the beta lipoprotein an equal volume of double strength nutrient broth was added before inoculation. An aliquot of broth was also added to the beta lipoprotein free supernatant before inoculation. Preparation and measurement of antistreptolysin factor (ASF). Antistreptolysin activity was obtained in neat serum or in serum broth mixtures (80 : 20 v/v) by incubation of cultures for 10-12 days at 37°. Generation of activity was slow usually with no increase demonstrable in the first 48 h. Maximum titres were usually obtained between 4-7 days incubation but in some sera only after 10-12 days. Sterile filtrates prepared from cultures were stored at -20° and retained activity over several months. Wte refer to this activity against streptolysin 0 as antistreptolysin factor (ASF) to distinguish it from antistreptolysin antibody (ASO). Antistreptolysin activity was measured by the standard method of Rantz and Randall (1945) or by microtitration in disposable plastic trays. In both 5000 haemolysis was detected in the presence of 1-0 i.u. of reduced streptolysin O (Burroughs Wellcome, Beckenham, Kent). Lipoprotein electrophoresis.-Electrophoresis
was carried out in a Corning-EEL cassette electrophoresis cell using fixed voltage (90 v ± 5% rectified direct current output). Separation was done on Agarose films (1% w/v) with sucrose (5% w/v) and EDTA disodium salt in 0-05 mol/l barbitone buffer, pH 8-6. Films were loaded with 1 1l of serum and electrophoresed for 30 min. The lipoproteins were stained with Fat Red 7B for 15 min, cleared in 5 % acetic acid and then dried in an oven for 20 min. Preparation of cholesterol esterase. Five hundred ml of brain heart infusion broth were inoculated with 10 ml of a 6-h broth culture of the Wood 46 strain of Staph. aureus. After 18 h incubation at 370 with continual shaking the bacteria were deposited by centrifugation and the supernatant serially filtered through Millipore membranes of 1-2 ,um, 0-45 ,um and 0-22 um porosity. Solid NH42SO4 (Analar grade) was added to saturation. After overnight storage at 40 the precipitate was recovered by centrifugation, redissolved in sterile 0 85% saline, dialysed for 24 h against running tap water and concentrated with polyethylene glycol (6000) to 20 ml volume. After centrifugation the supernatant fluid was again filtered through Millipore membranes of 0-45 ,um and 0-22 ,um porosity. DEAE-cellulose was equilibrated with 0-01 mol/l phosphate buffer (pH 7-5) and packed in a column 25 cm x 2 cm. The column was loaded with 10 ml of crude esterase preparation and washed through with the same buffer. Fractions were collected in 3 ml amounts. Adjacent tubes showing increased protein content were pooled and concentrated with polyethylene glycol to final volumes of 3 ml and then sterilized by membrane
filtration. Preparation of staphylococcal fraction binding antistreptolysin factor.-Following elution of cholesterol esterase from the DEAE-cellulose column, the column was washed through with McIlvaine's citric acid-phosphate buffer (0-1 mol/l, pH 7-5). Adjacent fractions were pooled in 30-ml amounts and concentrated down to 5 ml with polyethylene glycol. Fractions were then dialysed for 72 h against running tap water. The binding activity for ASF was determined by incubation of aliquot volumes of pooled concentrated fraction with an ASF preparation (titre 2560) for 1 h at 37°. Residual ASF activity was then measured as above. Demonstration of cholesterol esterase activity.Cholesterol linoleate, stearate and palmitate were dissolved in ether at concentrations of 400 mg/ml. One 0-02 ml volume was then added to 3 ml of sterile nutrient broth and the ether driven off by gentle heat, giving a finely divided suspension containing 2660,ug of ester. One-ml volumes were incubated with 0-02 ml volumes of the crude column esterase prepara-
107
STUDIES ON ANTISTREPTOLYSIN 0 ACTIVITY
tion suitably diluted for 24 h at 37°. Other aliquot volumes of cholesterol linoleate in broth were incubated with Staph. aureus or with a suitable strain of Pseud. aeruginosa. Antistreptolysin activity of released cholesterol was determined as above. Esterase activity was also demonstrated by mixing aliquot 0 5 ml volumes of normal serum and esterase preparation, either crude or derived from DEAE-cellulose columns, and incubating for 7 days at 37°. Effect of bacterial growth on 14C-labelled cholesterol. Ten mg of 14C-labelled cholesterol was added to 5 ml of sterile human serum (specific activity 1 mCi/ml) and allowed to equilibrate with lipoprotein cholesterol for 24 h. Three drops of an overnight culture of Staph. aureus (Wood 46 strain) were added and cultures incubated for 7 days at 37°. After removal of bacteria by centrifugation and sterilization by membrane filtration, the supernatant fluid was tested for the presence of labelled cholesterol to determine whether bacterial growth had produced catabolic breakdown.
