EGG YOLK FACTOR OF STAPHYLOCOCCUS AUREUS

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1 EGG YOLK FACTOR OF STAPHYLOCOCCUS AUREUS I. NATURE OF THE SUBSTRATE AND ENZYME INVOLVED IN THE EGG YOLK OPACITY REACTION D. B. SHAH' AND J. B. WILSON Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication 20 September 1962 ABSTRACT SHAH, D. B. (University of Wisconsin, Madison) AND J. B. WILSON. Egg yolk factor of Staphylococcus aureus. I. Nature of the substrate and enzyme involved in the egg yolk opacity reaction. J. Bacteriol. 85: Some pathogenic staphylococci produce an opacity reaction when grown in media containing egg yolk. The egg yolk factor is a lipase with a requirement for a fatty acid acceptor, rather than any of the three lecithinases. Highest amounts of egg yolk factor were obtained in Heart Infusion broth rather than nutrient broth or Casman's synthetic medium. The lowest density lipoprotein (lipovitellenin) isolated by ultracentrifugation of egg yolk has been identified as the opacity-producing substrate. The lipase acts on the lipid moiety, resulting in alterations in the solubility of lipovitellenin. Lipolysis is optimal at ph 8. The optimal ph for the opacity reaction is at ph 5.5 and 8. The protein (vitellenin) in lipovitellenin has an isoelectric point at ph 5.5. It is proposed that slight alterations in the stabilizing lipid due to lipolysis and the very low solubility of vitellenin may drastically alter the solubility of lipovitellenin at ph 5.5. the opacity reaction is not, per se, evidence for the lecithinase C activity. A possible association of the staphylococcus egg yolk factor with virulence was suggested by the findings of Alder, Gillespie, and Herdan (1953) and Reid and Wilson (1959). The experiments described in this paper were designed to obtain information on the nature of the enzyme involved in the egg yolk opacity reaction and identification of the opacityproducing substrate in the yolk. MATERIALS AND METHODS Preparation of the egg yolk factor. Staphylococcus aureus strain was grown in Heart Infusion broth (Difco) on a rotary shaker for 3 to 5 days. The enzyme had been purified from the cell-free culture filtrates by acid precipitation, two cycles of ethanol fractionation, and zone electrophoresis as described by Blobel, Shah, and Wilson (1961). Because of the instability of the zone electrophoresis-purified egg yolk factor, the ethanolfractionated enzyme preparation was used in all experiments. The lyophilized preparation was stored at -20 C. Using antiserum prepared against the ethanol-fractionated enzyme, gel diffusion precipitation analysis showed only one band with the zone electrophoresis-purified egg yolk factor and three bands with the homologous The ability of certain staphylococci to produce an opacity reaction in egg yolk media was first ethanol-fractionated enzyme. Staphylocoagulase described by Gillespie and Alder (1952). A was one of the three enzyme proteins present in variety of other microorganisms such as clostridia this preparation (Blobel et al., 1961). (Macfarlane, 1955) and aerobic sporeforming Studies on lecithinases. Lecithinase C activity bacilli (McGaughey and Chu, 1948) are known to was measured by determining the rate of increase produce such opacity reaction in egg yolk media. of acid-soluble phosphorus (Macfarlane and Macfarlane and Knight (1941) demonstrated Knight, 1941). For use in the reaction mixtures, that the production of opacity in egg yolk media stable aqueous emulsions of partially purified by a-toxin of Clostridium perfringens is due to its egg lecithin and soybean lecithin (Nutritional lecithinase C activity, but they pointed out that Biochemicals Corp., Cleveland, Ohio) and purified egg lecithin (Hanahan, Turner, and Jayko, I Present address: Johns Hopkins-LWM, School 1951) were prepared by treatment in a Raytheon 10-kc sonic oscillator for 10 min. After of Hygiene, Baltimore, Md. 516

