Egg Yolk Factor of Staphylococcus

JOURNAL QF BACTERIOLOGY, Apr., 1965 Copyright 1965 American Society for Microbiology Vol. 89, No. 4 Printed in U.S.A. Egg Yolk Factor of Staphylococcus II. Characterization of the Lipase Activity aureus D. B. SHAH AND J. B. WILSON Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication 28 October 1964 ABSTRACT SHAH, D. B. (University of Wisconsin, Madison), AND J. B. WILSON. Egg yolk factor of Staphylococcus aureus. II. Characterization of the lipase activity. J. Bacteriol. 89: 949-953. 1965.-The staphylococcal egg yolk factor was characterized as a lipase. The enzyme had an optimal ph of 7.8, but the optimal ph of stability was 7. Substrate specificity data showed that the relative rate of hydrolysis was lowest with triacetin as substrate, was maximal with tributyrin, and decreased as the chain length of the acyl moieties increased. The enzyme showed an absolute requirement for a fatty acid acceptor like calcium, when the acyl moiety of triglyceride was water-insoluble. Magnesium, strontium, and barium functioned equally well as fatty acid acceptors. The enzyme was able to hydrolyze coconut oil, peanut oil, olive oil, and egg yolk oil. The egg yolk opacity reaction given by certain staphylococci has been used to aid in characterizing different isolates of Staphylococcus aureus from a variety of sources. A possible association of the staphylococcus egg yolk factor with virulence was suggested by the findings of Alder, Gillespie, and Herdan (1953) and of Reid and Wilson (1959). Burns and Holtman (196) reported that all cultures isolated from lesions of undisputed staphylococcal etiology give a positive egg yolk opacity reaction, but they suggested that quantitative tests for a large number of biochemical reactions should be carried out to determine the total biochemical activity of virulent and avirulent strains. Rosendal (1962) found that the egg yolk reaction could not be correlated with virulence of the culture. Shah and Wilson (1963) identified the low-density lipoprotein (lipovitellenin) as the substrate which produces the opacity in egg yolk. It was also shown that the egg yolk factor of S. aureus is a lipase, which had been suggested earlier by Gillepsie and Alder (1952). The experiments described here were designed to characterize the enzyme involved in the production of egg yolk opacity. MATERIALS AND METHODS The egg yolk factor preparation described by Shah and Wilson (1963), which was obtained by ethanol fractionation and then lyophilized, was used in these studies. Substrates. Aqueous (4%) emulsions of various synthetic triglycerides (Eastman Kodak Co., Rochester, N.Y.) and oils, containing.5% 949 Tween 6, were prepared. Fresh emulsions were prepared for each experiment. Coarse emulsions were prepared by treating substrate mixtures in a Waring Blendor for 5 min. Fine emulsions were prepared by further treating a sample of coarse emulsion for 1 min in a sonic oscillator. Potentiometric assay of lipase activity. The initial velocity of lipolysis was followed by continuous titration at a constant optimal ph, as described by Marchis-Mouren, Sarda, and Desnuelle (1959). The reaction mixture contained, unless otherwise specified, 4.5 ml of substrate emulsion, 1. ml of 1 M CaC12, enzyme solution and water, in a total volume of 3 ml in a 5-ml beaker. The reaction mixture (except enzyme) was brought to 38 C, and a combination electrode from a direct-reading ph meter (Beckman Instruments, Inc., Fullerton, Calif.) was dipped into the mixture. Mixing was achieved by a glass-rod stirrer and by passing a stream of CO2-free nitrogen through a fine capillary tube in the reaction vessel. The ph of the reaction mixture was brought to 8.2 with carbonate-free.1 N NaOH, and the enzyme solution was added. As soon as the ph reached 7.8, a chronometer was started, and small amounts of.1 N NaOH were added from a 2.-ml burette to keep the ph value constant at 7.8. Titration was continued for 5 to 7 min in all cases. Control runs were performed to check nonenzymatic hydrolysis of each substrate. RESULTS Since the natural substrates for lipases are water-insoluble triglycerides, triolein was selected as an assay substrate for the enzyme. Instead of titrating the acids liberated after a standard time of incubation, a continuous titration at a Downloaded from http://jb.asm.org/ on June 27, 218 by guest