TABLE I.-Antistreptolysin Titres in Human Sera Inoculated with Staph. aureus (Wood 46 Strain) Antistreptolysin titres measured after Serum 1 2 3 4 5 6 7 8 9 10 11 12 13
Type Normal Normal Normal Normal Normal Normal l A* IlA* I IA* I lB* llB* I lB* IlB*
2 dlays 100 100 100 100 100 100 200 100 200 200 100 100 100
7 days 6400 6400 3200 3200 6400 6400 12800 6400 12800 25600 25600 25600 100
10 days 6400 6400 6400 3200 6400 12800 25600 12800 25600 25600 25600 25600 100
* Sera from patients with hyperbetalipoproteinaemia. Titres expressed as reciprocals of final dilutions showing 50% haemolysis. Broth culture control titre less than 20.
EXPERIMENTAL AND RESULTS
Generation of antistreptolysin activity (ASF) from beta lipoprotein8 Previously we noted that different normal sera varied in the levels of ASF activity when measured at 4 days after inoculation with a suitable organism (Watson and Kerr, 1975, unpublished). Such variations were not correlated with levels of total cholesterol in the sera. However, Table I shows that levels of ASF were related to the concentration of beta lipoprotein. With one exception, all the hyperbetalipoproteinaemic sera gave high titres of ASF activity but in both groups titres were slow to rise, showing that there was no correlation with the exponential phase of bacterial growth. The exception, serum No. 13, failed to develop any ASF activity in spite of good growth (vide infra). Figure 1 shows the electrophoretic changes in a serum in which Staph. aureus was grown. The pattern produced by a strain of Esch. coli incapable of generating ASF activity is included for comparison. The pattern of lipoprotein remained essentially unchanged in the control serum
Column fraction numbers
FIG. 1. Changes in lipoproteins of normal serum incubated with Staph. aureus (Wood 46 strain). Blurring and fusion of pre-beta and beta fractions is followed by almost complete disappearance. Note no change in lipoproteins in which Esch. coli has been grown.
inoculated with Esch. coli over the 6-day period of incubation. With the staphylococcus little change was noted over the first 18 h in spite of heavy growth in this time. This suggests that either cholesterol esterase is produced slowly only after the
108
K. C. WATSON AND E. J. C. KERR
exponential phase of growth or, more likely, that the esterase is initially bound to an inhibitor and that a certain critical concentration of free enzyme must first be available to split ester cholesterol. The findings with serum No. 13 in Table I support the latter view. The earliest changes consist of a broadening and increased diffusion of the pre-beta and beta bands which become less distinguishable from each other. After 24 h there is increased mobility of the bands, presumably due to charge alterations and possibly due to proteolytic enzyme activity. After 30 h the staining becomes very indistinct in the pre-beta and beta regions but there is still evidence of staining in the alpha lipoprotein region. A corresponding pattern at 30 h stained with amido black showed marked proteolytic activity as compared with the same serum uninoculated or inoculated with a strain of Esch. coli. The staining noted at the origin with Fat Red 7B appears to be associated with products of bacterial growth. After 50 h there was no detectable lipoprotein staining in any area. Similar appearances were obtained with serum cultures of Pseud. aeruginosa. In both cases the rise of ASF activity appears to lag behind the visual changes in lipoprotein staining. Study of many different serum cultures suggests that ASF activity is generated slightly more rapidly with pseudomonas cultures than with staphylococci but that the final titres tend to be higher with the latter. The rate of ASF production may be also a function of proteolysis which may precede cholesterol esterase activity. Pollard, Scanu and Taylor (1969) have shown that the less polar lipids are likely to occupy the core of low density lipoprotein molecules. If so, some mechanism for their exposure to esterase may be required. Growth of Staph. aureus in beta lipoprotein fraction Table II shows the ASF titres developing in the low density lipoprotein fraction (LDL) of normal human serum precipi-
TABLE II. Generation of Antistreptolysin Activity in Serum Fractions Inoculated with Staph. aureus (Wood 46 Strain) 500o haemolytic titre measured after 7 (lays Serum fraction Whole serum 6400 LDL component* 6400 Supernatant* 400 Whole serumt < 100 * Mixed with aliquot of nutrient broth. t Uninoculated control.
