Chemistry and Physics of Lipids 181 (2014) 34–39
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Interactions of a bacterial trehalose lipid with phosphatidylglycerol membranes at low ionic strength José A. Teruel, Antonio Ortiz, Francisco J. Aranda ∗ Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, E-30100 Murcia, Spain
a r t i c l e
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Article history: Received 27 February 2014 Received in revised form 21 March 2014 Accepted 26 March 2014 Available online 4 April 2014 Keywords: Trehalose lipids Phosphatidylglycerol DSC Light scattering Fluorescence polarization FT-IR
a b s t r a c t Trehalose lipids are bacterial biosurfactants which present interesting physicochemical and biological properties. These glycolipids have a number of different commercial applications and there is an increasing interest in their use as therapeutic agents. The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action and therefore the study of the interaction between these biosurfactants and biological membranes is critical. In this study, we examine the interactions between a trehalose lipid (TL) from Rhodococcus sp. and dimyristoylphosphatidylglycerol (DMPG) membranes at low ionic strength, by means of differential scanning calorimetry, light scattering, fluorescence polarization and infrared spectroscopy. We describe that there are extensive interactions between TL and DMPG involving the perturbation of the thermotropic intermediate phase of the phospholipid, the destabilization and shifting of the DMPG gel to liquid crystalline phase transition to lower temperatures, the perturbation of the sample transparency, and the modification of the order of the phospholipid palisade in the gel phase. We also report an increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer. These changes would increase the monolayer negative spontaneous curvature of the phospholipid explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. The observations contribute to get insight into the biological mechanism of action of the biosurfactant and help to understand the properties of the intermediate phase display by DMPG at low ionic strength. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glycolipids of bacterial origin are widespread biosurfactants which contain carbohydrates in combination with long-chain aliphatic or hydroxyl aliphatic acids. An important group of glycolipid biosurfactants is formed by trehalose-containing glycolipids (Asselineau and Asselineau, 1978). These trehalose lipids are mainly produced by rhodococci and present such interesting physicochemical and biological properties that a number of different commercial applications have been proposed for them (Lang and Philp, 1998). There is an increasing interest in the use of biosurfactants as therapeutic agents (Banat et al., 2000; Rodrigues et al., 2006). Trehalose lipids have been reported to have antiviral properties (Azuma et al., 1987; Hoq et al., 1997), and it has also been shown that trehalose lipids have excellent growth inhibition and differentiation-inducing activities against human leukemia cells
∗ Corresponding author. Tel.: +34 868884760; fax: +34 868884147. E-mail address: [email protected] (F.J. Aranda). http://dx.doi.org/10.1016/j.chemphyslip.2014.03.005 0009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.
such as myelogenous leukemia cell K562, promyelocytic ceukemia cell HL60 and basophilic leukocyte KU812 (Isoda et al., 1996, 1997b; Sudo et al., 2000). In addition, trehalose lipids inhibit the activity of phospholipids- and calcium-dependent protein kinase C of HL60 cells (Isoda et al., 1997a), and show immunomodulating activity (Kuyukina et al., 2007). The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action, thus, the study of the interaction between these biosurfactants and biological membranes is very important. We have found that a trehalose lipid from Rhodococcus sp. (TL) (Fig. 1) permeabilizes phospholipid membranes (Zaragoza et al., 2009) and induces red blood cells hemolysis (Zaragoza et al., 2010). We have studied the effect of TL on the most important membrane phospholipids in order to get insight into the molecular interaction between these biosurfactants and the lipidic component of biological membranes. We have shown that TL increases the fluidity of phosphatidylcholine membranes forming domains in the fluid state (Aranda et al., 2007), and that it exhibits and important dehydrating effect on the interfacial region of saturated phosphatidylethanolamines (PE) and greatly promotes the formation of the inverted hexagonal HII phase in
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2.2. Trehalose lipids production and purification
Fig. 1. The chemical structure of the most abundant trehalose lipid produced by Rhodococcus sp. (TH–succ–C11 –C10 –C7 ).
Strain 51T7 was isolated from an oil-contaminated soil sample after culture enrichment with kerosene, and was identified as Rhodococcus sp. (Espuny et al., 1995). This strain was maintained by fortnightly cultures on Trypticase Soy Agar (Pronadisa, Spain) and preserved in cryovials at −20 ◦ C. Biosurfactants were produced, purified and it structure was characterized as described before (Espuny et al., 1995, 1996). The TL surfactants are a mixture of several components containing four acyl substituents located at position 2, 3, 4 and 2 , these substituents were identified as succinic, heptanoic, decanoic and undecanoic acids. Fig. 1 depicts a representative most abundant component TH–succ–C10 –C11 –C7 . 2.3. Differential scanning calorimetry (DSC)
unsaturated phosphatidylethanolamines (Ortiz et al., 2008). We have also shown that TL was able to affect the thermotropic phase transition of phosphatidylserine in the absence and presence of calcium (Ortiz et al., 2009). Recently, we have studied the effects of TL on the gel and fluid phases of phosphatidylglycerol (PG), a predominant phospholipid of the cytoplasmic membrane of bacteria. Phosphatidylglycerol has been used extensively as model for acidic phospholipid membranes (Eklund and Kinnunen, 1986; Alakoskela and Kinnunen, 2007; Pabst et al., 2008). The physicochemical properties of phospholipids present in membranes of microorganisms are of interest because it has been suggested that the lipid composition of bacterial membranes plays an important role in the interaction with antimicrobial compounds (Lohner, 2001). We showed that TL did not affect the macroscopic bilayer organization of DMPG, but the presence of the biosurfactant produced a small decrease of the bilayer thickness together with an increase in the fluidity of the phospholipids acyl chains (Ortiz et al., 2011). DMPG has received particular attention because of its unusual phase properties. It appears to form single bilayers when dissolved in water at low lipid and low salt concentration (Gershfeld et al., 1986), exhibiting a very unusual thermal profile, with a broad transition with a width greater than 15 ◦ C. The structure of DMPG aggregates along this remarkable transition region is still a matter of debate, recent studies suggest that DMPG forms leaky vesicles at both gel and fluid phases (Barroso et al., 2012) which are highly perforated with large holes (Enoki et al., 2012). In order to understand the influence of this biosurfactant on this unusual phase behavior of DMPG, we have purified TL from Rhodococcus sp. (Fig. 1) and carried out a study of the effect of the glycolipid on the thermotropic and structural properties of DMPG membranes at low ionic strength, using differential scanning calorimetry (DSC), light scattering, steady-state fluorescence polarization and infrared spectroscopy (FT-IR).
2. Materials and methods 2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phospo-rac-glycerol, sodium salt (dimyristoylphosphatidylglycerol, DMPG), was purchased from Avanti Polar Lipids Inc. (Birmingham, AL). 1,6-Diphenyl-1,3,5hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl1,3,5-hexatriene (TMA-DPH) were from Sigma–Aldrich (Spain). All the other reagents were of the highest purity available. Purified water was deionized in a Milli-Q equipment from Millipore (Bedford, MA), and filtered through 0.24 m filters prior to use. Stock solutions of DMPG and TL were prepared in chloroform/methanol (8:1) and stored at −20 ◦ C. Phospholipid concentrations were determined by phosphorous analysis (Böttcher et al., 1961).