95 SHAH AND WILSON J. BACTERIOL. 2; 4 z 5 25 1 2 3 TIME. sec. FIG. 1. Potentiometric test for the determination of lipase activity (triolein substrate). The figures indicate the relative amounts of lipase used in each assay. The open circles and closed circles along curve 1 correspond to different experiments with the same amount of lipase. constant optimal ph was performed, thus giving true initial rates of triglyceride hydrolysis. Experimental conditions were such that lipolysis was zero-order during the assay; under the conditions described, the initial reaction rates were proportional to the enzyme concentration (Fig. 1). Requirement for a fatty acid acceptor for lipase activity. In view of the earlier observation (Shah and Wilson, 1963) that calcium is required, not only for the egg yolk opacity reaction but also for lipolysis, various cations were tested for their role as fatty acid acceptors. The effect of increasing concentrations of Ca ions on lipolysis is shown in Fig. 2. The optimal concentration of calcium under the test conditions was between 5 X 1-4 to 13 M CaCl2. Since some hydrolyzed products of the esters were present in the commercial substrates, 1-3 M CaCl2 was adapted for the assay. Magnesium, strontium, and barium added as chlorides stimulated the lipase activity to the same extent. Taurocholate, however, did not have any effect on lipase activity either in the presence or absence of calcium. Effect of ph on lipase activity. During the earlier studies on the egg yolk factor, lipolysis was 2 5 1 15 2-4 Co,2 M x 1 FIG. 2. Effect of calcium concentration on lipolysis by the egg yolk factor of Staphylococcus aureus. The reaction mixture contained 4.6 ml of 4% triolein emulsion, enzyme preparation, and varying concentrations of CaC12, in a total volume of 3 ml. Continuous titration for $ sec. followed by determining the rate of formation of glycerol, by the procedure of Lambert and Neish (195) and as described by Shah and Wilson (1963). The results describing the effect of ph on lipolysis (Fig. 3) indicate that the optimal ph of the reaction was about 7.8 To study the effect of ph on the stability of the enzyme, 5-ml samples of enzyme solution in distilled water containing.2 mg of protein per ml were adjusted to ph 6., 6.5, 7., 7.5, and 8. with.5 N HC1 or NaOH. The total volume was adjusted to 6 ml with distilled water. After incubation at 38 C for 9 min, the samples were assayed for lipase activity by the potentiometric method. The optimal ph for the stability of the enzyme was about 7, rather than 7.8, the optimal ph for lipolysis (Fig. 3). Substrate specificity. To determine the influence of the length of the acyl group linked to the glycerol moiety, a series of synthetic triglycerides were used to measure their relative rates of hydrolysis. A few complex oils (coconut oil, egg yolk oil, olive oil, and peanut oil) also were investigated. These substrates were not purified further to remove any mono- or diglycerides. Optimal emulsion concentration for enzyme saturation was determined separately for each substrate. All the reaction rates were carried out at ph 7.8, the known optimal ph for triolein hydrolysis, and in the presence of Ca++. The initial rates of hydrolysis rose with increase in the length of the acyl group, became maximal Downloaded from http://jb.asm.org/ on June 27, 218 by guest

VOL. 89, 1965 EGG YOLK FACTOR OF S. AUREUS 951 1. /.r,2 / )PI-_ 6 7 S ph oz 2 -C, -C ~ o (OLD 5.5 6. 6.5 7. 7.5 8. 8.5 FIG. 3. Effect of ph on lipolysis by the egg yolk factor. The reaction vessels contained.2 ml of 4% triolein emulsion, 8.5 X 1c5 moles of CaCl2,.3 ml of.2 m maleate buffer (between ph 5.5 and 6.5) or.2 m glycylglycine buffer (between ph 7. and 8.5), and enzyme preparation. Total volume, 1. ml. The insert shows the effect of ph on the stability of the enzyme. for C4, and then dropped with increasing chain length (Fig. 4). Since the simple triglycerides above C1 are solid at the temperature of the experiment, no attempt was made to determine the initial rates of hydrolysis for these substrates. The initial rates of hydrolysis of the four complex oils and methyl butyrate were as follows: triolein, 1; coconut oil, 83; egg yolk oil, 11; olive oil, 93; peanut oil, 78; methyl butyrate, 7. Inhibitors. The two established esterase inhibitors, atoxyl and tetraethylpyrophosphate, were used in the present study. Enzyme solutions containing.2 mg of protein per ml were incubated at 38 C for 9 min with an equal volume of inhibitor. Controls were incubated at 38 C for 9 min in the absence of the inhibitors. Tetraethylpyrophosphate solutions were adjusted to ph 7 with NaOH. The results of the inhibitor studies indicate that atoxyl, up to a final concentration of 5 X 1-2 M, and tetraethylpyrophosphate, up to a final concentration of 2.5 X 1-3 M, do not have any effect on the lipase. Effect of temperature on lipase activity. The ph 2 4 6 8 1 1 a NUMBER OF CARBONS 1IN CHAIN FIG. 4. Initial rate of lipolysis as a function of the chain length of the acyl group linked to glycerol (simple triglycerides). The rates are expressed by comparison with triolein (18). 