Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly-(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.     

Pure lipid vesicles can induce channel-like conductances in planar bilayers

Summary Typical channel-like current fluctuations were observed in planar lipid bilayers following brief exposure to large concentrations of lipid vesiclesdevoid of protein. Vesicles, formed by sonication of pure lipids suspended in 150mm salt solutions, were ejected 0.5 mm from a planar bilayer with a pipette. Over the next several minutes the bilayer conductance changed in ways usually considered to be indicative of reconstituted protein channels including step conductance changes (both up and down), flickering, ion selectivity, and inactivation. This observation demonstrates the need for caution in interpreting conductance changes which occur following ejection of channel-containing vesicles near a membrane.     

Large cation-selective pores from rat liver peroxisomal membranes incorporated to planar lipid bilayers

Summary Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. TheP _K/P _Cl ratio of these pores, estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3m KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3m KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1m KCl.     

Interaction of alamethicin pores in DMPC bilayers

We have investigated the x-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide alamethicin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius corresponding to the geometric radius of the pore, we find a repulsive lipid-mediated interaction with a range of approximately 30 A and a contact value of 2.4 k(B)T. This result is in qualitative agreement with recent theoretical models.     

Three-dimensional structure of Bax-mediated pores in membrane bilayers

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.     

Heat shock proteins induce pores in membranes

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, byS. aureus toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.     

New cationic lipids form channel-like pores in phospholipid bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Estimation of the radius of the pores formed by the Bacillus thuringiensis Cry1C delta-endotoxin in planar lipid bilayers

Pore formation constitutes a key step in the mode of action of Bacillus thuringiensis delta-endotoxins and various activated Cry toxins have been shown to form ionic channels in receptor-free planar lipid bilayers at high concentrations. Multiple conductance levels have been observed with several toxins, suggesting that the channels result from the multimeric assembly of a variable number of toxin molecules. To test this possibility, the size of the channels formed by Cry1C was estimated with the non-electrolyte exclusion technique and polyethylene glycols of various molecular weights. In symmetrical 300 mM KCl solutions, Cry1C induced channel activity with 15 distinct conductance levels ranging from 21 to 246 pS and distributed in two main conductance populations. Both the smallest and largest conductance levels and the mean conductance values of both populations were systematically reduced in the presence of polyethylene glycols with hydrated radii of up to 1.05 nm, indicating that these solutes can penetrate the pores formed by the toxin. Larger polyethylene glycols had little effect on the conductance levels, indicating that they were excluded from the pores. Our results indicate that Cry1C forms clusters composed of a variable number of channels having a similar pore radius of between 1.0 and 1.3 nm and gating synchronously.     

Colicin E1 in planar lipid bilayers

The channel formed by the C-terminal domain of colicin E1 in planar lipid bilayers has proven to be more complex than one might have guessed for such a simple system. The protein undergoes a pH-dependent rearrangement which transforms it from a water soluble form to a much different membrane bound form. There are at least two bound states which don't form a channel. The process by which the channel opens and closes is regulated by the pH and the transmembrane voltage. The voltage is probably sensed by at least 3 (and more likely 4 or more) lysine residues which must be driven through the field to open the channel. The process appears to be hindered by particular carboxyl groups when they are in the unprotonated state. The open channel has several substates and several superstates. Very large positive voltage catalyzes a transition of the open channel to an inactivated state, and may be able to drive the channel-forming region of the protein across the membrane. Little is known about the structure of any of these states, but the open channel is large enough to allow NAD to traverse the membrane and appears to be formed by one colicin molecule. This single polypeptide mimics many of the properties found in channels of mammalian cell membranes, but it may prove more relevant as a model for the transport of proteins across membranes. The comparative ease with which the protein can be manipulated chemically and genetically, along with the complexity of its behavior, promises to keep several laboratories busy for some time.     

Resistance of planar bilayers of gangliosides

The value of electrical resistance of planar bilayers (black membranes) of gangliosides was found to be sensitive to pH, the composition of ganglioside as well as the presence of phospholipids in bilayers. The pH-dependence of resistance of black mambranes prepared from porcine brain gangliosides was determined in the presence of K⁺, Na⁺, and Ca⁺². In the absence of added phospholipids gangliosides obtained from brain of a patient with syndrom of Tay-Sachs disease are not capable to form stable black membranes, although in the presence of phospholipids the membranes are formed. The pH-dependences of resistance of black membranes obtained from mixture of phospholipids with gangliosides of porcine or human brain are compared.     

Supported planar bilayers from hexagonal phases

In this work the presence of inverted hexagonal phases H(II) of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca(2+) were observed via (31)P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H(II) phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being approximately 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H(II) phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates ( approximately 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H(II) phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.     

Supported planar bilayers from hexagonal phases

In this work the presence of inverted hexagonal phases H_II of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca²⁺ were observed via ³¹P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H_II phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H_II phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H_II phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.     

Microbial lipopeptides induce the production of IL-17 in Th cells

Naive Th cells can be directed in vitro to develop into Th1 or Th2 cells by IL-12 or IL-4, respectively. In vivo, chronic immune reactions lead to polarized Th cytokine patterns. We found earlier that Borrelia burgdorferi, the spirochaete that causes Lyme disease, induces Th1 development in alpha beta TCR-transgenic Th cells. Here, we used TCR-transgenic Th cells and oligonucleotide arrays to analyze the differences between Th1 cells induced by IL-12 vs those induced by B. burgdorferi. Transgenic Th cells primed with peptide in the presence of B. burgdorferi expressed several mRNAs, including the mRNA encoding IL-17, at significantly higher levels than Th cells primed with peptide and IL-12. Cytometric single-cell analysis of Th cell cytokine production revealed that IL-17 cannot be categorized as either Th1 or Th2 cytokine. Instead, almost all IL-17-producing Th cells simultaneously produced TNF-alpha and most IL-17(+) Th cells also produced GM-CSF. This pattern was also observed in humans. Th cells from synovial fluid of patients with Lyme arthritis coexpressed IL-17 and TNF-alpha upon polyclonal stimulation. The induction of IL-17 production in Th cells is not restricted to B. burgdorferi. Priming of TCR-transgenic Th cells in the presence of mycobacterial lysates also induced IL-17/TNF-alpha coproduction. The physiological stimulus for IL-17 production was hitherto unknown. We show here for the first time that microbial stimuli induce the expression of IL-17 together with TNF-alpha in both murine and human T cells. Chronic IL-17 expression induced by microbes could be an important mediator of infection-induced immunopathology.     

Fusion of liposomes with planar lipid bilayers

The fusion of liposomes with planar lipid bilayers was monitored by two different methods. (a) Liposomes consisting of phospholipids and cholesterol were added to the aqueous phase bathing the cholesterol-deficient planar lipid bilayers in the presence of nystatin. The resulting increase in the planar lipid bilayer's electrical conductance was considered indicative of fusion. (b) Transplanar lipid bilayer injection of ³⁵SO₄^2? trapped inside the liposomes.It is shown by both methods that fusion is specifically dependent on the presence of negatively charged phospholipids both in the liposomes and the planar lipid bilayers and on Ca²⁺ in the aqueous phase of the fusion system.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.