Amphiphilic properties of bacterial lipopeptides: Self-association and monolayer studies of iturins

Amphiphilic Properties of Bacterial Lipopeptides: Self-Association and Monolayer Studies of Iturins ISABELLE HARNOIS, Rt~GINE MAGET-DANA, AND MARIUS PTAK Centre de Biophysique Moldculaire (CNRS) and Universitd d'Orl&ns, 1A, avenue de la Recherche Scientifique, 45071 Orl&ns c~dex 2, France Received March 19, 1987; accepted June 30, 1987 Iturins are a special class of bacterial lipopeptides with antifungal activity. They are amphiphilic compounds with a heptapeptidic cycle linked to a C~0, C~2 alkyl chain. We have studied the self-association in aqueous solution of iturin A and its O-methyltyrosine derivative that is biologically inactive. We have determined the concentration at which aggregates appear by using diphenylhexatriene as a fluorescent probe or by following the intrinsic fluorescence of the tyrosyl residue. The methylated derivative aggregated at a concentration 10 times lower than iturin A. Iturins formed monolayers at the air-water interface, isotherms of which have been studied. The monolayers of iturin A were not very stable. The isotherm curve presented a transition region that was interpreted as the passage to a tridimensional structure. The monolayers of O-methyltyrosine iturin A were more stable: the equilibrium spreading pressure 7re was 27 m N m -~ as opposed to 9 m N m -1 for iturin A. For both compounds, compression-expansion cycles resulted in nonreproducible hysteresis. © 1988AcademicPress,Inc.

INTRODUCTION

phiphiles. Their polar moiety is formed by a cyclic heptapeptide in which an invariant DTyr residue in position 2 plays a strategic role. The hydrophobic part is formed by a C10, C12 alkyl chain of the r-amino acid closing the peptide cycle, the length of which is comparable to the dimensions of the cycle. Nothing is known until now about the amphiphilic behavior of such antifungal compounds. We report here the first study on their self-association in aqueous medium and on their capacity to form monolayers at the airwater interface. For this we used iturin A, which is one active member of the family whose action on BLM permeability (5, 6) and conformation (7) were previously analyzed. The methylation of the OH group of its Tyr residue considerably modifies the biological activity (8) as well as the effects on polar membranes (9). Because the two samples have the same hydrophobic chains composition, we can determine the effect of a small modification of a crucial peptide residue on the amphiphilic properties.

Iturins are lipopeptides extracted from the culture media of different strains of Bacillus subtilis which exhibit interesting antifungal properties (1, 2). These properties are thought to be related to the interactions between lipopeptides and cytoplasmic membranes (3) although the molecular mechanism of action is not yet elucidated. The activity of iturins sharply depends on their constitution and also on the composition of the target membrane (3). In order to establish structure-activity relationships, we have undertaken several physicochemical studies of these compounds. NMR studies show that their conformations in solution sharply depend on the peptide sequence (4). Studies of their action on planar bilayer lipid membranes show that lipopeptides induce conducting pores (5, 6), the characteristics of which depend on both the peptide sequence and the membrane composition. Considering the primary structure of iturins (see Fig. 1 for iturin A) one can assume that they form a family of special nonionic am85

0021-9797/88 $3.00 Journal of Colloid and Interface Science, Vol. 123, No. 1, May 1988

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

86

HARNOIS, M A G E T - D A N A , A N D P T A K

CO- L A s n _ E) T y r _ D A s n =

?H2 CH3-(CH 211 )--CH-(CH_).-CH I zo I CH3 NH_L n=0

I

L Gin Ser- DAsn_

I L Pro

or1

FIG. 1. Primary structure of iturin A.

MATERIALS A N D M E T H O D S

Materials Iturin A was prepared as described by Peypoux et aL (10) and O-methyltyrosine iturin A according to Ref. (8). The purity of the lipopeptides was attested by 2D N M R experiments. The N M R spectra did not reveal minor compound abundance higher than 2% (7) and prove that these compounds are chemically stable under our experimental conditions. Diphenylhexatriene (DPH) was from KochLight, pyridine and KC1 were from Merck. Distilled water was obtained from a Millipore apparatus.