tated with dextran and redissolved in nutrient broth and in the supernatant serum fraction after inoculation with the Wood 46 strain of Staph. aureus. The small amount of activity in the supernatant fraction probably derives from alpha lipoprotein. Harvie (1974) noted a reaction of staphylococcal cholesterol esterase with alpha lipoprotein but it is clear that the beta and possible pre-beta (VLDL) fractions contribute most of the activity.
Effect of different strains of Staph. aureus on ASF production Table III illustrates variations in titre of ASF produced in 1-0 ml volumes of pooled human serum when inoculated with different strains of Staph. aureus. The results showed that ASF production in serum tends to be a feature of alpha toxin producing strains of Staph. aureus but the correlation is not complete. TABLE III. Generation of Antistreptolysin Activity in Same Serum by Different Strains of Staph. aureus Antistreptolysin titres after Staphylococcus NCTC 5661 NCTC 5662 NCTC 5663 NCTC 5664 NCTC 7121 Local 713 Local 562 Local 818 Local 613 *
Designated
Toxin produced f and AR* ai1 and Of2 f and y not ay ft and y not oa (x
(x
"
6400 6400
STUDIES ON ANTISTREPTOLYSIN 0 ACTIVITY GENERATED IN SERUM BY MICROORGANISMS K. C. WATSON AND E. J. C. KERR From the Central Mificrobiological Laboratories, Western General Hospital, Edinburgh EH4 2XU Received for publication September 1, 1975
Summary.-An antistreptolysin factor (ASF) was generated in normal human serum by the growth of Staph. aureus and Pseud. aeruginosa. Alpha toxin producing strains of the former were usually positive but activity was not restricted to such strains. Positive strains produce cholesterol esterase which was obtained from DEAE-cellulose column fractions of 18 h broth cultures. Antistreptolysin factor develops slowly in serum, being maximal between the 5th and 10th days and is associated with alterations and disappearance of beta lipoproteins on gel electrophoresis. Activity also appeared in beta lipoproteins precipitated from normal serum with dextran sulphate and redissolved in nutrient broth before inoculation with Staph. aureus. The slow appearance of antistreptolysin activity in serum appears to be due to an esterase inhibitor which is present in high concentration in some sera. Activity is also modified by the production of a staphylococcal fraction capable of binding to the antistreptolysin factor and reducing its activity. It is suggested that antistreptolysin factor which can be demonstrated in small amounts in normal human serum represents a readily available non-specific defence mechanism capable of binding to certain bacterial products and possibly to other foreign protein molecules.
NON-ANTIBODY inhibition of streptolysin 0 has been noted in sera from certain patients with jaundice (Packalen, 1948) and in hypercholesterolaemia (Winblad, 1966; Watson, Rose and Kerr, 1972). In addition, Hewitt and Todd (1939) showed that sera contaminated with bacteria could become inhibitory and we have shown previously that such activity occurs mainly amongst strains of Staphylococcus aureUs and Pseudomonas aeruginosa (Watson and Kerr, 1 974a). Preliminary evidence suggested that the mechanism involves production of cholesterol esterase by the organism, acting in association with a proteolytic enzyme, to produce lipoprotein fragments with molecular weights between 25,000 and 100,000. In these fragments the cholesterol moiety appears to be spatiallv oriented in a manner that exposes the hydroxyl group
at C3 and the hydrophobic chain at C17, required for inactivation of streptolysin 0 (Howard, Wallace and Wright, 1953; Watson and Kerr, 1 974b). The present investigation was undertaken to study some of the complex changes in sera contaminated with bacteria and the light which this may shed on our previously stated hypothesis which attributes a homoeostatic protective function to catabolic breakdown products of lipoprotein metabolism (Watson and Kerr, 1975). MATERIALS AND METHODS Bacteria. Most investigations were carried out with suitable strains of Staph. aureus. In general, these produced higher levels of antistreptolysin activity in serum than did Pseud. aeruginosa. Also, the latter tended to increase serum viscosity after 4-6 days incubation, mnaking filtration difficult. Cultures of Staph.