Samples for DSC were prepared by mixing the appropriate amounts of DMPG and TL in chloroform/methanol (8:1). The solvent was gently evaporated under a stream of dry N2 to obtain a thin film at the bottom of a glass tube. Last traces of solvent were removed by a further 3 h desiccation under high vacuum. To the dry samples 2 ml of a buffer containing 10 mM Hepes, 0.1 mM EDTA pH 7.4 (the pH was balanced with small aliquots of 5 M NaOH, and the final [Na+ ] was estimated to be approximately 2.2 mM) was added, and vesicles were formed by vortexing the mixture essentially as described previously (Alakoskela and Kinnunen, 2007). The suspensions were incubated for 90 min at 60 ◦ C with continuous shaking and vortexed vigorously every 30 min three times. The lipid suspensions so obtained were used in the measurements during the same day without any cold incubation. Experiments were performed using a MicroCal MC2 calorimeter (MicroCal, Northampton, USA). The final phospholipid concentration was 2 mg ml−1 . The heating scan rate was 60 ◦ C h−1 . 2.4. Light scattering Samples containing 2 mg/mL DMPG prepared as described above were measured at 550 nm (angle of 90 ◦ C) using a PTI Quantamaster spectrofluorometer (Photon Technology, NJ, USA). The cell holder was thermostated using a Peltier device, and the measurements were taken under continuous stirring. 2.5. Steady-state fluorescence polarization Steady-state fluorescence polarization measurements were carried out with samples prepared as described above, containing 0.5 mg/mL DMPG and 1% fluorescence probes and performed with a PTI Quantamaster spectrofluorometer (Photon Technology, NJ, USA) equipped with motorized polarizers. Quartz cuvettes with a path length of 10 mm were used. The cell holder was thermostated using a Peltier device, and the measurements were taken under continuous stirring. For monitoring DPH and TMA-DPH fluorescence, the excitation wavelength was set at 358 nm, and emission was monitored at 430 nm. The sample temperature was allowed to equilibrate for 5 min before fluorescence was recorded for 60 s, and then the excitation shutter was kept closed during heating to the next temperature, in order to minimize any photoisomerization of DPH and TMA-DPH. Steady-state fluorescence polarization values were calculated from the following equation: P=
IVV − GIVH IVV + GIVH
where IVV and IVH are the fluorescence intensities with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. G is the grating
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Fig. 3. Changes in 90◦ light scattering intensity relative to initial value at 4 ◦ C (RSI) for DMPG containing TL at different molar fractions.
Fig. 2. DSC heating thermograms for DMPG containing TL at different molar fractions.
factor, calculated as the ratio of the efficiencies of the detection system for vertically and horizontally polarized light, and is equal to IHV /IHH . 2.6. Fourier Transform infrared spectroscopy (FT-IR) For the infrared measurements multilamellar vesicles containing 10 mol DMPG were prepared as described above, in 50 l of the same buffer prepared in D2 O. Samples were placed in between two CaF2 windows (25 mm × 2 mm) separated by 25 m Teflon spacers and transferred to a Symta cell mount. Infrared spectra were acquired in a Nicolet 6700 FT-IR (Madison, WI). Each spectrum was obtained by collecting 256 interferograms with a nominal resolution of 2 cm−1 . The equipment was continuously purged with dry air in order to minimize the contribution peaks of atmospheric water vapor. The sample holder was thermostated using a Peltier device (Proteus system from Nicolet). Spectra were collected at 2 ◦ C intervals, allowing 5 min equilibration between temperatures. The D2 O buffer spectra taken at the same temperatures were subtracted interactively using either Omnic or Grams (Galactic Industries, Salem, NH) software. 3. Results and discussion We first characterized the effects of TL on the thermal phase behavior of DMPG by DSC. Fig. 2 shows heating thermograms of DMPG at increasing TL concentrations. In contrast to the very sharp and intense peak associated with the chain melting transition of pure DMPG at high ionic strength (Pabst et al., 2007; Zhang et al., 1997; Ortiz et al., 2011), pure DMPG at low ionic strength (Fig. 2) shows a complex thermogram with a pretransition peak around 10 ◦ C (Tp ), and a melting transition peak with four components: three peaks closely spaced, with the first sharp peak at approximately 17 ◦ C (Ton ), followed by two wide peaks, with the first centered at approximately 20 ◦ C (T1 ) and the second at 23 ◦ C (T2 ). A fourth component is at a higher temperature of approximately 40 ◦ C (Toff ). This complex behavior agrees well with previous characterization of DMPG phase transition at low ionic strength (Riske et al., 2002; Alakoskela and Kinnunen, 2007; Riske et al., 2009; Alakoslela et al., 2010), with Ton defining the beginning of
the melting process and Toff setting its end. The gel–fluid transition region, which extends in the range of 17–40 ◦ C, has been called intermediate phase (IP) and the presence of broad peaks (T1 and T2 ) superimposed in this region indicates the occurrence of structural changes between Ton and Toff (Lamy-Freund and Riske, 2003). The presence of low concentration of TL, such as 0.05 mol fraction, has profound effects on the thermotropic behavior of the DPMG: it produces the disappearing of the pretransition peak and considerably alters the melting region profile. The enthalpy of Ton peak is decreased and the peak is shifted to lower temperatures, T1 and T2 peaks seem to merge in a broader peak and Tof peak is also shifted to lower temperatures. Increasing concentrations of TL makes Ton and Toff peaks to disappear and finally only a unique broad peak is found starting at lower temperature (around 10 ◦ C) and extending over a shorter range of 10–20 ◦ C. One of the most characteristic features of the intermediate phase of DMPG at low ionic strength is the sample transparency (Heimburg and Biltonen, 1994; Riske et al., 1997). Fig. 3 shows the relative 90◦ light scattering intensity for pure DMPG membranes and those containing TL. The beginning and the end of the pure DMPG intermediate phase are characterized by a sharp decrease in scattering at Ton and a sharp increase at Toff . Contrary to the turbid gel and fluid phases (below Ton and above Toff ), the intermediate phase is optically transparent. We were able to detect the small decrease of scattering corresponding to the pretransition around 10 ◦ C, but, similarly to early reports (Riske et al., 1997; Lamy-Freund and Riske, 2003) we were not able to detect the higher temperature additional scattering shift (Tpost ) recently described (Alakoskela and Kinnunen, 2007; Alakoskela et al., 2010). There is considerably hysteresis for this additional increase in scattering (Alakoskela and Kinnunen, 2007) and most probably we cannot detect it due to the difference in phospholipid concentration and heating rate of our set up. The 90◦ scattering scan in the presence of low concentration of TL establishes the disappearing of the pretransition and the shift of both Ton and Toff to lower temperatures. The presence of high concentration of TL evidences a further shift of Ton to lower temperatures and a clear decrease of the temperature span of the intermediate state, which presents only minor changes in light scattering. To monitor the acyl chain mobility of DMPG membranes, fluorescence polarization measurements were carried out using DPH and TMA-DPH probes, which report changes in membrane order in the deep inner phospholipid palisade and the interfacial region of the membrane respectively. Fig. 4 shows the fluorescence polarization of DPH (Fig. 4A) and TMA-DPH (Fig. 4B) incorporated into pure DMPG systems and those containing TL as a function of temperature. In the case of pure DMPG membranes, the polarization values of both probes display a similar behavior to those reported previously for DPH (Lamy-Freund and Riske, 2003), showing upon heating a continuously decreasing acyl chain order within the intermediate phase. However, as compared to the DPH anisotropy data reported previously, our DPH and TMA-DPH
