THE PHYSICOCHEMICAL CHARACTERIZATION OF MICROVESICLES SECRETED BY SULFOLOBUS ACIDOCALDARIUS

Abstract

Microvesicles secreted from the thermoacidophilic archaeon S. acidocaldarius (Sa-MVs) contain a membrane made exclusively of tetraether lipids and covered by crystalline surface layer proteins known as the S-layer. While tetraether lipids and S-layer proteins are known to be useful biomaterials, little has been done to exploit Sa-MVs for any scientific applications. In the present study, as the start point to explore this area, we isolated Sa-MVs and used dynamic light scattering, laser Doppler electrophoresis, and cryo-transmission electron microscopy (cryo-TEM) to characterize the particle size, size distribution, zeta potential, and morphology of Sa-MVs and tested their stabilities against temperature, pH, autoclaving, and the detergent Triton X-100. We found that, at the cell’s growth pH (~2.6) and growth temperature (75-80oC), Sa-MVs in the growth medium are ~180-183 nm in diameter with a polydispersity index (PDI) ≤ 0.15 and have a zeta potential of -0.5 mV. Sa-MVs in buffer exhibited long-term (at least 137 days) stability with no signs of vesicle disintegration or fusion. When the pH was decreased from 7.2 to 2.6, the average size of Sa-MVs was increased by ~40-45 nm, which probably came from conformational changes of S-layer proteins, in concomitant with vesicle aggregation, but not due to conformational changes in tetraether lipid headgroups. The isoelectric point (pI) for Sa-MVs in 1 mM KCl is 3.0 while that for the reconstituted liposomes (LUVMV) is estimated to be below 2.0. Sa-MVs dispersed in buffer at pH 2.6 change little in size over five autoclaving cycles, despite becoming slightly less spherical after autoclaving, while at this pH liposomes made of diester lipids cannot sustain multiple cycles of autoclaving. In addition, compared to diester and PLFE liposomes, Sa-MVs and LUVMV exhibit unusual resistance against the surfactant Triton X-100. Although some man-made liposomes such as PLFE liposomes are also stable against temperature, pH, and other environmental stressors, Sa-MVs are unique in that they are naturally occurring nanoparticles with a native membrane environment suitable for inserting additional lipids and membrane-bound proteins as needed. With their great stability presented here and the lack of cytotoxicity known in the literature, Sa-MVs hold great promise for technological applications. In addition to these biophysical techniques employed to characterize these microvesicles, a series of fluorescence experiments have also been conducted to gain further insight into how the membrane packing of these vesicles compares to tetraether as well as diester liposomes. Intrinsic protein fluorescence of native microvesicles was examined to characterize the dynamics of the S. acidocaldarius MVs. We have used the probe 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) to monitor membrane packing and dynamics within the water-membrane interfacial region of the Sa-MVs. Specifically, we measured Laurdan’s generalized polarization (GP), which depends on the probe’s local polarity, probe location and nearby viscosity. We also measured Laurdan’s red edge excitation shift (REES), which depends on the dynamics of solvent relaxation around the fluorophore compared to the probe’s fluorescence lifetime. As temperature increased from 18 to 66.7 °C, GP decreased from 0.026 to -0.118. Comparing the GP values of reconstituted vesicles to that of the native Sa-MVs, it appears that the two curves are similar in both GP value and trend over increasing temperature range (values decrease from 0.112 to -0.215), which suggests that Laurdan in Sa-MVs resides in the lipid membrane, not in proteins and that Laurdan’s GP is not affected much by the presence of Sa-MV proteins. It is well known that, for liposomes made of diester lipids, the GP value of Laurdan fluorescence is high in the gel state and low in the liquid-crystalline state, with an abrupt change during the phase transition. However, Laurdan’s GP values obtained from liposomes comprised of tetraether lipids such as PLFE and Sa-MV lipids cannot be compared directly to those obtained from diester liposomes and cannot be interpreted simply based on membrane packing because probe location and chromophore orientation in tetraether liposomes could be distinctly different from those in diester liposomes. Our data show that the REES effect in PLFE LUVs is most pronounced among all the membranes examined showing a value of 10.58 nm at 24oC compared to 0.9 nm for DMPC LUVs at the same temperature. It is already known that membrane packing in PLFE liposomes is extraordinarily tight partly due to the strong hydrogen bond network in the polar head group regions where the phosphate and sugar moieties are abundant and partly due to the rigid and ordered dibiphytanyl hydrocarbon chains. Since the chromophore of Laurdan in PLFE liposomes is most likely located in the membrane-water interfacial region, it is not surprising that solvent tumbling around Laurdan in PLFE LUVs is much more restricted in PLFE LUVs than in DMPC LUVs, giving rise to a much higher REES value in PLFE LUVs than in DMPC LUVs. The REES values (10.3-14.1 nm) of Laurdan fluorescence in LUVs reconstituted from the extracted MV lipids are higher than those (9.3-10.6 nm) in PLFE LUVs, which suggests that the mobility of solvent molecules (including water and lipid polar groups) in the membrane-water interfacial regions of LUVMV is much less restricted than that in PLFE LUVs. Like PLFE, Sa-MV lipids are tetraethers. However, as mentioned earlier, PLFE lipids are different from Sa-MV lipids in their hydrophobic core composition. It is likely that their polar head groups are also different despite that the zeta potentials of LUVPLFE and LUVMV are virtually identical (-43 mV in 50 mM Tris buffer containing 10mM EDTA and 0.02% NaN3 at pH 7.2-7.6 and 25oC). The REES values (14.5-18.9 nm) of Laurdan fluorescence in Sa-MVs are higher still than those of both LUVPLFE and LUVMV, which is reasonable because tetraether lipids in Sa-MVs are covered with S-layer proteins. As a result, the mobility of solvent molecules around Laurdan’s chromophore in the lipid polar head group regions is more restricted than that in LUVPLFE or LUVMV. Free-standing planar membrane made of MV lipids built on a cellulose acetate partition and mounted onto a Teflon device sustained a nearly constant capacitance (~36-39 pF) for 8 h. Thereafter, the membrane collapsed as evidenced by a zero capacitance. In contrast, the planar membrane made of the diester lipid POPC had much lower stability, showing large fluctuations in capacitance before its collapse at 1.5 h, a very short lifetime typical for free-standing planar membranes made of diester lipids. The planar membrane made of the diester lipid DMPC also showed a short lifetime ~3h. In comparison, the planar membrane made of PLFE showed remarkable stability, exhibiting a constant capacitance for at least 11 days. Similar high stability of PLFE free-standing planar membranes over micro-pores on PDMS thin films in microchip platform was previously reported. Our data suggest that lipids extracted from S. acidocaldarius MVs are able to form fairly stable free-standing planar membranes across a pinhole on a solid support. However, even though both MV lipids and PLFE lipids are tetraethers, the planar membrane made of MV lipids is not as stable as that made of PLFE lipids. The molecular basis for the differential stability between planar membranes of MV lipids and PLFE lipids is not clearly understood at present, but the difference in stability is likely to originate from the chemical structure differences between PLFE lipids and MV lipids. As mentioned earlier, in terms of the hydrophobic cores, PLFE contains ~90% GDNT and ~10% GDGT, whereas MV lipids are mainly GDGT and GTGT, without any GDNT, and their headgroup structures are not known. We have also demonstrated the ability to observe channel activity in PLFE monolayers at a range of voltages from -200 to 200 mV. However, this was property was not replicated in lipids extracted from S. acidocaldarius microvesicles. In any case, our past and present data showed that archaeal tetraether lipids are excellent materials to make stable and yet biologically relevant free-standing planar membranes.