106
K. C. WATSON AND E. J. C. KERR
aureus included (a) 30 freshly isolated strains from wound swabs and all shown to produce alpha toxin as well as antistreptolysin activity in serum, (b) a non-alpha toxin producing strain isolated from a patient with staphylococcal septicaemia and whose serum had a non-antibody antistreptolysin ) titre of 1280 and (c) the following NCTC strains: No. 5661, producing beta lysin and an abnormal lysin for rabbit cells but not alpha lysin; 5663 and 5664, producing beta and gamma lysin but not alpha; 5662, producing alpha1 and alpha2 lysins and 7121 (Wood 46 strain), producing alpha lysin, and a local strain of Pseud. aeruginosa (strain 3). Strains were maintained on nutrient agar slopes and incubated in nutrient broth for 18 h before inoculation into serum. Sera.-Normal human sera were obtained from volunteers and from specimens submitted for syphilis serology. Pools of 40-50 ml were prepared and tested for antistreptolysin activity. Only those with titres less than 100 were employed. In addition, 15 sera from patients with hyperbetalipoproteinaemia (either Type llA or 1lB) were kindly supplied by Professor T. D. V. Lawrie, Royal Infirmary, Glasgow. Sera were stored at -700 and before use were filtered through Millipore membranes of 0 45 ,Im and 0-22 ,m porosity. Precipitation of beta lipoproteins.-To each ml of serum 0-02 ml of 10% dextran sulphate was added along with 0-1 ml of M CaCl2. After standing at room temperature for 30 min the precipitate was removed by centrifugation. Precipitates were redissolved in an aliquot of sterile buffered saline, pH 7-2. To investigate the effect of organismal growth on the beta lipoprotein an equal volume of double strength nutrient broth was added before inoculation. An aliquot of broth was also added to the beta lipoprotein free supernatant before inoculation. Preparation and measurement of antistreptolysin factor (ASF). Antistreptolysin activity was obtained in neat serum or in serum broth mixtures (80 : 20 v/v) by incubation of cultures for 10-12 days at 37°. Generation of activity was slow usually with no increase demonstrable in the first 48 h. Maximum titres were usually obtained between 4-7 days incubation but in some sera only after 10-12 days. Sterile filtrates prepared from cultures were stored at -20° and retained activity over several months. Wte refer to this activity against streptolysin 0 as antistreptolysin factor (ASF) to distinguish it from antistreptolysin antibody (ASO). Antistreptolysin activity was measured by the standard method of Rantz and Randall (1945) or by microtitration in disposable plastic trays. In both 5000 haemolysis was detected in the presence of 1-0 i.u. of reduced streptolysin O (Burroughs Wellcome, Beckenham, Kent). Lipoprotein electrophoresis.-Electrophoresis
was carried out in a Corning-EEL cassette electrophoresis cell using fixed voltage (90 v ± 5% rectified direct current output). Separation was done on Agarose films (1% w/v) with sucrose (5% w/v) and EDTA disodium salt in 0-05 mol/l barbitone buffer, pH 8-6. Films were loaded with 1 1l of serum and electrophoresed for 30 min. The lipoproteins were stained with Fat Red 7B for 15 min, cleared in 5 % acetic acid and then dried in an oven for 20 min. Preparation of cholesterol esterase. Five hundred ml of brain heart infusion broth were inoculated with 10 ml of a 6-h broth culture of the Wood 46 strain of Staph. aureus. After 18 h incubation at 370 with continual shaking the bacteria were deposited by centrifugation and the supernatant serially filtered through Millipore membranes of 1-2 ,um, 