2 VOL. 85, 1963 EGG YOLK FACTOR OF S. AUREUS 517 incubation of the enzyme, substrate, buffer, and varying amounts of CaCi2 (10-1 to 10-4 M) at 37 C for 3 hr, the reaction was terminated by adding cold 20% trichloroacetic acid to a final concentration of 5% in the mixture. The mixture was allowed to stand at room temperature for 30 min and was filtered. The clear filtrate was assayed for acid-soluble phosphorus by the method of King (1932). Lecithinase D activity was investigated according to the procedure of Davidson, Long, and Penny (1955). The discovery of Kates (1953, 1954) that degradation of lecithin by lecithinase D from cabbage and spinach is markedly stimulated by the presence of ether and CaC12 was also investigated. Free choline was measured by the enneaiodide method of Shapiro (1953). Lecithinase A activity was measured by extraction of the fatty acids liberated by the enzyme in the reaction mixture (Fairbairn, 1945) and determined by titration with standard alkali with phenolphthalein as an indicator. Turbidimetric assay for the egg yolk factor. Because of the insolubility of some components of the yolk in water, 5% egg yolk suspensions were made in increasing NaCl concentrations. The suspensions were adjusted to ph 5 and 8 with 0.1 N HCl or 0.1 N NaOH. Tubes (10 by 78 mm) containing 1.0 ml of the egg yolk-saline solutions and 1.0 ml of sterile Heart Infusion broth were incubated at 37 C for 30 min. The lowest concentration of NaCl in the egg yolk suspension that gave a stable and clear mixture as determined by optical density at 625 m,u in a Coleman Junior spectrophotometer was used. For the quantitative assay for the egg yolk factor, the enzyme solution was serially diluted in matched tubes (10 by 78 mm) by adding 1.0 ml of the enzyme solution to 1.0 ml of 0.1 M tris(hydroxymethyl)aminomethane (tris) buffer at ph 8, or 1.0 ml of 0.1 M phthalate puffer at ph 5.5. After mixing, 1.0 ml was transferred to the second tube containing 1.0 ml of the buffer, and the same procedure was repeated. The tubes were warmed to 37 C, and 1.0 ml of 5% egg yolk in 3.5% NaCl solution was added. Optical density measurements were made at 10-min intervals at 625 m, in a Coleman Junior spectrophotometer. The highest dilution of enzyme which gave a change of 0.4 optical density units at 60 min of incubation was used as an end point. Fractionation of egg yolk. Two lipoproteins, lipovitellin and lipovitellenin, were prepared from the yolk by Sharples centrifugation at 40,000 rev/min (ca. 32,000 X g), and by ether extraction of the subnatant as described by Alderton and Fevold (1945) and Fevold and Lausten (1946). To isolate the lipoproteins from the yolk without the use of organic solvents, egg yolk was fractionated in 0.45 M MgSO4 solution by ultracentrifugation at 30,000 rev/min (rotor 30; ca. 80,000 X g) for 24 hr in a Spinco model-l ultracentrifuge. The firm yellow gel, which constitutes the low-density fraction (LDF), was separated from the high-density fraction (HDF) by cutting the tubes. The HDF was resolved into four components by varying the ionic strength of the solutions and by (NH4)2SO4 precipitation according to the procedure of Bernardi and Cook (1960). Cholesterol estimation. The low-density fraction isolated from 15 ml of yolk was diluted to 100 ml with 3.5%, saline. Duplicate 1.9-ml samples were incubated with 0.5 mg of egg yolk factor at 38 C for 3 hr. A control containing no enzyme was also included. Samples of 1 ml each were used for extraction, precipitation, and colorimetric assay for cholesterol, as described by Sperry and Webb (1950). Preparation of lipoprotein and assay for lipoprotein lipase. Lipoprotein from coconut oil was prepared by incubating human serum and coconut oil emulsion, as described by Korn (1955a). After preliminary experiments to test the optimal ph, the reaction mixture in all subsequent experiments contained 2.0 ml of 407 oil emulsion, or "activated" coconut oil emulsion, 3.0 ml of 0.2 M glycylglycine buffer (ph 7.8), and enzyme solution in a total volume of 10 ml. To test the requirement for a fatty acid acceptor, 0.5 ml of 10% bovine serum albumin (Armour and Co.) and varying amounts of CaCl2 (10-' to 104 M) were used. Duplicate 0.5-ml samples were removed from the incubation mixture at 0-, 15-, 30-, and 60-min intervals, and added directly to 0.1 ml of cold 1 N H2SO4 in 15-ml conical-tip centrifuge tubes. Glycerol was determined by the procedure of Lambert and Neish (1950) as modified by Korn (1955a). RESULTS Production of the egg yolk factor. Comparative studies of media including Heart Infusion broth, nutrient broth, and Casman's (1958) synthetic

