The Inhibition of Lysozyme by Bile Acids G. VANTRAPPEN, MD, AggHO, Y. GHOOS, PhD, and T. PEETERS, PhD

The effect of bile acids on the bacteriolytic activity of lysozyme towards Micrococcus lysodeikticus was studied in vitro. All bile acids tested inhibited lysozyme activity. Conjugated bile acids were better inhibitors than their unconjugated homologs and sulfation resulted in still stronger inhibition. A study o f UV-difference spectra of bile acid--lysozyme mixtures suggests that bile acids distort the tertiary structure of the enzyme. The inhibitionconcentration curves of micelle-forming bile acids were bell-shaped, and peak inhibition was apparently related to the critical micellar concentration. The inhibition--concentration curves of sulfated bile acids, which do not form micelles, are characterized by a plateau of maximal inhibition. A mechanism of lysozyme activation by bile acid micelles is proposed. Our results illustrate the complex interactions between antibacterial compounds in the gut. As bile acids are known to inhibit lipase activity as well, these studies suggest that bile acids" may have an important influence on intestinal enzyme activity in general.

The factors which control the growth of the microflora in the small intestine are multiple and not fully understood (1). Gastric acidity (2, 3), the propulsive motor activity of the gut (4, 5), and components of the succus entericus (6) are some of the factors which play a role in this regulation. Bile acids not only have an important function in lipid digestion and absorption but they also interact with the intestinal flora in many ways. Certain intestinal bacteria are capable of deconjugating bile salts (7, 8). Intestinal microorganisms may also alter the structure of bile acids by dehydroxylation, oxidation, and reduction (9-11). On the other hand, bile acids have antibacterial properties against some gram-positive microorganisms (12-15) and may in this way exert a selective pressure on the bacterial flora in the small bowel. Intestinal as well as lacrimal, nasal, and vaginal secretions contain lysozyme (mucopeptide N-acetylmuramyl hydrolase, EC 3.2.1.t7) which hydrolyzes the glucoaminopeptide component of the bacterial cell wall (16). Biochemical (17), histochemi-

cal (18, 19), and immunohistochemical (20) studies indicate that intestinal lysozyme is stored in the granules of the Paneth cell, secreted into the crypt lumen, and transferred up to the top of the villus (21, 22). In a few bacterial species the enzymatic attack by lysozyme ultimately results in lysis and death of the bacteria. The antibacterial activity of lysozyme is influenced by detergents. Lysozyme and detergents may act synergistically (23, 24), and Tween 20 is known to activate lysozyme (25). There is little doubt that the antibacterial properties of bile acids are related to their detergent-like activity. Bile acids, however, may also influence the antibacterial activity of lysozyme by interaction with the enzyme rather than with the bacteria, because it is conceivable that a lowering of the surface tension of a lysozyme solution may cause denaturation of the enzyme. To test this hypothesis we studied the in vitro effect of various bile acids on the lysis of Micrococcus lysodeikticus by lysozyme.

From the Laboratory of Gastrointestinal Pathophysiology, Department of Medical Research, University of Leuven, Leuven, Belgium. Address for reprint requests: Prof. Dr. G. Vantrappen, Laboratory of Gastrointestinal Pathophysiology, A. Z. St. Rafa61, 3000 Leuven, Belgium.

Hen's egg white lysozyme, lot 4CC-8010, was obtained from Sigma Chemical Co., St. Louis, Missouri; Micrococcus lysodeikticus, lot U3962, from Mann research Lab., Orangeburg, New York, and all bile acids with the exception of the sulfated derivates from Supelco Inc., Belle-

Digestive Diseases, Vol. 21, No. 7 (July 1976)

MATERIALS AND METHODS

547

VANTRAPPEN ET AL % INHIBITION 100 i

9

50

60 MIN

B0

GDC lrnM

60 40 20 ,

I

I

i

10

20

30

40

~

I

Fig 1. Effect of the duration of incubation of lysozyme with different bile acids, at the concentration indicated, upon the lyric activity of lysozyme towards Microeoccus lysodeikticus. Glycodeoxycholic acid, 1 mM (.); taurochenodeoxycholic acid, 0.5 mM (A); cholic acid, 1 mM (1); cholic acid monosulfate, 0.05 mM (C~).

fonte, Pennsylvania. The sulfated bile acids were prepared according to the method of Palmer and Bolt (26). An additional purification was achieved by preparative thin-layer chromatography in the solvent ethylacetatebutanol-acetic acid-water (80:60:30:30) (27). This solvent system resulted in a good separation of the various sulfated derivatives. The Rr values for cholic acid and its derivatives, for instance, were 0.93 for the unsulfated acid, 0.64 for the monosulfate, 0.30 for the disulfate, and 0.14 for the trisulfate. The identity of the sulfated derivatives was checked by elementary analysis, and their purity by gas chromatography after solvolysis. Purity amounted to approximately 98%. Enzyme Assay, Lysozyme activity was assayed by following the change in turbidity at 450 nm of a suspension of Micrococcus lysodeikticus cells (28). To 0.2 ml of a freshly prepared lysozyme solution (50 /xg/ml) 2.7 ml of bile acid was added to yield the concentration of bile acid to be tested, and this mixture was incubated for given lengths of time. The bile acid solutions were made up in S6rensen buffer pH 7.6. Whenever necessary the solubility of the bile acids was checked enzymatically (29). Sulfated bile acids were also determined enzymatically after solvolysis (26). At the time of the assay 0.1 ml of the bacterial suspension (final concentration 100/xg/ml) was added to the lysozyme-bile acid mixture. All assays and the preincubations were carried out at 37 +- 0.5 ~ C in a constant temperature block mounted in a Unicam SP 800 spectrophotometer. The initial rate of lysis was recorded as A A/min, and the percentage inhibition was calculated from the initial rate in the presence and in the absence of bile acid. Under the experimental conditions the initial absorbance was 0.4, and the rate of lysis in the absence of bile acid, which was checked in every run, was approximately 0.016 A A/min. Absorbance always decreased linearly with time during the first 10 min of the experiments,

548

and therefore this period was used for the calculation of the initial rate. Difference spectra, The difference spectra of (lysozyme-bile acid)-(lysozyme) at pH 7.6 (0.066 M phosphate buffer) were measured in a SP 800 spectrophotometer at a temperature of 37 + 0.5 ~ C. The lysozyme concentration was 0.1%, the bile acid concentration as indicated in the legends to the figures. The solutions were filtered through a membrane filter with 0.5-/xm pore size, type VM. (Millipore Corp., Bedford, Massachusetts) to remove any solid matter. The solutions were used within 30 min after being mixed. The baseline was recorded with 0.1% lysozyme in both beams. The spectra of the pure inhibitors at the appropriate concentration were also recorded with buffer in the reference cell. Where required the difference spectra were corrected for deviations in the baseline and for absorption of the inhibitor. Lysozyme concentrations were checked spectrophotometrically using an extinction coefficient of 26.9 at 280 nm for a l% solution in a cuvette with 1.0-cm optical path (30). Critical Micellar Concentration (CMC). The CMC was determined spectrophotometrically using the spectrum change of Rhodamine 6G as an indicator (31). RESULTS All bile acids tested inhibited the lytic activity of lysozyme towards Micrococcus lysodeikticus. The degree of i n h i b i t i o n d e p e n d e d not o n l y on the bile acid c o n c e n t r a t i o n a n d the d u r a t i o n of its inc u b a t i o n with l y s o z y m e b u t also on the n a t u r e of the bile acid i n v o l v e d . T h e effect of the i n c u b a tion time is s h o w n in F i g u r e 1. T h e i n h i b i t i o n of the l y s o z y m e a c t i v i t y b y bile acids i n c r e a s e d as the i n c u b a t i o n time p r o g r e s s e d . After 40 m i n a stable v a l u e was o b t a i n e d a n d , a c c o r d i n g l y , all o t h e r studies were p e r f o r m e d after an i n c u b a t i o n

TABLE I.

OF LYSOZYMEBY B I L E ACIDSATA CONCENTRATIONOF 0.5 MM*

INHIBITION

Nonsulfated

Cholic Chenodeoxycholic Deoxycholic Lithocholic

Free

Conjugated Glyco Tauro

I1 11 3 N.D.

15 N .D. 60 N.D.

22 58 N.D. N.D.

Monosulfated Conjugated Free Tauro 40 80 N.D. 90

25 74 N .D. 89

* Except for free chenodeoxycholic and deoxycholic acid which reach saturation at 0.4 and 0.3 raM, respectively. The final concentration in the incubation mixture was therefore 0.35 mM for chenodeoxycholic acid and 0.25 mM for deoxycholic acid. Results shown are mean values of triplicate determinations. Digestive Diseases, Vol. 21, No. 7 (July 1976)

LYSOZYME INHIBITION BY BILE ACIDS period of 1 hr. Different bile acids resulted in different degrees of inhibition (Table 1). Conjugation of bile acids increased the inhibitory power, and sulfation had an even greater effect. For cholic acid the percentage depended upon the degree of sulfation. Thus, at a concentration of 0.5 mM cholic acid monosulfate resulted in a 40% inhibition, the disulfate in 49%, and trisulfate in 63%. Hydroxylation apparently has the opposite effect. In the conjugated and sulfated form, cholic acid, which has three OH groups, is less potent than deoxy- and chenodeoxycholic acid, which have two OH groups. The monohydroxyl compound, lithocholic acid, which is almost insoluble, could not be tested. However, the more soluble lithocholic acid sulfate was the strongest inhibitor of all sulfated bile acids tested. Conjugation apparently has little effect with sulfated acids. When the effect of bile acid concentration was studied, a typical curve was obtained with taurochenodeoxycholic acid monosulfate (Figure 2). Maximal inhibition was reached at a concentration of 1 mM, and higher concentrations of bile acid did not influence the percent inhibition appreciably. In contrast, the inhibition by the nonsulfated homolog first increased to a peak value of 84% at 1.0 mM, declined at higher concentrations, and increased again at still higher concentrations. A similar phenomenon was observed with two other nonsulfated bile acids tested. The concentrations at which these bile acids produced a peak inhibition are well within the range of concentrations at which one would expect these bile acids to reach their critical micellar concentration. Table 2 tabulates the concentrations at which peak inhibition is observed, together with the critical micellar concentrations found under the conditions of the lysozyme assays. For all bile acids tested and for all concentrations, control experiments were run in which the lysozyme solution was replaced by buffer. No lysis

TABLE 2. CRITICALMICELLARCONCENTRATIONS AND CONCENTRATIONS OF PEAK INHIBITION

I % INHIBITION 1001 80

t

~

Bile acid

Glycocholic Taurochenodeoxycholic Glycodeoxycholic

Digestive Diseases, Vol. 21, No. 7 (July 1976)

Peak inhibition (raM)

4.0 2.5 2.2

3.0 1.0 1.0

[]

q

TCDC1S TCDC GDC

60

GC

4O 2O

1

2

3

~

5 mM

Fig 2. Concentration-inhibition curves for different

bile acids: taurochenodeoxycholicacid monosulfate (m); taurochenodeoxycholicacid ( ,5); glycodeoxycholic acid (e); glycocholicacid (&).

was detected under these conditions even after prolonged incubation. Figure 3 shows typical difference spectra of lysozyme produced by cholic acid, taurocholic acid, and glycodeoxycholic acid. In each case a peak at 286 and 294 nm was observed. The intensity of the taurocholic acid peak at 294 nm was almost linearly related to the concentration of bile acid in the range 0.5-4.0 raM. It was not possible to obtain difference spectra with the sulfated bile acids, due to the precipitation of lysozyme they produced. Precipitation could be avoided by lowering the concentration of sulfated bile acids to 0.02 mM, but the difference

0.10

O,OC 250

CMC (raM)

u

~

.

.

.

.

'o

3 0

~'m

i

i

Fig 3. Difference spectra of lysozyme and mixtures of lysozyme and bile acids. The lysozyme concentration was 1 mg/ml, in 0.066 M phosphate buffer, pH 7.6. The bile acid concentration in the sample cuvette was 2 mM for cholic acid (curve 1), 2 mM for taurocholic acid (curve 2), and 0.5 mM for glycodeoxycholic acid (curve 3).

549

VANTRAPPEN ET AL spectra thus obtained were diffuse and not interpretable. It should be noted that the lysozyme concentration in these experiments is about 1000 times higher than in the inhibition studies. DISCUSSION This study indicates that bile acids inhibit the bacteriolytic activity of lysozyme in vitro. Conjugated bile acids are stronger inhibitors than their unconjugated homologs, and sulfation of bile acids, particularly of the unconjugated acids, also results in greater inhibitory activity. The lysis of Micrococcus lysodeikticus by lysozyme is a multistage process which may be influenced by several factors. Oligosaccharides inhibit lysis by competition with the lysozyme substrate present in the bacterial wall (32). On the other hand, high concentrations of basic amines such as spermine and spermidine do not interfere with enzyme-substrate interactions, but stabilize the protoplast membranes and prevent lysis of the protoplast formed by the enzyme (33, 34). Little is known about the interactions of bile with intestinal enzymes. Inhibition of lipase by bile acids has been described and was attributed to a distortion of the tertiary structure of lipase by bile acids (35). A similar phenomenon may be operative in the case of lysozyme. Thermal and chemical denaturation of lysozyme results in changes of its UV spectrum (30, 36). We found that incubation of bile acids with lysozyme produces perturbation peaks at 294 and 286 nm. Such peaks have also been observed in the presence of substrate-like compounds and have been interpreted as evidence for tryptophan perturbations (37). This is not surprising since three tryptophan residues are located in the active center of lysozyme: residues 62, 63, and 108. Apparently incubation with bile acids results in a distortion of the active center of the enzyme. The rate of denaturation may be appreciated from the increase in inhibition with the duration of incubation. The mechanism of the bile acid-lysozyme interaction is unknown. A tentative mechanistic model can be envisaged. In the roughly ellipsoidal lysozyme molecule (axis 30, 40, 45 A), polar amino acid residues are mostly located at the outside, nonpolar residues at the inside of the molecule. A deep cleft partially lined by hydrophobic residues runs up one side and houses the active center of the enzyme. The tryptophan residues mentioned above also are found along this cleft (16, 35). Bile acids have a

550

rather cylindrical shape (20 A_ long, 2.5 A radius) with all polar residues on one half of the molecu!e (39). It is thus quite possible that a bile acid molecule penetrates the cleft, with the nonpolar half undergoing Van der Waals interactions with the nonpolar environment inside the cleft. Polar groups on the other half of the cylinder may form hydrogen bonds or undergo ionic attraction and thus lock the bile acid in place. In this way bile acids could distort the tertiary structure of the active center and block it as well. The proposed mechanism is speculative but can partially explain the differences in inhibitory activity of different bile acids. As the nonpolar portion of the molecule is similar in all bile acids, nonpolar interactions will be almost identical for all bile acids. Polar interactions, however, will be stronger when the bile acids are conjugated and still stronger after sulfation, particularly for the unconjugated bile acids. Inhibitory activity may be seen to increase in this order (Table 1). Still, the effect of hydroxylation is difficult to explain. The decrease in inhibitory power observed with increasing concentrations beyond a certain concentration is surprising. Apparently another mechanism, involving a stimulation of lysozyme, counteracts the inhibitory effect. Bile acids are known to lyse pneumococci because they stimulate the release of autolytic enzymes (14). However, no such effect was observed in our control experiments. Detergents could also dissolve the outer lipolytic layers of the bacterial wall exposing the lysozyme substrate. In our model such an effect is excluded as the substrate of choice for lysozyme; Micrococcus lysodeikticus is devoid of an outer lipolytic layer. The decrease in surface tension caused by all detergents may also have an antibacterial effect and may partially explain the bacteriostatic properties of bile salts towards streptococci (15). However, in a detailed study of the enhancement of lysozyme activity by the nonionic detergent Tween 20 (25), it was concluded that lysozyme was bound to detergent micelles in a favorable orientation with respect to substrate particles, and that surface tension was not connected in any way to this phenomenon. Tween 20 probably binds the polar residues of the lysozyme molecule by hydrogen bonds, thus eliminating nonproductive collisions. An important physicochemical characteristic of bile acids is the formation of micelles. In bile acid micelles, 6-10 molecules are joined together by their nonpolar halves to form particles about 20 ]k long and with a radius of 7-15 A (39). Such a miDigestive Diseases, Vol. 21, No. 7 (July 1976)

LYSOZYME INHIBITION BY BILE ACIDS celle could bind lysozyme molecules in a favorable orientation because in micelles all polar groups of the bile acids are on the outer mantle. We speculate that at concentrations above the CMC, two opposing mechanisms are active; inhibition by bile acid monomers and enhancement by micelles. As the monomer concentration remains constant above the CMC, the effect would be a decrease in inhibition, the concentration corresponding to the peak inhibition being the CMC. Although the experimentally defined values for the CMC were all somewhat higher than the peak inhibition values, this is not necessarily an objection to this hypothesis, first, because the transition from a monomer solution to a solution containing micelles is not a sharp one (40), and second, because an accurate determination of the CMC is almost impossible. The involvement of micelle formation might also be deduced from the fact that sulfated bile acids, which do not form micelles, do not show a decrease in inhibition at increasing concentrations. According to this hypothesis one would expect that a progressive increase in micelle concentration would result in a progressive activation of lysozyme. This is inconsistent with our data which show that the initial activation is followed at higher concentrations by increasing inhibition. This phenomenon may be related to the formation of secondary micelles, but more elaborate physicochemical studies are needed to clarify this point. Although the bactericidal properties of lysozyme are limited to a small number of bacteria, increasing evidence is accumulating for a much wider spectrum of synergistic combinations of lysozyme and other compounds. Particularly interesting in this respect is the in vitro bactericidal action of lysozyme and IgA antibodies against gram-negative microorganisms (41). In our study it was shown that bile acids inhibit the enzymatic activity of lysozyme. Floch et al (12) demonstrated that unconjugated bile acids inhibit anerobic human intestinal bacteria, whereas conjugated bile acids do not have this effect. Our studies indicate that unconjugated bile acids inhibit lysozyme to a much lesser extent than the conjugated homologs. If the in vitro results can be applied to the in vivo situation in the gut, deconjugation of bile acids enhances the antibacterial properties of the intestinal contents in at least two different ways. Paradoxically, unconjugated bile salts are present in the large bowel where huge bacterial populations are found, whereas the less-active conjugated forms are present in the upper small Digestive Diseases', Vol. 21, No. 7 (July 1976)

bowel where there is a sparse flora. This stresses the complex nature of the mechanisms that control the growth of bacterial populations in the gut. Although sulfated bile acids are found in the serum and urine of patients with hepatic diseases (42), little is known about the presence and role (if any) of sulfated bile acids in intestinal disease. Sulfated bile acids are such potent inhibitors of lysozyme activity that it may be justified to search these bile acids in patients with inflammatory bowel diseases. Bile salts have mostly been related to enzymes in terms of bile acid-lipid interactions. Thus the influence of bile acids on lypolytic enzymes is well documented. Although Lippel and Olson (43) found no influence of bile acids on nonlipolytic digestive enzymes of the pancreas, inhibition of pepsin has been reported (44). The inhibition of lysozyme demonstrated in this paper justifies further efforts in this direction. Preliminary evidence from our laboratory suggests amylase and elastase activities also are influenced by bile acids. REFERENCES 1. Donaldson RM Jr: Small bowel bacterial overgrowth. Adv Intern Med 16:191-212, 1970 2. Gray JDA, Shiner M: Influence of gastric pH on gastric and jejunal flora. Gut 8:574-581, 1967 3. Drasar BS, Shiner M, McLeod GM: Studies on the intestinal flora. I. The bacterial flora of the gastrointestinal tract in healthy and achlorhydric persons, Gastroenterology 56:7179, 1969 4. Dixon JM: The fate of bacteria in the small intestine. J Pathol Bacteriol 79:131-140, 1960 5. Summers RW, Kent TH: Effects of altered propulsion on rat small intestinal flora. Gastroenterology 59:740-744, 1970 6. Goldsworthy NE, Florey H: Some properties of mucus, with special reference to its antibacterial functions. Br J Exp Pathol 11:192-208, 1930 7. Drasar BS, Hill MJ, Shiner M: The deconjugation of bile salts by human intestinal bacteria. Lancet 1:1237-1238. 1966 8. Shimada K, Bricknell KS, Finegold SM: Deconjugation of bile acids by intestinal bacteria: Review of literature and additional studies. J Infect Dis 119:273-280, 1969 9. Midtvedt T, Norman A: Anaerobic bile acid transforming microorganisms in rat intestinal content. Acta Pathol Microbiol Scand 72:33%344, 1968 10. Aries V, Crowther JS, Drasar BS, Hill MJ: Degradation of bile salts by human intestinal bacteria. Gut 10:575-576, 1969 11. Garbutt JT, Wilkins RM, Lack L, Tyor MP: Bacterial modification of taurocholate during enterohepatic recirculation in normal man and patients with small intestinal disease. Gastroenterology 59:553-566, 1970 12. Floch MH, Gershengoren W, Elliott S, Spiro HM: Bile acid inhibition of the intestinal microflora--a function for simple bile acids? Gastroenterology 61:228-233, 1971 13. Downie AW, Stent L, White SM: The bile solubility ofpneu55 l

VANTRAPPEN

14.

15.

16. 17.

18.

19. 20.

21. 22. 23.

24.

25.

26.

27.

28.

29.

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30.

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44.

ET A L

en Analyse, Band I1. HV Bergmeyer (ed). Weinheim, Vetlag-Chorale, 1970, p 1824 Shimaki N, lkeda K, Hamaguchi K: Interaction of alcohols with lysozyme. 1I. Studies on difference absorption spectra. J Biochem 68:795-803, 1970 Corrin ML, Harkins WD: Determination of the critical concentration for micelle formation in solutions of colloidal electrolYtes by the change of a dye. J Am Chem Soc 69:679-683. 1947 Sharon N: The chemical structure of lysozyme substrates and their cleavage by the enzyme. Proc R Soc London. Set B 167:402-415, 1967 Grossowicz N, Ariel M: Mechanism of protection of cells by spermine against lysozyme induced lysis. J Bacteriol 85:293300, 1963 Tabor CW: Stabilization of protoplasts and speroplasts by spermine and other polyiamines. J Bacteriol 83:1101-1111, 1962 Morgan RG, Hoffman NE: The interaction of lipase, lipase cofactor and bile salts in triglyceride hydrolysis. Biochim Biophys Acta 248: 143-148, 1971 Gerlsma SY, Stuur ER: The effect of polyhydric and monohydric alcohols on the heat-induced reversible denaturation of lysozyme and ribonuclease, lnt J Pept Protein Res 4:377383, 1972 Neuberger A, Wilson BM: Inhibition of lysozyme by derivatives of D-glucosamine. Biochim Biophys Acta 147:473-486, 1967 Johnson L: The structure and function oflysozyme. Sci Prog Oxford 54:367-385, 1966 Small DM: The physical chemistry of cholanic acids. The Bile Acids. PP Nair, D Kritchevsky (eds). New York, Plenum Press, 1971, pp 302-326 Makino S, Reynolds JA, Tanford C: The binding of deoxycholate and Triton X-100 to proteins. J Biol Chem 248:49264932, 1973 Adinolfi M, Glynn AA, Lindsay M, Milne CM: Serological properties of A antibodies to Escherichia coli present in human colostrum. Immunology 10:517-526, 1966 Makino L: Sulfated bile acids in urine of patients with hepatobiliary disease. Lipids 8:47-49, 1973 Lippel K, Olson JA: The activity of non-lipolytic digestive enzymes of the pancreas in the presence of conjugated bile salts. Biochim Biophys Acta 127:243-245, !966 Tompkins RK, Hayashi RM: Investigations into the reduction of pepsin activity by bile. Am J Surg 128:633-637, 1974

Digestive Diseases, Vol. 21, No. 7 (July 1976)