0-45 ,um and 0-22 um porosity. Solid NH42SO4 (Analar grade) was added to saturation. After overnight storage at 40 the precipitate was recovered by centrifugation, redissolved in sterile 0 85% saline, dialysed for 24 h against running tap water and concentrated with polyethylene glycol (6000) to 20 ml volume. After centrifugation the supernatant fluid was again filtered through Millipore membranes of 0-45 ,um and 0-22 ,um porosity. DEAE-cellulose was equilibrated with 0-01 mol/l phosphate buffer (pH 7-5) and packed in a column 25 cm x 2 cm. The column was loaded with 10 ml of crude esterase preparation and washed through with the same buffer. Fractions were collected in 3 ml amounts. Adjacent tubes showing increased protein content were pooled and concentrated with polyethylene glycol to final volumes of 3 ml and then sterilized by membrane
filtration. Preparation of staphylococcal fraction binding antistreptolysin factor.-Following elution of cholesterol esterase from the DEAE-cellulose column, the column was washed through with McIlvaine's citric acid-phosphate buffer (0-1 mol/l, pH 7-5). Adjacent fractions were pooled in 30-ml amounts and concentrated down to 5 ml with polyethylene glycol. Fractions were then dialysed for 72 h against running tap water. The binding activity for ASF was determined by incubation of aliquot volumes of pooled concentrated fraction with an ASF preparation (titre 2560) for 1 h at 37°. Residual ASF activity was then measured as above. Demonstration of cholesterol esterase activity.Cholesterol linoleate, stearate and palmitate were dissolved in ether at concentrations of 400 mg/ml. One 0-02 ml volume was then added to 3 ml of sterile nutrient broth and the ether driven off by gentle heat, giving a finely divided suspension containing 2660,ug of ester. One-ml volumes were incubated with 0-02 ml volumes of the crude column esterase prepara-
107
STUDIES ON ANTISTREPTOLYSIN 0 ACTIVITY
tion suitably diluted for 24 h at 37°. Other aliquot volumes of cholesterol linoleate in broth were incubated with Staph. aureus or with a suitable strain of Pseud. aeruginosa. Antistreptolysin activity of released cholesterol was determined as above. Esterase activity was also demonstrated by mixing aliquot 0 5 ml volumes of normal serum and esterase preparation, either crude or derived from DEAE-cellulose columns, and incubating for 7 days at 37°. Effect of bacterial growth on 14C-labelled cholesterol. Ten mg of 14C-labelled cholesterol was added to 5 ml of sterile human serum (specific activity 1 mCi/ml) and allowed to equilibrate with lipoprotein cholesterol for 24 h. Three drops of an overnight culture of Staph. aureus (Wood 46 strain) were added and cultures incubated for 7 days at 37°. After removal of bacteria by centrifugation and sterilization by membrane filtration, the supernatant fluid was tested for the presence of labelled cholesterol to determine whether bacterial growth had produced catabolic breakdown.
TABLE I.-Antistreptolysin Titres in Human Sera Inoculated with Staph. aureus (Wood 46 Strain) Antistreptolysin titres measured after Serum 1 2 3 4 5 6 7 8 9 10 11 12 13
Type Normal Normal Normal Normal Normal Normal l A* IlA* I IA* I lB* llB* I lB* IlB*
2 dlays 100 100 100 100 100 100 200 100 200 200 100 100 100
7 days 6400 6400 3200 3200 6400 6400 12800 6400 12800 25600 25600 25600 100
10 days 6400 6400 6400 3200 6400 12800 25600 12800 25600 25600 25600 25600 100
* Sera from patients with hyperbetalipoproteinaemia. Titres expressed as reciprocals of final dilutions showing 50% haemolysis. Broth culture control titre less than 20.