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Fig. 5. Temperature dependence of the maximum of the symmetric CH2 stretching absorption band exhibited by pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
Fig. 4. Steady state fluorescence polarization as a function of temperature of DPH (A) and TMA-DPH (B) incorporated into membranes composed of pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
experiments reveal a steeper decline upon the disappearance of the intermediate state. The data agree with the electron spin resonance results for DMPG including a phosphatidylcholine probe with paramagnetic label (Lamy-Freund and Riske, 2003) and with recent DPH-phosphatidylcholine anisotropy values (Alakoskela and Kinnunen, 2007). The presence of TL makes the second steeper decrease in polarization to disappear and the transition is shifted to lower temperatures in accordance to DSC and scattering data reported above. Interestingly, the final polarization value, i.e. disorder, reached at high temperature in the fluid phase is similar in all the systems. However, before the phase transition, in the gel phase a small decrease in polarization, i.e. disordering effect, is observed for both probes in the presence of TL. The shift of the onset of the phase transition to lower temperatures together with the disordering effect in the gel phase, indicates that the hydrophobic moieties of TL incorporate into the PG acyl chain palisade and, like in the case of high ionic strength (Ortiz et al., 2011), perturb the acyl chains and shift the phase transition to lower values. A number of hypotheses have been proposed to explain the characteristics of the intermediate phase. These hypothesis include the formation of a sponge phase (Heimburg and Biltonen, 1994; Schneider et al., 1999) and secondary changes in the aggregation state of DMPG (Riske et al., 1997), but on the light of recent evidence their contribution seems unlikely (Riske et al., 2001, 2002, 2004; Lamy-Freund and Riske, 2003; Alakoskela and Kinnunen, 2007). Based in new experimental evidence, it has been proposed that the DMPG intermediate phase would be structured as vesicles containing multiple holes in the bilayer with an associated positively curved defect to cover the free edge of the bilayer (Riske et al., 2004; Alakoskela and Kinnunen, 2007). Recently it has been shown that the thermal stability of the intermediate phase can be changed in a highly predictable way by modifying the spontaneous curvature of DMPG by additives (Alakoskela et al., 2010), providing evidence that the intermediate phase is a structure with positive curvature (defects) and supporting the perforated vesicle structure suggested before. According to evidence, all compounds increasing the monolayer negative spontaneous curvature decrease the temperature span of the intermediate phase and lower the temperature of its dissolution. An attractive relation was found between the ability of a compound to destabilize the intermediate phase and its ability to
promote the formation of inverted non-lamellar phases (Alakoskela et al., 2010). In this context, it is interesting to note that we have found that TL was able to promote the formation of inverted hexagonal HII phases in dielaidoylphosphatidylethanolamine systems by producing a more effective cone shape phosphatidylethanolamine molecule, i.e. increasing its negative spontaneous curvature (Ortiz et al., 2008). Moreover, we have recently described that, at high ionic strength, TL increases the fluidity of DMPG acyl chains and dehydrates the interfacial region of the phosopholipid, both effects leading to an increase of the negative spontaneous curvature of the membrane (Ortiz et al., 2011). In order to get insight into the molecular mechanism of destabilization of the intermediate state by TL, we studied the interaction between TL and DMPG by infrared spectroscopy at low ionic strength. The infrared spectra of glycerophospholipids contain useful information regarding the intermolecular interactions that occur in the different domains of the molecule. In this way, the CH2 stretching region includes information about the conformational disposition of the hydrocarbon chains, whereas the C O stretching region includes information about lipid interfacial hydration–hydrogen bonding interaction. The CH2 symmetric stretching band near 2850 cm−1 is of remarkable importance because of its sensitivity to changes in the mobility and in the conformational disorder of the hydrocarbon chains (Mendelson and Mantsch, 1986). Fig. 5 presents the temperature dependence of the frequency at the absorbance maximum of the symmetric CH2 stretching vibration band of the infrared spectra of pure DMPG and DMPG/TL systems. For pure DMPG the data show a sharp increase of about 2 cm−1 in the frequency of the band maximum at temperature that coincide with the onset of the intermediate state. This frequency increase is characteristic of the chain melting transition of hydrated phospholipids (Mantsch and McElhaney, 1991) and results from the increased conformational disorder in the hydrocarbon as a consequence of the introduction of a high population of gauche conformers (Lewis and McElhaney, 1996). This sharp increase in the frequency of the methylene band span over a range of temperature which coincides with that of Ton , T1 and T2 , i.e. from 17 to 29 ◦ C, and from that point the increase is continuous with temperature. This data support the previous suggestion that at Toff the bilayer is practically completely fluid, and that at Toff there is effectively a transition between two fluid phases (Alakoskela et al., 2010). In the presence of TL, the chain melting phase transition starts at lower temperatures than that of the pure DMPG, in accordance with the DSC experiments shown above. At temperatures at which pure DMPG is found in the gel state, the presence of TL produces an increase of the frequency of the band, indicating and increase in gauche conformers and thus an increase in hydrocarbon chain conformational disorder in the gel phase. The C O stretching band of pure DMPG is a fairly broad band in the 1750–1700 cm−1 , and seems to be a summation of at least two
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Fig. 6. Temperature dependence of the maximum of the carbonyl stretching absorption band exhibited by pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
subcomponents centered near 1741 and 1727 cm−1 (Zhang et al., 1997). The relative intensities of these component bands reflect the contribution of subpopulations of dehydrated and hydrated carbonyl groups (Blume et al., 1988). Fig. 6 shows the temperature dependence of the frequency at the absorbance maximum of the C O stretching band of the infrared spectra of pure DMPG and DMPG/TL systems. For pure DMPG, the onset of the intermediate state produces a shift of the maximum to lower frequencies, reflecting an increase in intensity of the underlying component band at 1727 cm−1 , attributed to a higher amount of hydrogen bonded carbonyl groups resulting from a phase state-induced increase in the hydration of the polar–apolar interface (Zhang et al., 1997). Interestingly, an additional shift to lower frequencies is found at the Toff transition. The latter suggests that a further hydration of the aqueous interface is taken place even when no change in the population of the CH2 gauche conformers is detected (Fig. 5). This change at the interfacial phase could explain the steeper decline in fluorescence polarization upon the disappearance of the intermediate state shown in Fig. 4. The presence of TL produces a disappearing of the additional decrease in frequency at Toff and a broadening of the initial shift in frequency at Ton . It is interesting to note that the presence of TL produced a net shift of the maximum of the C O band to higher frequencies than that of the pure DMPG, during the intermediate phase. This increase in frequency suggests an increase in the proportion of dehydrated C O component and that TL, at low ionic strength, interacts with the interfacial region of the DMPG bilayer, decreasing the hydrogen bonding of the C O groups with the water molecules of the hydration layer, in an overall way similar to that found at high ionic strength (Ortiz et al., 2011). However, in spite of this dehydration effect exerted by TL the fluorescence polarization value reached in the liquid crystalline phase is similar to that of the pure phospholipid (Fig. 4). It seems that the presence of TL produces different effects on the DMPG molecule; it causes conformational disorder in the methylene region in the gel phase, while dehydrates the interfacial region in the intermediate phase. These two effects gives rise to an increase of the monolayer negative spontaneous curvature of DMPG, and according to the hypothesis that the intermediate state is structured in perforated vesicles with positive curvature defects (Alakoskela et al., 2010) explain why TL behaves as a destabilizer of the intermediate state. 