3 518 SHAH AND WILSON J. BACTERIOL. ph FIG. 1. The ph optimum for the egg yolk opacity reaction by the egg yolk factor of Staphylococcus aureus. Reaction mixture contained 1.0 ml of 6% egg yolk saline, 0.5 ml of 0.2 M tris-maleate buffer at various ph values, and 0.5 ml of enzyme in tubes (10 by 78 mm). Incubation at 37 C for 60 min. medium showed that the highest yields of egg yolk factor were produced in Heart Infusion broth. The amount of the egg yolk factor produced in Heart Infusion broth was four times that obtained in nutrient broth, and in Casman's synthetic medium even less egg yolk factor was found. Lecithinase activities. Since the egg yolk factor of staphylococci produces an opacity reaction in egg yolk saline grossly indistinguishable from that produced by lecithinase C of C. perfringens, attempts were made first to demonstrate similar activity in egg yolk factor preparations using soybean lecithin, ovalecithin, and one sample of egg lecithin which had been purified further on an alumina column (Hanahan et al., 1951). Stimulation of lecithinase C activity (Costlow, 1958) by CaCl2 was also investigated. The results failed to indicate any lecithinase C activity in egg yolk factor preparations. The opacity production in egg yolk saline by lecithinase C is due to the hydrolysis of lecithin with the loss of its emulsifying properties. However, the opacity reaction, per se, is not evidence for lecithinase C activity (Macfarlane, 1955). Hence, further attempts were made to test for the presence of lecithinase D and lecithinase A activity in egg yolk factor preparations. The marked stimulation of lecithinase D from cabbage chloroplasts and the requirement for Ca ions by lecithinase A and lecithinase D were also investigated. The results also failed to indicate such lecithinase activities in the egg yolk factor preparations. Assay for the egg yolk factor. The complexity of egg yolk precluded the ready identification of an opacity-producing component, and, during the early part of this investigation, the whole egg yolk was used as a substrate. A turbidimetric assay for the egg yolk factor was devised using whole egg yolk as the substrate. Effect of NaCI concentration on the stability of the egg yolk suspensions. Egg yolk suspensions were stable between ph 5.0 and 8.0, when made in 3.5% NaCl solution rather than in distilled water. The effect of this increased ionic strength on the stability of the yolk suspensions is due to the dissolution of yolk granules, which are made up of a phosphoprotein-lipovitellin complex (Schjeide and Urist, 1960). The dissolution of phosvitin, a polyelectrolyte protein that is present in the yolk as an insoluble calcium salt and is partly converted to the soluble sodium salt with the loss of calcium when dissolved in NaCl solution (Cook, 1961), may also contribute to the stability and clarity of the egg yolk suspension in sodium chloride solution. Effect of ph on the egg yolk opacity reaction. The determination of the optimal ph for the opacity reaction showed two ph optima: ph 5.5 and 8.0 (Fig. 1). These two ph optima were observed when phthalate or acetate buffer at ph 5.5 or tris or glycylglycine buffer at ph 8 were used. When the rate of change of optical density at ph 5.5 and 8 were plotted (Fig. 2), a long lag period was observed at ph 8 before the optical density (OD) changes. A slight lag was also observed at ph 5.5. The effect of substrate concentration in the range of 1 to 15% of yolk in 3.5% saline did not eliminate the lag period observed in the opacity reaction. Fractionation of egg yolk and identification of the opacity-producing substrate. During the incubation of the egg yolk factor with egg yolk saline, it was observed that there was production of acidity in the medium. Linoleic, linolenic, oleic, and traces of palmitic acid were identified chromatographically (Schlenk et al., 1957) as the acids produced during the opacity reaction. Since the results indicated absence of the three lecithinases, and because lipolytic activity was not demonstrated through use of higher fatty acid fats, attempts were made to identify the opacityproducing substrate in the yolk. Two lipoproteins (lipovitellin and lipovitellenin), isolated by Sharples centrifugation and

4 VOL. 85, 1963 EGG YOLK FACTOR OF S. AUREUS O. * t o Time, minutes FIG. 2. Rate of change of optical density in 6% egg yolk saline at ph 6.6 and 8 by the egg yolk factor of staphylococci. Experimental conditions a-s in the text. ether extraction of the sediment, did not show lipolytic activity with egg yolk factor. The solubility of these lipoproteins is drastically altered by ether treatment (Fevold and Lausten, 1946). To isolate these lipoproteins without the use of organic solvents, ultracentrifugation was used. The HDF containing lipovitellin did not give an opacity reaction. Addition of egg yolk oil to the HDF also did not result in the opacity reaction. The LDF gave a pronounced opacity reaction with the egg yolk factor. The opacity-producing scum isolated from the