The role of acyl chain character and other determinants on the bilayer activity of A21978C an acidic lipopeptide antibiotic

An acidic lipopeptide A21978C has previously been shown to have a powerful antibiotic activity against Gram-positive organisms. Due to its ability to increase the K+ permeability of bacterial cells and its specific calcium requirement, which is similar to a previously described ionophore CDA, its effect on planar bilayer membranes has been studied. Although it produces significant increases in the conductivity of lipid bilayers it is shown that this alone cannot account for its in vivo activity. Similarly, unlike the in vivo results, the Ca2+-induced increases in bilayer conductivity can be mimicked by Mg2+ and charged lipids. Results from a series of homologues differing in the length of the acyl moiety show a close similarity between bilayer conductance and LD50 trends from in vivo studies. A complex activity is proposed which depends upon incorporation in, rather than disruption of, the bilayer membrane.     

A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers

Iturin A is a lipopeptide extracted from the culture media of Bacillus subtilis which shows a strong antifungal action. The interaction of iturin A with multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) induced structures which did not sediment during centrifugation. Electron microscopy after negative staining showed that, at 30 mol%, iturin A/DMPC vesicles were visible but smaller than those formed by pure DMPC. Thermograms of DMPC/iturinA obtained after differential scanning calorimetry, at low concentrations of iturin A, were interpreted as indicating the presence of two laterally separated phases, one formed by pure phospholipid and the other by lipopeptide-phospholipid complexes, these two separated phases being already detected even at low concentrations such as 2 mol%. Fluorescence quenching experiments showed that the D-Tyr residue of the lipopeptide was fully accessible to the aqueous medium, indicating that the polar part of iturin A is located outside of the membrane hydrophobic palisade. It was concluded that the membrane barrier properties are likely to be damaged in the area where the lipid complexes are accumulated, due to structural fluctuations, and this may be one of the bases of its biological activity. Iturin-A was also able to greatly destabilize dielaidoylphosphatidylethanolamine (DEPE) membranes in the fluid form, producing a new structure which had a poor correlation in X-ray diffraction, and in 31P NMR spectroscopy gave rise to a spectrum containing a double isotropic signal. Iturin A was shown to induce DEPE to adopt phases other than H(II) inverted hexagonal, underlining that this lipopeptide is capable of modifying the curvature of the membrane, which may also be important in explaining the tendency of iturin A to create small vesicles and which may be another of the bases of its biological activity.     

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.     

The formation of daptomycin by supplying decanoic acid to Streptomyces roseosporus cultures producing the antibiotic complex A21978C

Antibiotic substance A21978C is a complex of compounds having a common cyclic polypeptide nucleus and different fatty acid sidechains. Daptomycin is a semi-synthetic antimicrobial substance derived from the A21978C complex by a very elaborate chemical process. To obtain the daptomycin, the A21978C complex was first isolated from culture filtrates of Streptomyces roseosporus by several resin procedures. The resulting material was then ‘blocked’ and added to an Actinoplanes culture for deacylation. The protected A21978C nucleus was subsequently isolated by the same procedure as the parent complex and reacylated with the desired fatty acid (decanoic acid). The acylated compound was deblocked to yield daptomycin. This report describes the experimentation undertaken to establish a strategy to supply a very toxic precursor to cultures of S. roseosporus and, thereby, produce daptomycin biosynthetically.     

A comparative monomolecular film study of antibiotic A21978C homologues of various lipid chain length

A21978C is a calcium-dependent lipopeptide antibiotic whose biological properties are modulated by changes in its lipid chain length. This article reports on the monolayer characteristics of this cyclic lipopeptide and of LY146032 a semi synthetic homologue. The equilibrium spreading pressure pi e increases linearly with the ionic concentration of the subphase and is higher with divalent cations. The nature of the divalent cation plays a crucial role in the spreading as indicated by the variation in the molecular free energy delta Gs.delta Gs decreases in the order K+ greater than Mg2+ greater than Ca2+, which indicates privileged interactions with Ca2+. Also, the larger the lipid chain, the easier the spreading of antibiotic molecules. The compression isotherm curves are shown. The mean area of the uncompressed molecules is around 220-240 A 2 which is compatible with the size of the peptide cycle lying at the interface. The isotherm curves of the natural compounds show a transition region where the molecules are more compressible. At a given area/molecule, the surface pressures increase with the acyl chain length. When the molecules are spread on various salt solutions, the surface pressures increase in the order K+ less than Mg2+ less than Ca2+. The isotherm curves are not reversible upon a compression-expansion cycle and a wide amplitude hysteresis is observed. If a second compression is done, the curve shape is that of a liquid-expanded state and the transition region is no longer observed. This implies a conformational change of the molecules during the first compression process.     

Conformational change on calcium binding by the lipopeptide antibiotic amphomycin. A C.D. and monolayer study

The acidic linear lipopeptide amphomycin is a calcium dependent antibiotic which is thought to bind to carrier lipids such as dolichol monophosphate. The actual role of Ca++ is not definitely established and in this article we have examined the peptides interactions with a range of divalent cations. By CD we have shown that a conformational change is induced by Ca++, Sr++ and Ba++ but not by Mg++, Zn++, Cd++ or Gd+++. Monolayer studies show a decrease in molecular area and an increase in film stability when the subphase contains Ca++. The ensemble of results provides preliminary evidence for the formation of a beta hairpin structure on ion binding (Ka (Ca++) = 2.4 x 10(3)M-1) which could enhance amphomycin's bilayer solubility.     

Pore-forming properties of iturin A, a lipopeptide antibiotic

The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.     

Production of a lipopeptide antibiotic surfactin with recombinantBacillus subtilis

Summary Production of a lipopeptide antibiotic surfactin was carried out using a recombinantBacillus subtilis. Surfactin yield of the recombinant strain was about one and half times as much as that ofBacillus subtilis RB 14, the strain in which the surfactin gene was originated. This system is especially noteworthy because a recombinant strain surpassed the original strain in the production of a bacterial antibiotic as a secondary metabolite of the bacterium.     

The binding of barium and calcium ions by the antibiotic beauvericin

The ion-transporting antibiotic beauvericin has been shown to have a high affinity for calcium and barium ions in addition to the more usual affinity for monovalent cations. As judged by crystallization, extraction into organic solvent, and U-tube transport the cation selectivity is Rb>Ba>K>Na?Ca?Li. For these studies an improved method for the synthesis of beauvericin has been developed.     

The interaction of calcium ions with glycocholate micelles in aqueous solution.

The formation of soluble complexes of Ca2+ ions and glycocholate has been demonstrated. The dissociation constant is 26 nmol/litre and a maximum of 2 Ca2+ ions are bound to each glycocholate micelle. The formation of this complex is shown to be reversible. Binding is increased by the introduction of phosphatidylcholine into the micelle; it is decreased by a decrease in pH and by increased counter-ion concentration. The biological significance of these effects is discussed.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum.

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.     

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

The kinetics of the Quin 2-Ca²⁺ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca²⁺ complex with a large excess of EGTA, EDTA or MgCl₂ resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s^?1 for the dissociation rate constant (k_?) at pH 7.2, 37°C, ± 3mM Mg²⁺. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k₊ was estimated to be ≥10⁹ M^?1s^?1, in agreement with the calculation from $\text{k}{}_{\mathrm{+}}\text{=}\text{k}{}_{\mathrm{?}}\text{K}$. These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca²⁺ transients.     

Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids

Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2. The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium. Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A. Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes. When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature. The methylated derivative has no effect. Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A. Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated. On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A. We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane.     

A survey of the interaction of calcium ions with mitochondria from different tissues and species

A survey was made of the capacity of mitochondria isolated from a number of different tissues and species to accumulate Ca(2+) from the suspending medium during electron transport. The species examined included the rat, mouse, rabbit, hamster, guinea pig, cow, chicken, turtle, blowfly, yeast and Neurospora crassa. The tissues examined included vertebrate liver, kidney, brain, heart, spleen, thyroid and adrenal cortex, and the flight muscle of the blowfly. The mitochondria from all vertebrate tissues examined showed: (a) stimulation of State 4 respiration by added Ca(2+) (Ca(2+)/~ activation ratio about 2.0), accompanied by accumulation of Ca(2+) and ejection of H(+), with a H(+)/Ca(2+) ratio about 1.0; (b) a requirement of phosphate for accumulation of large amounts of Ca(2+); (c) respiration-independent high-affinity binding sites for Ca(2+); (d) endogenous Ca(2+), which is largely released by uncoupling agents. However, mitochondria from yeast and blowfly flight muscle are unable to accumulate Ca(2+) in a respiration-dependent process and possess no high-affinity Ca(2+)-binding sites. These findings support the view that the high-affinity sites represent the ligand-binding sites of a specific Ca(2+) ;permease' or transport system in the membrane. The relatively high affinity for Ca(2+), which equals or exceeds the affinity for ADP, and the generally uniform characteristics of Ca(2+) transport in all the vertebrate mitochondria tested strongly suggest that respiration-linked Ca(2+) accumulation plays a general and fundamental role in vertebrate cell physiology.     

Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K(+) leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 A. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.