2.5. 2.. -.I 1. 31 32 33 34 3' T 1 FIG. 5. Arrhenius plot showing the effect of temperature on the rate of lipolysis of triolein. (Energy of activation = 11,8 cal/mole.) effect of temperature on lipolysis of triolein was investigated between 2 and 45 C. From the data of the Arrhenius plot (Fig. 5), an energy of activation of 11,8 cal/mole was calculated. DISCUSSION The term "lipase" usually covers a large series of enzymes which hydrolyze various soluble and insoluble aromatic or aliphatic esters. The term "lipase" also has been used in a synonymous manner with the term "esterase." Action on soluble substrates, such as p-nitrophenylacetate (Huggins and Lapides, 1947), does not define an esterase, since such compounds are hydrolyzed by the proteolytic enzymes, trypsin and chymotrypsin (Keller, Cohen, and Neurath, 1958). Sarda and Desnuelle (1958) showed that true substrates of pancreatic lipase are insoluble ester emulsions rather than soluble esters. For these reasons, triolein, a long-chain water-in- Downloaded from http://jb.asm.org/ on June 27, 218 by guest

952 SHAH AND WILSON J. BACTERIOL. soluble triglyceride, was selected as an assay substrate for investigating the substrate specificity of the staphylococcal lipase. In view of the insoluble nature of the substrates tested, substrate concentrations have not been given, but the optimal concentration of emulsion required for enzyme saturation was determined separately for each substrate. The necessity of this precaution was indicated by the observations that, when both coarse and fine emulsions were prepared from the same quantity of a substrate, the initial rates of lipolysis with the coarse emulsion were lower than with the fine emulsion. Hence, different reciprocal plots were obtained when measuring the initial rates against substrate weight. Desnuelle (1961) suggested that the site of action of pancreatic lipase is not on molecules dispersed in water but on substrate globules separated from water by an interface. He found that a single curve is obtained, irrespective of the state of the emulsion, when initial rates are plotted against interfacial area rather than substrate weight. Results of this study are consistent with the above observations, but no attempt was made to define Km in terms of interfacial area. The reported relative rates of hydrolysis of various synthetic triglycerides and complex oils differ from those reported by Drummond and Tager (1959a). It is noteworthy that these investigators found that tributyrin and monobutyrin were the only two substrates hydrolyzed by a purified staphylococcal coagulase preparation which also gave an egg yolk opacity reaction. The lack of a fatty acid acceptor such as calcium in the manometric assay used by these authors would explain the high rate of tributyrin hydrolysis and the consequent designation of the enzyme as "tributyrinase." No hydrolysis of other water-insoluble synthetic triglycerides or oils was observed (Drummond and Tager, 1959b). Our studies have shown that triacetin, tripropionin, and tributyrin are hydrolyzed in the absence of a fatty acid acceptor, with rates about three-fourths of that when calcium is added. The absolute requirement of a fatty acid acceptor becomes evident when the acyl moiety of the ester becomes water-insoluble. During the work on comparison of the relative rates of hydrolysis of various triglycerides, it was observed that the triacetin concentration required for maximal reaction rate was very high, compared with all other triglycerides. Triacetin is soluble in water (7 parts per 1 parts). Very little lipolysis was observed with triacetin until substrate saturation in water was reached. However, when the concentration was further increased, resulting in the formation of insoluble emulsion, there was marked increase in lipolytic activity. These observations suggest that staphylococcal lipase is able to hydrolyze triacetin emulsions, but acts very slowly on molecules in true solution. This property sharply differentiates staphylococcal lipase from