Methods The self-associative properties of the lipopeptides in solution were determined by using DPH as a fluorescent probe. A solution of DPH (10 -2 M) in freshly distilled tetrahydrofuran was diluted 5000-fold by injection into 10 -3 M Tris-C1 buffer, pH 7.4, with a few seconds sonication (11). The final concentration of D P H w a s 10 - 6 M for experiments with iturin A and 5 X 10-8 M for experiments with the methylated derivative. Iturin A and Omethyltyrosine iturin A exhibit a very low solubility in water and in most common solvents. Pyridine was found to be an adequate solvent for N M R studies in which iturin A remains in the monomeric state for concentrations ~ 4 × 10 -3 M at room temperature. To study the self-association of iturins in water we used the following procedure: the solution of lipopeptide in ethanol was evaporated under nitrogen in order to form a film on the tube wall. The film of lipopeptide was dissolved in the Tris buffer under sonication and the concentration Journal ofColloM and lnterfaceScience, Vol. 123,No. 1, May 1988

of the lipopeptide solution was then determined by measuring the absorbance at 275 nm. The concentration was 2 × 10-4 M for iturin A and 2.10 -5 M for the methylated derivative. Emission fluorescence spectra were recorded on a Kontron spectrofluorometer. The integral of the D P H spectra was used to follow the incorporation of DPH in micelles. Another determination was done by using the intrinsic fluorescence of the tyrosyl residue of the lipopeptide. Monolayer experiments were performed using a conventional Langmuir film balance system conceived in our laboratory by Alain Sanson. The information was stored on an Apple II microcomputer. The thermostated trough was made of Teflon; the mica frame and the sliding barrier were coated with Teflon. The lipopeptides dissolved in pyridine were spread onto distilled water or KCI solution with a 50-#1 Hamilton microsyringe. The rate of compression was routinely 5 ~2. molec -1. min -1. Equilibrium spreading pressure (Tre) was measured at constant area (57 cm 2) by depositing the lipopeptide in the solid state (powder) onto the subphase until no further increase in surface pressure was observed (12). KC1 was heated overnight at 500°C before use and the KC1 solution was filtered on glass wool. The absence of any contamination was checked by spreading 50 #1 of solvent before a compression process. Talc was used as a leak detector. RESULTS

1. Self-Association The self-association ofiturins in an aqueous medium as a function of concentration was demonstrated by two types of measurements. As seen on the semi-log plot of D P H fluorescence versus lipopeptide concentration (Fig. 2A), the intensity of fluorescence abruptly increased around concentrations of 3.5 × 10 -5 M f o r iturin A and 2.8 × 10 - 6 M f o r the methylated derivative. Such an effect is usually re-

AMPHIPHILIC PROPERTIES OF ITURINS

87

If

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i 1,0_6

~,

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i lb_S

~1,_5 lb_4 3.510 [iturin A] tool .I-1

~¢ lb- 6

Z/ I

I

5.10-6

2.10 -6

l- 4

FIG. 2. Self-associationofiturins in aqueous medium. Experiments were done in Tris-C1 buffer 10-3 M, pH 7, T = 20°C. (A) Integral intensity of fluorescenceof DPH (10 -7 M) versus the concentration ofiturins. Xex= 335 nm. The spectra were recorded between 400 and 500 nm. The representation is semi-logarithmic. (B) Fluorescence variation of the D-Tyr residue versus the concentration of iturins. Ordinate: IF/lo is the ratio of the fluorescencespectra at the concentrations C and Co of iturins. Co = 5 × 10-5 M for iturin A and 10-6 M for O-methyltyrosineiturin A. ~ex = 270 nm. The spectra were recorded between 290 and 400 nm. The representation is semi-logarithmic. (A) Iturin A; (©) O-methyltyrosineiturin A.

lated to the incorporation of D P H in a hydrophobic medium formed by aggregates of amphiphiles. This p h e n o m e n o n was confirmed by measuring the fluorescence of the D-Tyr residue. The plot of the fluorescence of the tyrosyl residue versus the concentration of the lipopeptide showed a transition between two states. The lower plateau when the iturin was highly diluted corresponded to the monomeric state. Increasing the concentration of iturin led to an oligomeric state where the tyrosyl residue was in a more hydrophobic environment. The break, around 2 × 10 -5 M for iturin A and 5 X 10 -6 M for the methylated derivative represents the lowest concentration at which aggregated structures appeared.

2. Monolayer Experiments The equilibrium spreading pressure a-e was determined as the time-dependent surface pressure exerted when an excess of solid material is placed onto the aqueous surface. As seen in Fig. 3A, the molecules of iturin A spread very quickly from the solid bulk until the monolayer was very condensed at 7r~.

