Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.     

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.     

Kinetics of erythrocyte swelling and membrane hole formation in hypotonic media

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the β-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl₂ demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t_memb. Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t_memb).     

Analysis of Glycerol-3H Transport in the Frog Oocyte by Extractive and Radioautographic Techniques

The efflux of glycerol-³H from mature R. pipiens oocytes was studied by extractive analysis and by quantitative radioautography using techniques suitable for diffusible solutes. Extractive analysis was used to determine the total cellular concentration of tracer, and radioautography, regional intracellular concentrations, at equilibrium and as a function of efflux time, t_E. The efflux was resolvable into four kinetic fractions: cytoplasmic fast and slow fractions, and nuclear fast and slow fractions. The fast fractions represent freely diffusible glycerol in the two compartments; the solvent space accessible to glycerol is unity in the nucleus (germinal vesicle), but only 0.73 in the cytoplasm. The efflux of both fast fractions from the cell is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The permeability at 13.6°C is 2.2 x 10^-5 cm/sec. The slow fractions leave the cell at about one-tenth the rate of the fast; the interpretation is that these fractions represent glycerol bound to impermeant cellular constituents. The size of these constituents is below the radioautographic resolution; in the cytoplasm, they appear not to be the yolk platelets. The extent of binding is about fourfold greater, per milliliter of compartment water, in the cytoplasm than in the germinal vesicle.     

TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

The rate of allelism of lethal genes in a geographically structured population

The expected rate of allelism, E[I(x)], of lethal genes between two colonies with distance x in a structured population is studied by using one- and two-dimensional stepping-stone models. It is shown that E[I(x)] depends on the magnitude of selection in heterozygous condition (h), the rate of migration among adjacent colonies (m), the number of loci which produce lethal mutations (n) and the effective population size of each colony (N).——E[I(x)] always decreases with distance x. The rate of decrease is affected strongly by the magnitude of m. The rate of decrease is faster when m is small. E[I(x)] also decreases with increasing N and n. The effect of h on E[I(x)] is somewhat complicated. However, E[I(0)] is always smaller when h is small than when it is large.——For large x, the following approximate formulae may be obtained: (see PDF) where q and Var (q) are the mean and the variance of gene frequencies in each colony, t is approximated as t=h, (see PDF), -h for the partially recessive, completely recessive, and overdominant lethals, respectively, and C₀ is a function of m and t. It is clear that E[I(x)] declines exponentially with x in a one-dimensional habitat. The decrease E[I(x)] is faster in a two-dimensional habitat than in a one-dimensional habitat. The present result is applied to some of the existing data and the estimation of population parameters is also discussed.     

Effect of Human Serum Albumin Upon the Permeabilizing Activity of Sticholysin II, a Pore Forming Toxin from Stichodactyla heliantus

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Lipid II-Based Antimicrobial Activity of the Lantibiotic Plantaricin C

We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i) lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.     

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.     

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Measuring single-virus fusion kinetics using an assay for nucleic acid exposure

The kinetics by which individual enveloped viruses fuse with membranes provide an important window into viral-entry mechanisms. We have developed a real-time assay using fluorescent probes for single-virus genome exposure than can report on stages of viral entry including or subsequent to fusion pore formation and prior to viral genome trafficking. We accomplish this using oxazole yellow nucleic-acid-binding dyes, which can be encapsulated in the lumen of target membranes to permit specific detection of fusion events. Since increased fluorescence of the dye occurs only when it encounters viral genome via a fusion pore and binds, this assay excludes content leakage without fusion. Using this assay, we show that influenza virus fuses with liposomes of different sizes with indistinguishable kinetics by both testing liposomes extruded through pores of different radii and showing that the fusion kinetics of individual liposomes are uncorrelated with the size of the liposome. These results suggest that the starting curvature of such liposomes does not control the rate-limiting steps in influenza entry.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Clonal analysis of a genetically caused duplication of the anterior wing in Drosophila melanogaster

Two recessive lethal mutations, l(2)cos_t and l(2)cos₂, in Drosophila melanogaster are described which in the double heterozygote lead to the formation of large duplications and occasional triplications and quadruplications of the anterior part of the wing blade in the region of the costa and triple row of bristles. Clonal analysis with marker mutants following induced mitotic recombination showed that disruption of clonal growth was confined to the area of the duplication. Comparisons were made between clone size in the duplication, clone size in the remainder of the wing, and the size of the duplication. The rate of cell division in the duplication was similar to that in the main wing but continued for a longer period. Variation in size of the duplication was dependent upon the initial number of cells in the duplication. Clone shape in the duplication resembled that in the normal wings. These results are discussed in terms of the possibility that localised cell death initiates the duplication.     

Physiochemical characterization of the nisin-membrane interaction with liposomes derived from Listeria monocytogenes.

Mechanistic information about the bacteriocin nisin was obtained by examining the efflux of 5(6)-carboxy-fluorescein from Listeria monocytogenes-derived liposomes. The initial leakage rate (percentage of efflux per minute) of the entrapped dye was dependent on both nisin and lipid concentrations. At all nisin concentrations tested, 5(6)-carboxyfluorescein efflux plateaued before all of the 5(6)-carboxyfluorescein was released (suggesting that pore formation was transient), but efflux resumed when more nisin was added. Isotherms for the binding of nisin to liposomes constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 6.2 x 10(5)M(-1) at pH 6.0. The critical number of nisin molecules required to induce efflux from liposomes at pH 6.0 was approximately 7,000 molecules per liposome. The pH affected the 5(6)-carboxyfluorescein leakage rates, with higher pH values resulting in higher leakage rates. The increased leakage rate observed at higher pH values was not due to an increase in the binding affinity of the nisin molecules towards the liposomal membrane. Rather, the critical number of nisin molecules required to induce activity was decreased (approximately 1,000 nisin molecules per liposome at pH 7.0). These data are consistent with a poration mechanism in which the ionization state of histidine residues in nisin plays an important role in membrane permeabilization.     

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) – in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and ³¹P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70–415?nm upon repeated extrusion through 400?nm pore-filters, eventually ending with a size range from ≈30 to 85?nm upon extrusion through 30?nm pore size filters. While for small pores sizes (50?nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (～?300?nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.     

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.