horseliver esterase, which readily hydrolyzes methyl butyrate in aqueous solution with no increase in activity when an insoluble emulsion of the substrate is used (Sarda and Desnuelle, 1958). That the staphylococcal enzyme is a lipase rather than an esterase is further supported by the fact that atoxyl and tetraethylpyrophosphate, both established esterase inhibitors (Dixon and Webb, 1958), do not have any effect on staphylococcal lipase. A previous report by Drummond and Tager (1959b) on a staphylococcus coagulase preparation, which also gave an egg yolk opacity reaction, showed that tributyrinase activity is not significantly affected by p-chloromercuribenzoate, atoxyl, or tetraethylpyrophosphate. It was suggested by Desnuelle (1961) that lipase substrates as well as inhibitors should be in an emulsified form. This interpretation was supported by the observation that diisopropylfluorophosphate, a powerful esterase inhibitor, did not inhibit pancreatic lipase. Diethyl-p-nitrophenylphosphate, an esterase inhibitor of low solubility, similarly did not inhibit pancreatic lipase when in true solution. However, dilute emulsions of diethyl-p-nitrophenylphosphate strongly inhibit pancreatic lipase (Desnuelle, Sarda, and Ailhaud, 196). Studies on the staphylococcal lipase with such an inhibitor may result in more information on the nature of this enzyme. Since staphylococcal lipase is able to hydrolyze lipoproteins of avian and plant origin, it seems likely that mammalian lipoproteins, particularly those concerned with intracellular structure and function, are possible substrates. The experimental demonstration of the production of staphylococcal lipase in the intracellular environment of the host system may elucidate the possible significance of this activity as a function of virulence in S. aureus. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant E-2962 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED ALDER, V. G., W. A. GILLESPIE, AND G. HERDAN. 1953. Production of opacity in egg-yolk broth by staphylococci from various sources. J. Pathol. Bacteriol. 66:25-21. Downloaded from http://jb.asm.org/ on June 27, 218 by guest

VOL. 89, 1965 EGG YOLK FACTOR OF S. AUREUS 953 BURNS, J., AND D. F. HOLTMAN. 196. Biochemical properties of virulent and avirulent staphylococci. Ann. N.Y. Acad. Sci. 88:1115-1124. DESNUELLE, P. 1961. Pancreatic lipase. Advanc. Enzymol. 23:129-161. DESNUELLE, P., L. SARDA, AND G. AILHAUD. 196. Inhibition de la lipase pancr6atique par le didthyl-p-nitroph6nyl phosphate en emulsion. Biochim. Biophys. Acta 37:57-571. DIXON, M., AND E. C. WEBB. 1958. Enzymes. Academic Press, Inc., New York. DRUMMOND, M. C., AND M. TAGER. 1959a. Enzymatic activity of staphylocoagulase. I. Characterization of an esterase associated with purified preparations. J. Bacteriol. 78:47-412. DRUMMOND, M. C., AND M. TAGER. 1959b. Enzymatic activity of staphylocoagulase. II. Dissociation of plasma clotting from tributyrinase activity. J. Bacteriol. 78:413-421. GILLESPIE, W. A., AND V. G. ALDER. 1952. Production of opacity in egg-yolk media by coagulasepositive staphylococci. J. Pathol. Bacteriol. 64:187-2. HUGGINS, C., AND J. LAPIDES. 1947. Chromogenic substrates. IV. Acyl esters of p-nitrophenol as substrates for the colorimetric determination of esterase. J. Biol. Chem. 17:467-482. KELLER, P. J., E. COHEN, AND H. NEURATH. 1958. Procarboxypeptidase. II. Chromatographic isolation, further characterization, and activation. J. Biol. Chem. 23:95-915. LAMBERT, M., AND A. C. NEISH. 195. Rapid method for estimation of glycerol in fermentation solutions. Can. J. Res. Sect. B 28:83-89. MARCHIS-MOUREN, G., L. SARDA, AND P. DES- NUELLE. 1959. Purification of hog pancreatic lipase. Arch. Biochem. Biophys. 83:39-319. REID, W. B., AND J. B. WILSON. 1959. A study of the staphylococci associated with the bovine udder. Amer. J. Vet. Res. 2:825-831. ROSENDAL, K. 1962. Correlation of virulence of staphylococci from patients with bacteraemia with various laboratory indices, p. 57-574. In N. E. GIBBONS [ed.], Recent progress in microbiology. Symp. Intern. Congr. Microbiol., 8th, Montreal. Univ. Toronto Press, Toronto. SARDA, L., AND P. DESNUELLE. 1958. Action de la lipase pancreatique sur les esters en 6mulsion. Biochim. Biophys. Acta 3:513-521. SHAH, D. B., AND J. B. WILSON. 1963. Egg yolk factor of Staphylococcus aureus. I. Nature of the substrate and enzyme involved in the egg yolk opacity reaction. J. Bacteriol. 85:516-521. Downloaded from http://jb.asm.org/ on June 27, 218 by guest