Then iturin A molecules either aggregated or desorbed (or both) until a new equilibrium was reached at a surface pressure that can be defined alternatively as 7re or as the monolayer stability limit (12). To diminish a possible desorption of molecules, experiments have been done upon 1 M KC1. In this case the surface pressures attained were higher (Tr~a = 18.5 _+ 0.5 m N m -1 compared to 15 _+0.5 m N m - l ; rre = 9 + 0.5 m N m -I compared to 7 + 0.5 m N m-], average of three experiments). The molecules of O-methyltyrosine iturin A behave quite differently. They spread readily too, but the surface pressure increased monotonously until it reached 27.5 _+ 0.5 m N m -1, defined as re. This pressure was stable although a slight decrease was observed that attained 5% 2 h after (Fig. 4A). The surface pressure-area ( r - A ) curve of iturin A spread onto aqueous subphase is shown in Fig. 3B. Changing the rate of compression from 3 to 14 ~2 molec-~ min-i did not affect the shape of the curve. The following results are the average of seven experiments. The mean area per molecule extrapolated to r = 0 m N m -~ (point a) was 122 + 4 ~2 when the subphase contained 1 M KCI (97 Journal of Colloid and Interface Science, Vol. 123, No. 1, May 1988

88

HARNOIS, MAGET-DANA, AND PTAK

n (mNm -1)

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='8

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FIG. 3. Monolayer of iturin A. (A) Spreading of iturin A molecules from solid material: variation with time of the surface pressure. The total area was constant (57 cm2). The subphase contained 1 M KC1. T = 23°C. 7rM,Maximum surface pressure attained; ~re,equilibrium spreading pressure. (B) Isotherm compression of iturin A upon 1 M KC1. Variation of the surface pressure versus the mean area per molecule A at 1 . 2 and 2 bis . 3 and 3 6 i s . . T = 23°C. --~ first compression; ) expansion; . . . . . ~second compression 2 h after the expansions 2 or 2 bis. (Note the correspondence between the surface pressure attained in b and ~reand the surface pressure attained in c and rM.)

+ 5 ~2 upon H20). The 7r-A curve presented an inflection at 9.5 + 0.5 m N m -x and 83.5 + 3.5 ~2 per molecule. The transition region was not horizontal: it ended toward 19.5 _+0.5

m N m -~ and 54 + 3 ~2. We noticed that these pressure values were respectively near the "monolayer stability limit" and the m a x i m u m pressure 7rM reached by the spread molecules

B

n(mNm -1)

40-

A

3: mN~-'l 30-

20-

10

111

20 60 100 ,

I

I

i

I

2'0

Time (rain)

~o

s'o

do

1~o

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1'20

FIG. 4. Monolayer of O-methyltyrosine iturin A. (A) Spreading of O-methyltyrosine iturin A from solid material: variation with time of the surface pressure. The total area was constant (57 cm2). The subphase contained 1 M KC1. T = 23°C. (B) Isotherm compression of O-methyltyrosine iturin A upon 1 M KC1. Variation of the surface pressure versus the main area p e r molecule at T = 23°C. -~ first compression; expansion;--3-~second compression 2 h after the expansion. Journal of Colloid and Interface Science, V o l .

123, No.

1, M a y

1988

AMPHIPHILIC PROPERTIES OF ITURINS as defined above. Furthermore these values were constant when the temperature varied from 16 to 26°C, indicating that this transition was not a phase transition but only an "apparent phase transition" in the terminology of Horn and Gershfeld (13). If we expanded the film from point c we observed a "hysteresis loop" which was nonreproducible over a second compression-expansion cycle done 2 h after the first. This suggested that the iturin A aggregates did not readily dissociate. If we compressed the film beyond point c, the surface pressure increased above ~'M while the molecular area reached the small value of 15 ~2 that could not be the limit area of a molecule of iturin A. This could indicate that iturin A aggregates were displaced from the surface in either the subphase or the air phase. This hypothesis was supported by the following observation: when the film was expanded from point d, the surface pressure fell very abruptly. If the film was compressed again 2 h after, the 7r-A curve was that of a condensed state and the mean area extrapolated at ~- = 0 m N m -1 was then about 50 ,~2 per molecule, i.e., the same order of magnitude as the area observed at the end of the transition. The 7r-A curve of O-methyltyrosine iturin A spread upon 1 M KC1 is shown in Fig. 4B. The mean area per molecule extrapolated to 7r = 0 m N m -1 was 99.5 + 3.5 A2 (average of six experiments). The shape of the isotherm was that of an expanded monolayer until it reached between 20 and 25 m N m -1. Then the monolayer became unstable and the ~r-A curve became progressively flatter. The kink observed at 15 m N m-~ was sometimes absent in freshly prepared solutions. However, it progressively reappeared in time although any chemical degradation could be observed. Thus the hypothesis of the coexistence of two species of O-methyltyrosine iturin A molecules differing either by their conformation or their aggregation state was reaffirmed. As observed for iturin A, a hysteresis loop was established under a compression-expansion cycle. But if the lipopeptide film was