EXPERIMENTAL AND RESULTS
Generation of antistreptolysin activity (ASF) from beta lipoprotein8 Previously we noted that different normal sera varied in the levels of ASF activity when measured at 4 days after inoculation with a suitable organism (Watson and Kerr, 1975, unpublished). Such variations were not correlated with levels of total cholesterol in the sera. However, Table I shows that levels of ASF were related to the concentration of beta lipoprotein. With one exception, all the hyperbetalipoproteinaemic sera gave high titres of ASF activity but in both groups titres were slow to rise, showing that there was no correlation with the exponential phase of bacterial growth. The exception, serum No. 13, failed to develop any ASF activity in spite of good growth (vide infra). Figure 1 shows the electrophoretic changes in a serum in which Staph. aureus was grown. The pattern produced by a strain of Esch. coli incapable of generating ASF activity is included for comparison. The pattern of lipoprotein remained essentially unchanged in the control serum
Column fraction numbers
FIG. 1. Changes in lipoproteins of normal serum incubated with Staph. aureus (Wood 46 strain). Blurring and fusion of pre-beta and beta fractions is followed by almost complete disappearance. Note no change in lipoproteins in which Esch. coli has been grown.
inoculated with Esch. coli over the 6-day period of incubation. With the staphylococcus little change was noted over the first 18 h in spite of heavy growth in this time. This suggests that either cholesterol esterase is produced slowly only after the
108
K. C. WATSON AND E. J. C. KERR
exponential phase of growth or, more likely, that the esterase is initially bound to an inhibitor and that a certain critical concentration of free enzyme must first be available to split ester cholesterol. The findings with serum No. 13 in Table I support the latter view. The earliest changes consist of a broadening and increased diffusion of the pre-beta and beta bands which become less distinguishable from each other. After 24 h there is increased mobility of the bands, presumably due to charge alterations and possibly due to proteolytic enzyme activity. After 30 h the staining becomes very indistinct in the pre-beta and beta regions but there is still evidence of staining in the alpha lipoprotein region. A corresponding pattern at 30 h stained with amido black showed marked proteolytic activity as compared with the same serum uninoculated or inoculated with a strain of Esch. coli. The staining noted at the origin with Fat Red 7B appears to be associated with products of bacterial growth. After 50 h there was no detectable lipoprotein staining in any area. Similar appearances were obtained with serum cultures of Pseud. aeruginosa. In both cases the rise of ASF activity appears to lag behind the visual changes in lipoprotein staining. Study of many different serum cultures suggests that ASF activity is generated slightly more rapidly with pseudomonas cultures than with staphylococci but that the final titres tend to be higher with the latter. The rate of ASF production may be also a function of proteolysis which may precede cholesterol esterase activity. Pollard, Scanu and Taylor (1969) have shown that the less polar lipids are likely to occupy the core of low density lipoprotein molecules. If so, some mechanism for their exposure to esterase may be required. Growth of Staph. aureus in beta lipoprotein fraction Table II shows the ASF titres developing in the low density lipoprotein fraction (LDL) of normal human serum precipi-
TABLE II. Generation of Antistreptolysin Activity in Serum Fractions Inoculated with Staph. aureus (Wood 46 Strain) 500o haemolytic titre measured after 7 (lays Serum fraction Whole serum 6400 LDL component* 6400 Supernatant* 400 Whole serumt < 100 * Mixed with aliquot of nutrient broth. t Uninoculated control.
tated with dextran and redissolved in nutrient broth and in the supernatant serum fraction after inoculation with the Wood 46 strain of Staph. aureus. The small amount of activity in the supernatant fraction probably derives from alpha lipoprotein. Harvie (1974) noted a reaction of staphylococcal cholesterol esterase with alpha lipoprotein but it is clear that the beta and possible pre-beta (VLDL) fractions contribute most of the activity.
Effect of different strains of Staph. aureus on ASF production Table III illustrates variations in titre of ASF produced in 1-0 ml volumes of pooled human serum when inoculated with different strains of Staph. aureus. The results showed that ASF production in serum tends to be a feature of alpha toxin producing strains of Staph. aureus but the correlation is not complete. TABLE III. Generation of Antistreptolysin Activity in Same Serum by Different Strains of Staph. aureus Antistreptolysin titres after Staphylococcus NCTC 5661 NCTC 5662 NCTC 5663 NCTC 5664 NCTC 7121 Local 713 Local 562 Local 818 Local 613 *
Designated
Toxin produced f and AR* ai1 and Of2 f and y not ay ft and y not oa (x
(x
"
6400 6400