LDF after the reaction with egg yolk factor, when extracted with alcohol-acetone (3:1) mixture, gave an insoluble protein residue and alcoholacetone-soluble lipid fraction. The comparison of dry weights of the protein and lipid in an average of three experiments gave a protein-to-lipid ratio of 63:37. There was no net increase in free cholesterol after the opacity reaction. Lipoprotein lipase activity. The specificity of the egg yolk factor towards a lipoprotein rather than free lipid was investigated according to the procedure of Korn (1955a, b). Evidence was presented by Korn for the formation of a lipoprotein complex when coconut oil was incubated with normal human serum. Since the LDF had not been resolved by physiological methods, and since the lipovitellenin isolated by ether extraction was degraded (Turner and Cook, 1958), a simpler substrate like "activated" coconut oil was used as a substrate to test the specificity of 0. ~0 Time, minutes FIG. 3. Hydrolysis of coconut oil and "activated" coconut oil (in the presence or absence of fatty acid acceptors) by the egg yolk factor. Experimental conditions as in the text. 0: "Activated" coconut oil + calcium; 0: coconut oil + calcium; A: coconut oil + albumin; and X: coconut oil alone. the egg yolk factor towards lipoprotein. The results (Fig. 3) demonstrate that the relative rates of hydrolysis of coconut oil and "activated" coconut oil are the same in the presence of Ca ions as the fatty acid acceptor. Albumin does not serve well as a fatty acid acceptor. The rates with coconut oil and activated coconut oil were the same in the absence of Ca ions. When the LDF was dialyzed against 3.5% NaCl and then with distilled water, using dialyzed LDF as a substrate, the opacity reaction was not observed with egg yolk factor. Ethylenediamine tetraacetic acid (EDTA) also inhibited the egg yolk opacity reaction. DISCUSSION Many species of the genus Clostridium and some aerobic sporeforming bacilli (Macfarlane, 1955; McGaughey and Chu, 1948) have been described as producing an opacity reaction in media containing egg yolk. In most cases, lecithinase C has been found to bring about such a reaction. The results of this study indicate that

5 520 SHAH AND WILSON J. BACTERIOL.. lecithinase A, lecithinase C, and lecithinase D are not present in the egg yolk factor enzyme preparations of S. aureus, and hence are not responsible for the opacity reaction in egg yolk by this organism. The nonidentity of the egg yolk factor with lecithinase C was also reported by Gillespie and Alder (1952). Studies on the identification of the opacityproducing substrate in yolk indicate that the lipoprotein present in the LDF is involved in the opacity reaction with staphylococcal enzyme. Ether extraction of the LDF removes 80% of the so-called "free" lipid, and a lipoprotein containing about 40% bound lipid is obtained (Turner and Cook, 1958). This lipoprotein has been termed lipovitellenin. After ether extraction, the solubility of the lipoprotein is drastically altered. The presence of only one lipoprotein-lipovitellenin in the LDF has been suggested by the above authors. The degraded solubility of the lipoprotein and nonsedimentability of the natural lipoprotein in LDF suggests the removal of a stabilizing lipid by ether extraction. The natural lipovitellenin contains higher amounts of bound lipid (Turner and Cook, 1958). Our data show that the opacity-producing scum isolated after opacity reaction (using LDF as a substrate) contains approximately 37% lipid. Hence, some lipid which may play a role as a stabilizing lipid in the natural lipoprotein is attacked by the egg yolk factor, resulting in the alterations of its solubility. This lipid may also be susceptible to cleavage by ether. The determination of the optimal ph for the opacity reaction showed two ph optima: ph 5.5 and 8. Gillespie and Alder (1952) reported only ph 5.5 as the optimal ph for the opacity reaction. However, their range of ph tested for such determination was only ph 5 to 7. It has been realized that what has been determined is the influence of the ph on the opacity production, which probably represents the end of a complex reaction rather than the primary action of the enzyme on the substrate. A possible explanation for the two ph optima obtained for the opacity reaction can be offered here. The lipovitellenin isolated from the LDF is least soluble at ph 5.5, but more soluble at ph 8 (Fevold and Lausten, 1946). Slight alterations in the stabilizing lipid in the natural lipovitellenin