89

compressed again 2 h after expansion, the 7rA curve corresponded still to an expanded state but more compressible than the fresh film (the compressibility coefficient B = -1/,4(0,4/ &r)T was then 0.021 m N - l m instead of 0.01 m N - l m at ~- = 9 m N m - l ) . The area per molecule at the "lift off" was again around 100 A2. We noticed too, that the kink observed on the ~--A curve of the fresh film had disappeared. Analogous behavior has been observed by Rolandi et al. when studying monolayers of ether lipids (14). Unfortunately, the authors have not discussed this peculiarity. DISCUSSION (1) The self-association ofiturins as a function of concentration is rather similar to that usually observed with nonionic amphiphiles which form micelles. The concentration values at which they appear are in the same range as the critical micelle concentrations (CMC) usually found for a number of nonionic detergents. For instance, CMC values around l0 -5 M a r e found for compounds with an alkyl chain length of 12 carbon atoms (e.g., saccharose monoester fatty acid (15)) to 14 carbon atoms (e.g., alkylglucosides (16)). The lower value found for the O-methylated derivative demonstrates the crucial role played by the OTyr residue in the amphiphilic character of iturins. However, at present the size and dispersity of aggregates have not been determined. (2) Both compounds, iturin A and Omethyltyrosine iturin A are able to form monolayers at the air-water interface, properties of which depend on the hydrophobicity of the tyrosyl side chain. The shape of the 7rA isotherm of iturin A was similar to that observed with polypeptides in a helical conformation (17, 18) where the plateau was interpreted as a passage from a monolayer to a bilayer. It was then plausible to consider that the transition observed in our study was, in this case also, a transition between a two-dimensional state to a more compact three-dimensional structure. Indeed it is noteworthy that Journal of Colloid and Interface Science, Vol. 123, No. 1, May 1988

90

HARNOIS, MAGET-DANA, AND PTAK

at the end of the transition A "~ 55 A2, groups is in the size of the molar moiety of i.e., about half the value at the lift off (A iturins: the peptide cycle closed by a/3-amino 120 ,~2). acid contains seven residues and is then conThe existence of a metastable organization siderably larger than the previously mentioned which could be a bilayer is compatible with groups. That explains the importance of the the hysteresis phenomena and the time de- change induced by a minor modification of pendence of the surface pressure (Fig. 3A). For the Tyr side chain. This structure contributes iturin A, an expansion starting from point c to the determination of the special properties of the ~r-A isotherm does not restore com- of the monolayers formed at the air-water inpletely a monolayer, and an expansion starting terface. The presence of several different lipidic from point d leads to a bilayer state. Then we chains prevents us from going further in the can suppose that the spreading of the solid interpretation. That is why we are now synmaterial forms first a bilayer and then a thesizing a series of compounds of well-defined chain constitution, in order to test the role of monolayer. The behavior of the O-methyltyrosine iturin this part of the molecule. It is premature to relate the amphiphilic A films differed greatly from that of iturin A. First, its stability was much higher. Second, properties ofiturins to their antifungal activity. we did not observe a pseudo-transition. Third, However, one point deserves special attention: while iturin A molecules apparently remained the CMC value of iturin A is around the conself-associated after a compression, the mol- centration which gives the maximum biologecules of O-methyltyrosine iturin A returned ical activity. Indeed the minimum inhibitory in a monomeric state with a unique confor- concentration (MIC) of iturin A upon the growth ofSaccharomyces cerevisiae is 3 × 10-5 mation. Some remarks have to be made: (i) the pres- M (2) and in the same way the concentration sure increase and the more expanded state of of iturin A giving 100% hemolysis on human the monolayer when the subphase contains erythrocytes is 2.5 × 10-5 M(3). However, the electrolytes, although not explained, is consis- lower CMC value does not explain, by itself, tent with the observations of Lange on non- the absence of biological activity of the methionic detergent films (19); (ii) the observed area ylated derivative. According to this low CMC of molecules in a monolayer has only a statis- value, the partition coefficient of the lipopeptical significance. However, the molecular area tide would be in favor of the hydrophobic core value at the lift off can give an estimation of of the lipid membrane, but the conformation the cross-sectional area of the peptide cycle of the lipopeptide and the structures formed lying at the air-water interface. And in fact, inside the membrane, with or without lipids, the value found (around 100 ~2) is in good might be different than that in the case of itagreement with the calculated conformation urin A. In the same way, the monolayer approach of the peptidic cycle in solution in pyrireveals that these closely related compounds dine. show great differences in their molecular packing. The study of mixed lipid-lipopeptide CONCLUSION monolayers that is now being undertaken Lipopeptides such as iturins exhibit a be- would give us further information concerning havior rather similar to that of classical am- the interaction of these two lipopeptides with phiphiles. They self-associate in a narrow range lipid membranes. of concentrations to form aggregates and they ACKNOWLEDGMENTS form monolayers at the air-water interfaces. A major difference with nonionic detergents We are indebted to Fran~oisePeypouxand Georges carrying hydroxyl, ether, ester, nitrile, etc. Michel from the laboratoire de BiochimieMicrobienne Journal of Colloid and Interface Science, Vol. 123, No. 1, May 1988

AMPHIPHILIC PROPERTIES OF ITURINS de l'Universit6 de Lyon for the gift of the lipopeptides studied. We thank Daniel Genest for hdpful advice in the fluorescence field. This work was supported by CNRS and Universit6 d'Od6ans.

REFERENCES 1. Delcambe, L, and Devignat, R., Acad. R. Sci. Colon. 6, 1 (1957). 2. Besson, F., Peypoux, F., and Michel, G., J. Antibiot. 8, 828!(1979). 3. Besson, E., Peypoux, F., Michel, G., and Delcambe, L., Biochem. Biophys. Res, Commun. 81, 297 (1978). 4. Genest, M., Marion, D., Caille, A., and Ptak, M., Eur. J, Biochem. 169, 389 (1987). 5. Maget-Dana, R., Ptak, M., Peypoux, F., and Michel, G., Biochim. Biophys. Acta 815, 405 (1985). 6. Maget-Dana, R., Heitz, F., Ptak, M., Peypoux, F., and Guinand, M., Biochem. Biophys. Res. Commun. 129, 965 (1985). 7. Marion, D., Genest, M., Caille, A., Peypoux, F., Michel, G., and Ptak, M., Biopolymers 25, 153 (1986).

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8. Peypoux, F., Besson, F., Michel, G., and Delcambe, L., J. Antibiot. 32, 136 (1979). 9. Maget-Dana, R., Ptak, M., Peypoux, F., and Michel, G., Biochim. Biophys. Acta 898, 1 (1987). 10. Peypoux, F., Guinand, M., Michel, G., Delcambe, L., Das, C., Varenne, P., and Lederer, E., Tetrahedron 29, 3455 (1973). 11. Shinitzky, M., and Barenholz, Y., .L Biol. Chem. 249, 2652 (1974). 12. Gaines, G. L., Jr., "Insoluble Monolayers at LiquidGas Interfaces," p. 136. Interscience, New York, 1966. 13, Horn, L. W., and Gershfeld, N. L., Biophys. J. 18, 302 (1977). 14. Rolandi, R., Schindler, H., De Rosa, M., and Gambacorta, A., Eur. Biophys. J. 14, 19 (1986). 15. De Grip, W, J., and Bovee-Geurts, P. H. M., Chem. Phys. Lipids 23, 321 (1979). 16. Wachs, W., and Hayano, J., Kolloid Z. 181, 169 (1962). 17. Malcolm, B. R., Proc. Roy. Soc. A 305, 363 (1968). 18. Baglioni, P., Dei, L., and Gabrielli, G., J. Colloid Interface Sci. 93, 402 (1983). 19. Lange, H., in "Nonionic Surfactants" (M. J. Schick, Ed.), p. 443. Dekker, New York (1966).

Journalof ColloidandInterfaceScience,Vol. 123.No. 1, May 1988