4. Conclusions We have carried out a study of the molecular interaction between a trehalose lipid bacterial biosurfactant and membranes composed of DMPG at low ionic strength. We have shown that TL is able to incorporate into DMPG bilayers and affect their structural properties. DSC data showed that TL produces the disappearing of the pretransition peak and considerably alters the melting region
profile with the different peaks merging in a broad peak which is shifted to lower temperatures. Light scattering studies confirmed the shift of the transitions to lower temperatures and a drastic decrease of the scattering change characteristic of the intermediate phase. Fluorescence polarization of membrane probes evidenced that TL did not change the final polarization value in the fluid phase but it induced a small disordering effect in the gel phase. Infrared experiments revealed that the biosurfactant increased the fluidity of the phospholipids acyl chains and decreased the hydration of the interfacial region of the membrane. These two effects would give rise to an increase of the monolayer negative spontaneous curvature of DMPG explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. Given the key structural and functional importance of PG in membranes and the wide diversity of biological actions played by trehalose lipids, the results presented here contribute to the knowledge of its underlying molecular mechanisms of action and also contribute to get insight into the properties of the DMPG intermediate phase. Conflict of interest The authors declare that there are no conflicts of interest. References Alakoskela, J.M., Kinnunen, P.K., 2007. Thermal phase behavior of DMPG: the exclusion of continuous network and dense aggregates. Langmuir 10, 4203–4213. Alakoskela, J.-M., Parry, M.J., Kinnunen, P.K.J., 2010. The intermediate state of DMPG is stabilized by enhanced positive spontaneous curvature. Langmuir 26, 4892–4900. Aranda, F.J., Teruel, J.A., Espuny, M.J., Marqués, A., Manresa, A., Palacios-Lidón, E., Ortiz, A., 2007. Domain formation by a Rhodococcus sp. biosurfactant trehalose lipid incorporated into phosphatidylcholine membranes. Biochim. Biophys. Acta 1768, 2596–2604. Asselineau, C., Asselineau, J., 1978. Trehalose-containing glycolipids. Prog. Chem. Fats Other Lipids 16, 59–99. Azuma, M., Suzutani, T., Sazaki, K., Yoshida, I., Sakuma, T., Yoshida, T., 1987. Role of interferon in the augmented resistance of trehalose 6,6 -dimycolate-treated mice to influenza virus infection. J. Gen. Virol. 68, 835–843. Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53, 495–508. Barroso, R.P., Perez, K.R., Cuccovia, I.M., Lamy, M.T., 2012. Aqueous dispersions of DMPG in low salt contain leaky vesicles. Chem. Phys. Lipids 165, 169–177. Blume, A., Hübner, W., Messner, G., 1988. Fourier transform infrared spectroscopy of 13 C O-labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry 27, 8239–8249. Böttcher, C.J.F., Van Gent, C.M., Pries, C., 1961. A rapid and sensitive sub-micro phosphorus determination. Anal. Chim. Acta 24, 203–204. Eklund, K.K., Kinnunen, P.K., 1986. Effects of polyamines on the thermotropic behaviour of dipalmitoylphosphatidylglycerol. Chem. Phys. Lipids 39, 109–117. Enoki, T.A., Henriques, V.B., Lamy, M.T., 2012. Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition. Chem. Phys. Lipids 165, 826–837. Espuny, M.J., Egido, S., Mercadé, M.E., Manresa, A., 1995. Characterization of trehalose tetraester produced by a waste lube oil degrader Rhodococcus sp. 51T7. Toxicol. Environ. Chem. 48, 83–88. Espuny, M.J., Egido, S., Rodón, I., Manresa, A., Mercadé, M.E., 1996. Nutritional requirements of a biosurfactant producing strain Rhodococcus sp. 51T7. Biotechnol. Lett. 18, 521–526. Gershfeld, N.L., Stevens Jr., W.F., Nossal, R.J., 1986. Equilibrium studies of phospholipid bilayer assembly. Coexistence of surface bilayers and unilamellar vesicles. Faraday Discuss. Chem. Soc. 81, 19–28. Heimburg, T., Biltonen, R.L., 1994. Thermotropic behavior of dimyristoylphosphatidylglycerol and its interaction with cytochrome c. Biochemistry 33, 9477–9488. Hoq Md, M., Suzutani, T., Toyoda, T., Horijke, G., Yoshida, I., Azuma, M., 1997. Role of ␥␦ TCR lymphocytes in the augmented resistance of trehalose 6,6 -dimycolatetreated mice to influenza virus infection. J. Gen. Virol. 78, 1597–1603. Isoda, H., Shinmoto, H., Kitamoto, D., Matsumura, M., Nakahara, T., 1996. Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages. Cryotechnology 19, 79–88. Isoda, H., Kitamoto, D., Shinmoto, H., Matsumura, M., Nakahara, T., 1997a. Microbial extracellular glycolipid induction of differentiation and inhibition of the protein kinase C activity of human promyelocytic leukaemia cell line HL60. Biosci. Biotechnol. Biochem. 61, 609–614. Isoda, H., Shinmoto, H., Kitamoto, D., Matsumura, M., Nakahara, T., 1997b. Differentiation of human promyelocytic leukaemia cell line HL60 by microbial extracellular glycolipids. Lipids 32, 263–271.
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Riske, K.A., Politi, M.J., Reed, W.F., Lamy-Freund, M.T., 1997. Temperature and ionic strength dependent light scattering of DMPG dispersions. Chem. Phys. Lipids 89, 31–44. Riske, K.A., Amaral, L.Q., Lamy-Freund, M.T., 2001. Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies. Biochim. Biophys. Acta 1511, 297–308. Riske, K.A., Döbereiner, H.-G., Lamy-Freund, M.T., 2002. Gel–fluid transition in dilute versus concentrated DMPG aqueous dispersions. J. Phys. Chem. 106, 239–246. Riske, K.A., Amaral, L.Q., Döbereiner, H.-G., Lamy, M.T., 2004. Mesoscopic structure in the chain-melting regime of anionic phospholipids vesicles: DMPG. Biophys. J. 86, 3722–3733. Riske, K.A., Amaral, L.Q., Lamy, M.T., 2009. Extensive perforation coupled with the phase transition region of an anionic phospholipids. Langmuir 25, 10083–10091. Rodrigues, L., Banat, I.M., Teixeira, J., Oliveira, R., 2006. Biosurfactants: potential applications in medicine. J. Antimicrob. Chemother. 57, 609–618. Schneider, M.F., Marsh, D., Jahn, W., Kloesgen, B., Heimburg, T., 1999. Network formation of lipid membranes: triggering structural transitions by chain melting. Proc. Natl. Acad. Sci. 96, 14312–14317. Sudo, T., Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Nakahara, T., Suzuki, A., Kobayashi, Y., Jin, C., Murata, T., Yokohama, K.K., 2000. Induction of the differentiation of human HL60 promyelocytic leukaemia cell line by succinoyl trehalose lipids. Cryotechnology 33, 259–264. Zaragoza, A., Aranda, F.J., Espuny, M.J., Teruel, J.A., Marqués, A., Manresa, A., Ortiz, A., 2009. Mechanism of membrane permeabilization by a bacterial trehalose lipid biosurfactant produced by Rhodococcus sp. Langmuir 25, 7892–7898. Zaragoza, A., Aranda, F.J., Espuny, M.J., Teruel, J.A., Marqués, A., Manresa, A., Ortiz, A., 2010. Hemolytic activity of a bacterial trehalose lipid biosurfactant produced by Rhodococcus sp.: evidence for a colloid-osmotic mechanism. Langmuir 26, 8567–8572. Zhang, Y.-P., Lewis, R.N.A.H., McElhaney, R.N., 1997. Calorimetric and spectroscopic studies of the thermotropic phase behaviour of the n-saturated 1,2-diacylphosphatidylglycerols. Biophys. J. 72, 779–793.
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Interactions of a bacterial trehalose lipid with phosphatidylglycerol membranes at low ionic strength José A. Teruel, Antonio Ortiz, Francisco J. Aranda ∗ Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, E-30100 Murcia, Spain
a r t i c l e
i n f o
Article history: Received 27 February 2014 Received in revised form 21 March 2014 Accepted 26 March 2014 Available online 4 April 2014 Keywords: Trehalose lipids Phosphatidylglycerol DSC Light scattering Fluorescence polarization FT-IR
a b s t r a c t Trehalose lipids are bacterial biosurfactants which present interesting physicochemical and biological properties. These glycolipids have a number of different commercial applications and there is an increasing interest in their use as therapeutic agents. The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action and therefore the study of the interaction between these biosurfactants and biological membranes is critical. In this study, we examine the interactions between a trehalose lipid (TL) from Rhodococcus sp. and dimyristoylphosphatidylglycerol (DMPG) membranes at low ionic strength, by means of differential scanning calorimetry, light scattering, fluorescence polarization and infrared spectroscopy. We describe that there are extensive interactions between TL and DMPG involving the perturbation of the thermotropic intermediate phase of the phospholipid, the destabilization and shifting of the DMPG gel to liquid crystalline phase transition to lower temperatures, the perturbation of the sample transparency, and the modification of the order of the phospholipid palisade in the gel phase. We also report an increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer. These changes would increase the monolayer negative spontaneous curvature of the phospholipid explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. The observations contribute to get insight into the biological mechanism of action of the biosurfactant and help to understand the properties of the intermediate phase display by DMPG at low ionic strength. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glycolipids of bacterial origin are widespread biosurfactants which contain carbohydrates in combination with long-chain aliphatic or hydroxyl aliphatic acids. An important group of glycolipid biosurfactants is formed by trehalose-containing glycolipids (Asselineau and Asselineau, 1978). These trehalose lipids are mainly produced by rhodococci and present such interesting physicochemical and biological properties that a number of different commercial applications have been proposed for them (Lang and Philp, 1998). There is an increasing interest in the use of biosurfactants as therapeutic agents (Banat et al., 2000; Rodrigues et al., 2006). Trehalose lipids have been reported to have antiviral properties (Azuma et al., 1987; Hoq et al., 1997), and it has also been shown that trehalose lipids have excellent growth inhibition and differentiation-inducing activities against human leukemia cells
∗ Corresponding author. Tel.: +34 868884760; fax: +34 868884147. E-mail address: [email protected] (F.J. Aranda). http://dx.doi.org/10.1016/j.chemphyslip.2014.03.005 0009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.
such as myelogenous leukemia cell K562, promyelocytic ceukemia cell HL60 and basophilic leukocyte KU812 (Isoda et al., 1996, 1997b; Sudo et al., 2000). In addition, trehalose lipids inhibit the activity of phospholipids- and calcium-dependent protein kinase C of HL60 cells (Isoda et al., 1997a), and show immunomodulating activity (Kuyukina et al., 2007). The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action, thus, the study of the interaction between these biosurfactants and biological membranes is very important. We have found that a trehalose lipid from Rhodococcus sp. (TL) (Fig. 1) permeabilizes phospholipid membranes (Zaragoza et al., 2009) and induces red blood cells hemolysis (Zaragoza et al., 2010). We have studied the effect of TL on the most important membrane phospholipids in order to get insight into the molecular interaction between these biosurfactants and the lipidic component of biological membranes. We have shown that TL increases the fluidity of phosphatidylcholine membranes forming domains in the fluid state (Aranda et al., 2007), and that it exhibits and important dehydrating effect on the interfacial region of saturated phosphatidylethanolamines (PE) and greatly promotes the formation of the inverted hexagonal HII phase in
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2.2. Trehalose lipids production and purification
Fig. 1. The chemical structure of the most abundant trehalose lipid produced by Rhodococcus sp. (TH–succ–C11 –C10 –C7 ).
Strain 51T7 was isolated from an oil-contaminated soil sample after culture enrichment with kerosene, and was identified as Rhodococcus sp. (Espuny et al., 1995). This strain was maintained by fortnightly cultures on Trypticase Soy Agar (Pronadisa, Spain) and preserved in cryovials at −20 ◦ C. Biosurfactants were produced, purified and it structure was characterized as described before (Espuny et al., 1995, 1996). The TL surfactants are a mixture of several components containing four acyl substituents located at position 2, 3, 4 and 2 , these substituents were identified as succinic, heptanoic, decanoic and undecanoic acids. Fig. 1 depicts a representative most abundant component TH–succ–C10 –C11 –C7 . 2.3. Differential scanning calorimetry (DSC)
unsaturated phosphatidylethanolamines (Ortiz et al., 2008). We have also shown that TL was able to affect the thermotropic phase transition of phosphatidylserine in the absence and presence of calcium (Ortiz et al., 2009). Recently, we have studied the effects of TL on the gel and fluid phases of phosphatidylglycerol (PG), a predominant phospholipid of the cytoplasmic membrane of bacteria. Phosphatidylglycerol has been used extensively as model for acidic phospholipid membranes (Eklund and Kinnunen, 1986; Alakoskela and Kinnunen, 2007; Pabst et al., 2008). The physicochemical properties of phospholipids present in membranes of microorganisms are of interest because it has been suggested that the lipid composition of bacterial membranes plays an important role in the interaction with antimicrobial compounds (Lohner, 2001). We showed that TL did not affect the macroscopic bilayer organization of DMPG, but the presence of the biosurfactant produced a small decrease of the bilayer thickness together with an increase in the fluidity of the phospholipids acyl chains (Ortiz et al., 2011). DMPG has received particular attention because of its unusual phase properties. It appears to form single bilayers when dissolved in water at low lipid and low salt concentration (Gershfeld et al., 1986), exhibiting a very unusual thermal profile, with a broad transition with a width greater than 15 ◦ C. The structure of DMPG aggregates along this remarkable transition region is still a matter of debate, recent studies suggest that DMPG forms leaky vesicles at both gel and fluid phases (Barroso et al., 2012) which are highly perforated with large holes (Enoki et al., 2012). In order to understand the influence of this biosurfactant on this unusual phase behavior of DMPG, we have purified TL from Rhodococcus sp. (Fig. 1) and carried out a study of the effect of the glycolipid on the thermotropic and structural properties of DMPG membranes at low ionic strength, using differential scanning calorimetry (DSC), light scattering, steady-state fluorescence polarization and infrared spectroscopy (FT-IR).
2. Materials and methods 2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phospo-rac-glycerol, sodium salt (dimyristoylphosphatidylglycerol, DMPG), was purchased from Avanti Polar Lipids Inc. (Birmingham, AL). 1,6-Diphenyl-1,3,5hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl1,3,5-hexatriene (TMA-DPH) were from Sigma–Aldrich (Spain). All the other reagents were of the highest purity available. Purified water was deionized in a Milli-Q equipment from Millipore (Bedford, MA), and filtered through 0.24 m filters prior to use. Stock solutions of DMPG and TL were prepared in chloroform/methanol (8:1) and stored at −20 ◦ C. Phospholipid concentrations were determined by phosphorous analysis (Böttcher et al., 1961).
Samples for DSC were prepared by mixing the appropriate amounts of DMPG and TL in chloroform/methanol (8:1). The solvent was gently evaporated under a stream of dry N2 to obtain a thin film at the bottom of a glass tube. Last traces of solvent were removed by a further 3 h desiccation under high vacuum. To the dry samples 2 ml of a buffer containing 10 mM Hepes, 0.1 mM EDTA pH 7.4 (the pH was balanced with small aliquots of 5 M NaOH, and the final [Na+ ] was estimated to be approximately 2.2 mM) was added, and vesicles were formed by vortexing the mixture essentially as described previously (Alakoskela and Kinnunen, 2007). The suspensions were incubated for 90 min at 60 ◦ C with continuous shaking and vortexed vigorously every 30 min three times. The lipid suspensions so obtained were used in the measurements during the same day without any cold incubation. Experiments were performed using a MicroCal MC2 calorimeter (MicroCal, Northampton, USA). The final phospholipid concentration was 2 mg ml−1 . The heating scan rate was 60 ◦ C h−1 . 2.4. Light scattering Samples containing 2 mg/mL DMPG prepared as described above were measured at 550 nm (angle of 90 ◦ C) using a PTI Quantamaster spectrofluorometer (Photon Technology, NJ, USA). The cell holder was thermostated using a Peltier device, and the measurements were taken under continuous stirring. 2.5. Steady-state fluorescence polarization Steady-state fluorescence polarization measurements were carried out with samples prepared as described above, containing 0.5 mg/mL DMPG and 1% fluorescence probes and performed with a PTI Quantamaster spectrofluorometer (Photon Technology, NJ, USA) equipped with motorized polarizers. Quartz cuvettes with a path length of 10 mm were used. The cell holder was thermostated using a Peltier device, and the measurements were taken under continuous stirring. For monitoring DPH and TMA-DPH fluorescence, the excitation wavelength was set at 358 nm, and emission was monitored at 430 nm. The sample temperature was allowed to equilibrate for 5 min before fluorescence was recorded for 60 s, and then the excitation shutter was kept closed during heating to the next temperature, in order to minimize any photoisomerization of DPH and TMA-DPH. Steady-state fluorescence polarization values were calculated from the following equation: P=
IVV − GIVH IVV + GIVH
where IVV and IVH are the fluorescence intensities with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. G is the grating
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Fig. 3. Changes in 90◦ light scattering intensity relative to initial value at 4 ◦ C (RSI) for DMPG containing TL at different molar fractions.
Fig. 2. DSC heating thermograms for DMPG containing TL at different molar fractions.
factor, calculated as the ratio of the efficiencies of the detection system for vertically and horizontally polarized light, and is equal to IHV /IHH . 2.6. Fourier Transform infrared spectroscopy (FT-IR) For the infrared measurements multilamellar vesicles containing 10 mol DMPG were prepared as described above, in 50 l of the same buffer prepared in D2 O. Samples were placed in between two CaF2 windows (25 mm × 2 mm) separated by 25 m Teflon spacers and transferred to a Symta cell mount. Infrared spectra were acquired in a Nicolet 6700 FT-IR (Madison, WI). Each spectrum was obtained by collecting 256 interferograms with a nominal resolution of 2 cm−1 . The equipment was continuously purged with dry air in order to minimize the contribution peaks of atmospheric water vapor. The sample holder was thermostated using a Peltier device (Proteus system from Nicolet). Spectra were collected at 2 ◦ C intervals, allowing 5 min equilibration between temperatures. The D2 O buffer spectra taken at the same temperatures were subtracted interactively using either Omnic or Grams (Galactic Industries, Salem, NH) software. 3. Results and discussion We first characterized the effects of TL on the thermal phase behavior of DMPG by DSC. Fig. 2 shows heating thermograms of DMPG at increasing TL concentrations. In contrast to the very sharp and intense peak associated with the chain melting transition of pure DMPG at high ionic strength (Pabst et al., 2007; Zhang et al., 1997; Ortiz et al., 2011), pure DMPG at low ionic strength (Fig. 2) shows a complex thermogram with a pretransition peak around 10 ◦ C (Tp ), and a melting transition peak with four components: three peaks closely spaced, with the first sharp peak at approximately 17 ◦ C (Ton ), followed by two wide peaks, with the first centered at approximately 20 ◦ C (T1 ) and the second at 23 ◦ C (T2 ). A fourth component is at a higher temperature of approximately 40 ◦ C (Toff ). This complex behavior agrees well with previous characterization of DMPG phase transition at low ionic strength (Riske et al., 2002; Alakoskela and Kinnunen, 2007; Riske et al., 2009; Alakoslela et al., 2010), with Ton defining the beginning of
the melting process and Toff setting its end. The gel–fluid transition region, which extends in the range of 17–40 ◦ C, has been called intermediate phase (IP) and the presence of broad peaks (T1 and T2 ) superimposed in this region indicates the occurrence of structural changes between Ton and Toff (Lamy-Freund and Riske, 2003). The presence of low concentration of TL, such as 0.05 mol fraction, has profound effects on the thermotropic behavior of the DPMG: it produces the disappearing of the pretransition peak and considerably alters the melting region profile. The enthalpy of Ton peak is decreased and the peak is shifted to lower temperatures, T1 and T2 peaks seem to merge in a broader peak and Tof peak is also shifted to lower temperatures. Increasing concentrations of TL makes Ton and Toff peaks to disappear and finally only a unique broad peak is found starting at lower temperature (around 10 ◦ C) and extending over a shorter range of 10–20 ◦ C. One of the most characteristic features of the intermediate phase of DMPG at low ionic strength is the sample transparency (Heimburg and Biltonen, 1994; Riske et al., 1997). Fig. 3 shows the relative 90◦ light scattering intensity for pure DMPG membranes and those containing TL. The beginning and the end of the pure DMPG intermediate phase are characterized by a sharp decrease in scattering at Ton and a sharp increase at Toff . Contrary to the turbid gel and fluid phases (below Ton and above Toff ), the intermediate phase is optically transparent. We were able to detect the small decrease of scattering corresponding to the pretransition around 10 ◦ C, but, similarly to early reports (Riske et al., 1997; Lamy-Freund and Riske, 2003) we were not able to detect the higher temperature additional scattering shift (Tpost ) recently described (Alakoskela and Kinnunen, 2007; Alakoskela et al., 2010). There is considerably hysteresis for this additional increase in scattering (Alakoskela and Kinnunen, 2007) and most probably we cannot detect it due to the difference in phospholipid concentration and heating rate of our set up. The 90◦ scattering scan in the presence of low concentration of TL establishes the disappearing of the pretransition and the shift of both Ton and Toff to lower temperatures. The presence of high concentration of TL evidences a further shift of Ton to lower temperatures and a clear decrease of the temperature span of the intermediate state, which presents only minor changes in light scattering. To monitor the acyl chain mobility of DMPG membranes, fluorescence polarization measurements were carried out using DPH and TMA-DPH probes, which report changes in membrane order in the deep inner phospholipid palisade and the interfacial region of the membrane respectively. Fig. 4 shows the fluorescence polarization of DPH (Fig. 4A) and TMA-DPH (Fig. 4B) incorporated into pure DMPG systems and those containing TL as a function of temperature. In the case of pure DMPG membranes, the polarization values of both probes display a similar behavior to those reported previously for DPH (Lamy-Freund and Riske, 2003), showing upon heating a continuously decreasing acyl chain order within the intermediate phase. However, as compared to the DPH anisotropy data reported previously, our DPH and TMA-DPH
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Fig. 5. Temperature dependence of the maximum of the symmetric CH2 stretching absorption band exhibited by pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
Fig. 4. Steady state fluorescence polarization as a function of temperature of DPH (A) and TMA-DPH (B) incorporated into membranes composed of pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
experiments reveal a steeper decline upon the disappearance of the intermediate state. The data agree with the electron spin resonance results for DMPG including a phosphatidylcholine probe with paramagnetic label (Lamy-Freund and Riske, 2003) and with recent DPH-phosphatidylcholine anisotropy values (Alakoskela and Kinnunen, 2007). The presence of TL makes the second steeper decrease in polarization to disappear and the transition is shifted to lower temperatures in accordance to DSC and scattering data reported above. Interestingly, the final polarization value, i.e. disorder, reached at high temperature in the fluid phase is similar in all the systems. However, before the phase transition, in the gel phase a small decrease in polarization, i.e. disordering effect, is observed for both probes in the presence of TL. The shift of the onset of the phase transition to lower temperatures together with the disordering effect in the gel phase, indicates that the hydrophobic moieties of TL incorporate into the PG acyl chain palisade and, like in the case of high ionic strength (Ortiz et al., 2011), perturb the acyl chains and shift the phase transition to lower values. A number of hypotheses have been proposed to explain the characteristics of the intermediate phase. These hypothesis include the formation of a sponge phase (Heimburg and Biltonen, 1994; Schneider et al., 1999) and secondary changes in the aggregation state of DMPG (Riske et al., 1997), but on the light of recent evidence their contribution seems unlikely (Riske et al., 2001, 2002, 2004; Lamy-Freund and Riske, 2003; Alakoskela and Kinnunen, 2007). Based in new experimental evidence, it has been proposed that the DMPG intermediate phase would be structured as vesicles containing multiple holes in the bilayer with an associated positively curved defect to cover the free edge of the bilayer (Riske et al., 2004; Alakoskela and Kinnunen, 2007). Recently it has been shown that the thermal stability of the intermediate phase can be changed in a highly predictable way by modifying the spontaneous curvature of DMPG by additives (Alakoskela et al., 2010), providing evidence that the intermediate phase is a structure with positive curvature (defects) and supporting the perforated vesicle structure suggested before. According to evidence, all compounds increasing the monolayer negative spontaneous curvature decrease the temperature span of the intermediate phase and lower the temperature of its dissolution. An attractive relation was found between the ability of a compound to destabilize the intermediate phase and its ability to
promote the formation of inverted non-lamellar phases (Alakoskela et al., 2010). In this context, it is interesting to note that we have found that TL was able to promote the formation of inverted hexagonal HII phases in dielaidoylphosphatidylethanolamine systems by producing a more effective cone shape phosphatidylethanolamine molecule, i.e. increasing its negative spontaneous curvature (Ortiz et al., 2008). Moreover, we have recently described that, at high ionic strength, TL increases the fluidity of DMPG acyl chains and dehydrates the interfacial region of the phosopholipid, both effects leading to an increase of the negative spontaneous curvature of the membrane (Ortiz et al., 2011). In order to get insight into the molecular mechanism of destabilization of the intermediate state by TL, we studied the interaction between TL and DMPG by infrared spectroscopy at low ionic strength. The infrared spectra of glycerophospholipids contain useful information regarding the intermolecular interactions that occur in the different domains of the molecule. In this way, the CH2 stretching region includes information about the conformational disposition of the hydrocarbon chains, whereas the C O stretching region includes information about lipid interfacial hydration–hydrogen bonding interaction. The CH2 symmetric stretching band near 2850 cm−1 is of remarkable importance because of its sensitivity to changes in the mobility and in the conformational disorder of the hydrocarbon chains (Mendelson and Mantsch, 1986). Fig. 5 presents the temperature dependence of the frequency at the absorbance maximum of the symmetric CH2 stretching vibration band of the infrared spectra of pure DMPG and DMPG/TL systems. For pure DMPG the data show a sharp increase of about 2 cm−1 in the frequency of the band maximum at temperature that coincide with the onset of the intermediate state. This frequency increase is characteristic of the chain melting transition of hydrated phospholipids (Mantsch and McElhaney, 1991) and results from the increased conformational disorder in the hydrocarbon as a consequence of the introduction of a high population of gauche conformers (Lewis and McElhaney, 1996). This sharp increase in the frequency of the methylene band span over a range of temperature which coincides with that of Ton , T1 and T2 , i.e. from 17 to 29 ◦ C, and from that point the increase is continuous with temperature. This data support the previous suggestion that at Toff the bilayer is practically completely fluid, and that at Toff there is effectively a transition between two fluid phases (Alakoskela et al., 2010). In the presence of TL, the chain melting phase transition starts at lower temperatures than that of the pure DMPG, in accordance with the DSC experiments shown above. At temperatures at which pure DMPG is found in the gel state, the presence of TL produces an increase of the frequency of the band, indicating and increase in gauche conformers and thus an increase in hydrocarbon chain conformational disorder in the gel phase. The C O stretching band of pure DMPG is a fairly broad band in the 1750–1700 cm−1 , and seems to be a summation of at least two
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Fig. 6. Temperature dependence of the maximum of the carbonyl stretching absorption band exhibited by pure DMPG (䊉) and DMPG/TL mixtures at 0.05 () and 0.20 () molar fraction.
subcomponents centered near 1741 and 1727 cm−1 (Zhang et al., 1997). The relative intensities of these component bands reflect the contribution of subpopulations of dehydrated and hydrated carbonyl groups (Blume et al., 1988). Fig. 6 shows the temperature dependence of the frequency at the absorbance maximum of the C O stretching band of the infrared spectra of pure DMPG and DMPG/TL systems. For pure DMPG, the onset of the intermediate state produces a shift of the maximum to lower frequencies, reflecting an increase in intensity of the underlying component band at 1727 cm−1 , attributed to a higher amount of hydrogen bonded carbonyl groups resulting from a phase state-induced increase in the hydration of the polar–apolar interface (Zhang et al., 1997). Interestingly, an additional shift to lower frequencies is found at the Toff transition. The latter suggests that a further hydration of the aqueous interface is taken place even when no change in the population of the CH2 gauche conformers is detected (Fig. 5). This change at the interfacial phase could explain the steeper decline in fluorescence polarization upon the disappearance of the intermediate state shown in Fig. 4. The presence of TL produces a disappearing of the additional decrease in frequency at Toff and a broadening of the initial shift in frequency at Ton . It is interesting to note that the presence of TL produced a net shift of the maximum of the C O band to higher frequencies than that of the pure DMPG, during the intermediate phase. This increase in frequency suggests an increase in the proportion of dehydrated C O component and that TL, at low ionic strength, interacts with the interfacial region of the DMPG bilayer, decreasing the hydrogen bonding of the C O groups with the water molecules of the hydration layer, in an overall way similar to that found at high ionic strength (Ortiz et al., 2011). However, in spite of this dehydration effect exerted by TL the fluorescence polarization value reached in the liquid crystalline phase is similar to that of the pure phospholipid (Fig. 4). It seems that the presence of TL produces different effects on the DMPG molecule; it causes conformational disorder in the methylene region in the gel phase, while dehydrates the interfacial region in the intermediate phase. These two effects gives rise to an increase of the monolayer negative spontaneous curvature of DMPG, and according to the hypothesis that the intermediate state is structured in perforated vesicles with positive curvature defects (Alakoskela et al., 2010) explain why TL behaves as a destabilizer of the intermediate state. 4. Conclusions We have carried out a study of the molecular interaction between a trehalose lipid bacterial biosurfactant and membranes composed of DMPG at low ionic strength. We have shown that TL is able to incorporate into DMPG bilayers and affect their structural properties. DSC data showed that TL produces the disappearing of the pretransition peak and considerably alters the melting region
profile with the different peaks merging in a broad peak which is shifted to lower temperatures. Light scattering studies confirmed the shift of the transitions to lower temperatures and a drastic decrease of the scattering change characteristic of the intermediate phase. Fluorescence polarization of membrane probes evidenced that TL did not change the final polarization value in the fluid phase but it induced a small disordering effect in the gel phase. Infrared experiments revealed that the biosurfactant increased the fluidity of the phospholipids acyl chains and decreased the hydration of the interfacial region of the membrane. These two effects would give rise to an increase of the monolayer negative spontaneous curvature of DMPG explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. Given the key structural and functional importance of PG in membranes and the wide diversity of biological actions played by trehalose lipids, the results presented here contribute to the knowledge of its underlying molecular mechanisms of action and also contribute to get insight into the properties of the DMPG intermediate phase. Conflict of interest The authors declare that there are no conflicts of interest. References Alakoskela, J.M., Kinnunen, P.K., 2007. Thermal phase behavior of DMPG: the exclusion of continuous network and dense aggregates. Langmuir 10, 4203–4213. Alakoskela, J.-M., Parry, M.J., Kinnunen, P.K.J., 2010. The intermediate state of DMPG is stabilized by enhanced positive spontaneous curvature. Langmuir 26, 4892–4900. Aranda, F.J., Teruel, J.A., Espuny, M.J., Marqués, A., Manresa, A., Palacios-Lidón, E., Ortiz, A., 2007. Domain formation by a Rhodococcus sp. biosurfactant trehalose lipid incorporated into phosphatidylcholine membranes. Biochim. Biophys. Acta 1768, 2596–2604. Asselineau, C., Asselineau, J., 1978. Trehalose-containing glycolipids. Prog. Chem. Fats Other Lipids 16, 59–99. Azuma, M., Suzutani, T., Sazaki, K., Yoshida, I., Sakuma, T., Yoshida, T., 1987. Role of interferon in the augmented resistance of trehalose 6,6 -dimycolate-treated mice to influenza virus infection. J. Gen. Virol. 68, 835–843. Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53, 495–508. Barroso, R.P., Perez, K.R., Cuccovia, I.M., Lamy, M.T., 2012. Aqueous dispersions of DMPG in low salt contain leaky vesicles. Chem. Phys. Lipids 165, 169–177. Blume, A., Hübner, W., Messner, G., 1988. Fourier transform infrared spectroscopy of 13 C O-labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry 27, 8239–8249. Böttcher, C.J.F., Van Gent, C.M., Pries, C., 1961. A rapid and sensitive sub-micro phosphorus determination. Anal. Chim. Acta 24, 203–204. Eklund, K.K., Kinnunen, P.K., 1986. Effects of polyamines on the thermotropic behaviour of dipalmitoylphosphatidylglycerol. Chem. Phys. Lipids 39, 109–117. Enoki, T.A., Henriques, V.B., Lamy, M.T., 2012. Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition. Chem. Phys. Lipids 165, 826–837. Espuny, M.J., Egido, S., Mercadé, M.E., Manresa, A., 1995. Characterization of trehalose tetraester produced by a waste lube oil degrader Rhodococcus sp. 51T7. Toxicol. Environ. Chem. 48, 83–88. Espuny, M.J., Egido, S., Rodón, I., Manresa, A., Mercadé, M.E., 1996. Nutritional requirements of a biosurfactant producing strain Rhodococcus sp. 51T7. Biotechnol. Lett. 18, 521–526. Gershfeld, N.L., Stevens Jr., W.F., Nossal, R.J., 1986. Equilibrium studies of phospholipid bilayer assembly. Coexistence of surface bilayers and unilamellar vesicles. Faraday Discuss. Chem. Soc. 81, 19–28. Heimburg, T., Biltonen, R.L., 1994. Thermotropic behavior of dimyristoylphosphatidylglycerol and its interaction with cytochrome c. Biochemistry 33, 9477–9488. Hoq Md, M., Suzutani, T., Toyoda, T., Horijke, G., Yoshida, I., Azuma, M., 1997. Role of ␥␦ TCR lymphocytes in the augmented resistance of trehalose 6,6 -dimycolatetreated mice to influenza virus infection. J. Gen. Virol. 78, 1597–1603. Isoda, H., Shinmoto, H., Kitamoto, D., Matsumura, M., Nakahara, T., 1996. Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages. Cryotechnology 19, 79–88. Isoda, H., Kitamoto, D., Shinmoto, H., Matsumura, M., Nakahara, T., 1997a. Microbial extracellular glycolipid induction of differentiation and inhibition of the protein kinase C activity of human promyelocytic leukaemia cell line HL60. Biosci. Biotechnol. Biochem. 61, 609–614. Isoda, H., Shinmoto, H., Kitamoto, D., Matsumura, M., Nakahara, T., 1997b. Differentiation of human promyelocytic leukaemia cell line HL60 by microbial extracellular glycolipids. Lipids 32, 263–271.
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