by the egg yolk factor may result in its precipitation at ph 5.5, whereas at ph 8 it is more soluble. At ph 8, the mono- and diglycerides produced during hydrolysis of the neutral lipid may tend to stabilize the lipoprotein, and only when all the fatty acids are removed may the lipoprotein become insoluble. Since there is a long lag period before glycerol appears as a product of tryglyceride hydrolysis during lipolysis, one would expect such a lag period in the change of solubility of the lipoprotein during egg yolk reaction. Figure 2 illustrates such a lag period at ph 8, before the optical density of the reaction mixture changes. The egg yolk factor is a lipase with a specific requirement for a fatty acid acceptor such as Ca ions. This is further supported by the fact that dialyzed LDF does not give an opacity reaction. EDTA also inhibits the egg yolk opacity reaction. The fatty acid acceptor in egg yolk saline is probably calcium. Calcium may also be contributed by phosvitin, a polyelectrolyte protein that is present in the yolk as an insoluble calcium salt and is converted to the soluble sodium salt with the loss of calcium (Cook, 1961). ACKNOWLEDGMENT This investigation was supported in part by grant E-2962 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. LITERATURE CITED ALDER, V. G., W. A. GILLESPIE, AND G. HERDAN Production of opacity in egg yolk broth by staphylococci from various sources. J. Pathol. Bacteriol. 66: ALDERTON, G., AND H. L. FEVOLD Preparation of the egg yolk lipoprotein, lipovitellin. Arch. Biochem. 8: BERNARDI, G., AND W. H. COOK An electrophoretic and ultracentrifugal study on the proteins of the high density fraction of egg yolk. Biochim. Biophys. Acta 44: BLOBEL, H., D. B. SHAH, AND J. B. WILSON Serologic studies on the egg yolk factor isolated from coagulase-positive staphylococci. J. Immunol. 87: CASMAN, E. P Serologic studies of staphylococcal enterotoxin. Public Health Rept. U.S. 73: CooK, W. H Proteins of hen's egg yolk. Nature 190: COSTLOW, R. D Lecithinase from Bacillus anthracis. J. Bacteriol. 76: DAVIDSON, F. M., G. LONG, AND I. F. PENNY Enzymic degradation of ovolecithin and re-

6 VOL. 85, 1963 EGG YOLK FACTOR OF S. AUREUS 521 lated substances by phospholipases A and D, p In G. Popjak and E. LeBrenton [ed.], Biochemical problems of lipids. Butterworths Scientific Publications, London. FAIRBAIRN, D The phospholipase of the venom of the cottonmouth moccasin (Agkistrodon psicivorus L). J. Biol. Chem. 157: FEVOLD, H. L., AND A. LAUSTEN Isolation of a new lipoprotein, lipovitellenin from egg yolk. Arch. Biochem. 11:1-7. GILLESPIE, W. A., AND V. G. ALDER Production of opacity in egg-yolk media by coagulase-positive staphylococci. J. Pathol. Bacteriol. 64: HANAHAN, D. J., M. B. TURNER, AND M. E. JAYKO The isolation of egg phosphatidyl choline by an adsorption column technique. J. Biol. Chem. 192: KATES, M Lecithinase activity of chloroplasts. Nature 172: KATES, M Lecithinase systems in sugar beet, spinach, cabbage and carrot. Can. J. Biochem. Physiol. 32: KING, E. J The colorimetric determination of phosphorus. Biochem. J. 26: KORN, E. D. 1955a. Clearing factor, a heparinactivated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart. J. Biol. Chem. 215:1-14. KORN, E. D. 1955b. Clearing factor, a heparin-activated lipoprotein lipase. II. Substrate specificity and activation of coconut oil. J. Biol. Chem. 215: LAMBERT, M., AND A. C. NEISH Rapid method for estimation of glycerol in fermentation solutions. Can. J. Res. 28: MACFARLANE, M. G On the biochemical mechanism of action of gas-gangrene toxins. Symp. Soc. Gen. Microbiol. 5: MACFARLANE, M. G., AND B. C. J. G. KNIGHT The biochemistry of bacterial toxins. I. The lecithinase activity of Clostridium welchii toxins. Biochem. J. 35: MCGAUGHEY, G. A., AND H. P. CHU The egg yolk reaction of aerobic sporing bacilli. J. Gen. Microbiol. 2: REID, W. B., AND J. B. WILSON A study of the staphylococci associated with the bovine udder. Am. J. Vet. Res. 20: SCHJEIDE, 0. A., AND M. R. URIST Proteins induced in plasma by oestrogens. Nature 188: SCHLENK, H., J. L. GELLERMAN, J. A. TILLOSTON, AND H. K. MANGOLD Paper chromatography of lipides. J. Am. Oil Chemist's Soc. 34: SHAPIRO, B Purification and properties of a lysolecithinase from pancreas. Biochem. J. 53: SPERRY, W. M., AND M. WEBB A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187: TURNER, K. J., AND W. H. COOK Molecular weight and physical properties of a lipoprotein from the floating fraction of egg yolk. Can. J. Biochem. Physiol. 36: