Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

A nuclear magnetic resonance study of the metal binding sites in bacitracin.

Carbon-13 nuclear magnetic resonance spectroscopy has been used to identify sites in bacitracin which bind Cu2+ and Mn2+. Results are presented which implicate the free carboxyl groups of the aspartic and glutamic acid residues and the imidazole ring of the histidine residue as metal complexation sites between pH 6 and 8. Evidence is presented which also indicates that the thiazoline ring of bacitracin binds Mn2+. Bacitracin does not bind Cu2+ or Mn2+ at pH values of 2.5 or less.     

Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants

The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-[³H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[¹⁴C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [¹⁴C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.     

Atomic force microscopy of cell growth and division in Staphylococcus aureus

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.     

EXAFS study of zinc coordination in bacitracin A.

Bacitracin is a dodecapeptide antibiotic produced by Bacillus sp. The antibacterial activity depends upon the peptide binding a divalent metal. Hitherto, the exact coordination of the cation has not been established. In particular the role played by the sulphur and nitrogen atoms of the thiazoline ring of bacitracin A has not been clear. Here the coordination of Zn2+ by bacitracin A has been studied using extended X-ray absorption fine structure. The experimental data are consistent with a model in which zinc is coordinated by one oxygen and three nitrogen atoms with the sulphur atom of the thiazoline ring not being directly involved in the zinc coordination.     

Effects of various drugs on hemolytic activity of Streptococcus zymogenes

Drugs known to interrupt the chain of protein synthesis (streptomycin, chloramphenicol, ethidium bromide) appeared to affect production of the group D lysin by Streptococcus zymogenes by inhibiting growth. Drugs which block cell wall synthesis (vancomycin, bacitracin, d-cycloserine, and phosphonomycin) inhibited lytic activity by a mechanism independent of growth. Bacitracin appeared not to have a major effect on lysin production but elicited the production of an inhibitor of lytic activity.     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.     

Inhibition by bacitracin of rat adipocyte plasma membrane degradation of 125I-insulin is associated with an increase in plasma membrane bound insulin and a potentiation of glucose oxidation by adipocytes

The present study demonstrated that at physiological concentrations of insulin bacitracin inhibited the degradation of specifically bound insulin by enzymes located in the rat adipocyte plasma membrane. Bacitracin increased the amount of intact insulin specifically bound to the plasma membrane and potentiated the stimulation of adipocyte glucose oxidation by submaximal concentrations of the hormone. In contrast to agents such as chloroquine, which inhibit lysosomal degradation of internalized insulin, bacitracin was shown by two approaches to inhibit a degradative process localized to the adipocyte plasma membrane. Cyanide and 2,4-dinitrophenol, agents which inhibit energy requiring endocytosis, had no effect on the bacitracin inhibition of cellular degradation of 125I-insulin. Bacitracin directly inhibited 125I-insulin degradation by isolated plasma membranes at similar concentrations and to a similar extent as found with cells. The degradative process inhibited by bacitracin accounted for the majority of cellular degradation of the hormone. The increased 125I-insulin bound to adipocytes was shown to be intact by gel chromatographic analysis and was localized to the plasma membrane by direct and indirect approaches. Bacitracin increased 125I-insulin specifically bound to isolated plasma membranes as early as 2 min. The 125I-insulin bound to adipocytes in the presence of bacitracin was completely dissociable by the addition of 8 microM unlabeled insulin whereas a significant portion of 125I-insulin bound to chloroquine-treated cells could not be dissociated. Bacitracin slowed dissociation of 125I-insulin from the cells. Bacitracin increased the 125I-insulin binding to cells in the presence and absence of cyanide and 2,4-dinitrophenol. Bacitracin potentiated the stimulation of adipocyte glucose oxidation at submaximal concentrations of insulin.     

Metal binding ability of glutathione transferases conserved between two animal species, the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II)>V(IV)>Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.     

Inhibition of staphylococcal enterotoxin B formation by cell wall blocking agents and other compounds

Enterotoxin B formation by Staphylococcus aureus S6 was inhibited by Tween 80, oleic acid, sodium deoxycholate, penicillin, d-cycloserine, or bacitracin. Toxin formation by strain 243 was sensitive to oleic acid, sodium deoxycholate, sodium lauryl sulfate, d-cycloserine, or bacitracin. The effect of d-cycloserine was reversed by d-alanine with strain 243 but not with strain S6. Neither penicillin nor bacitracin inhibited alpha-hemolysin or coagulase activity of strain S6; however, 0.118 mumoles of d-cycloserine per ml increased the alpha-hemolysin titer more than eightfold. Pigmentation of strain 243 was reduced by oleic acid, sodium deoxycholate, or methicillin, and was completely inhibited by d-cycloserine or bacitracin. Glucose was required for the inhibition by spermine of (14)C-valine incorporation into cellular protein of strain S6. These data indicate that the cell surface may contain sites important to the synthesis of enterotoxin B.     

Antibacterial cobalt(II), nickel(II) and zinc(II) complexes of nicotinic acid-derived Schiff-bases

Nicotinic acid derived Schiff bases and their transition metal [cobalt(II), nickel(II) and zinc(II)] complexes have been prepared and characterized by physical, spectral and analytical data. The Schiff bases act as deprotonated tridentate ligands for the complexation of the above mentioned metal ions. These complexes, possessing the general formula [M(L)2] [where M = Co(II), Ni(II) and Zn(II) and L = HL1-HL4] showed an octahedral geometry of the metal ions. For determining the effect of metal ions upon chelation, the Schiff bases and their complexes have been screened for antibacterial activity against several pathogenic strains of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The new metal derivatives reported here were more bactericidal against one or more bacterial species as compared to the uncomplexed Schiff bases.     

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids

Background

Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA.

Methods

In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5′ ends with ³²P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined.

Results and conclusions

Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg^2 +, Mn^2 + or Zn^2 + are absolutely necessary.

General significance

The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.     

Biosynthesis of bacitracin on a protein thiotemplate.

The dodecapeptide bacitracin A is the major constitutent of a family of antibacterial peptides produced by Bacillus licheniformis. The non-ribosomal biosynthesis of bacitracin has been studied in cell-free extracts. Bacitracin synthetase has been fractionated on Sephadex G 200 column into two fractions; both fractions were required for bacitracin biosynthesis. On the other hand, on a Sepharose affinity chromatography column, using L-leucine as ligand, three fractions were obtained; all three were required for bacitracin biosynthesis. During bacitracin synthesis, the enzyme components contain a number of thioester bound peptides. The nature of the peptides suggested that the synthesis proceeds towards the C-terminal end of the molecule. It is assumed that by sequential addition of thioester-bound amino acids, bacitracin A could be synthesized on the surface of the enzyme containing phosphopantetheine.     

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations

Abstract Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo' -deficient mutants of Escherichia coli K-12 with the following order of potency: Zn ( II ) > Cd ( II ) > > Co ( II ). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.     

Concurrent sorption of Zn(II), Cu(II) and Co(II) by Oscillatoria angustissima as a function of pH in binary and ternary metal solutions

This paper reports biosorption of Zn(II), Cu(II) and Co(II) onto O. angustissima biomass from single, binary and ternary metal solutions, as a function of pH and metal concentrations via Central Composite Design generated by statistical software package Design Expert 6.0. The experimental design revealed that metal interactions could be best studied at lower pH range i.e. 4.0-5.0, which facilitates adequate availability of all the metal ions. The sorption capacities for single metal decreased in the order Zn(II)>Co(II)>Cu(II). In absence of any interfering metals, at pH 4.0 and an initial metal concentration of 0.5 mM in the solution, the adsorption capacities were 0.33 mmol/g Zn(II), 0.26 mmol/g Co(II) and 0.12 mmol/g Cu(II). In a binary system, copper inhibited both Zn(II) and Co(II) sorption but the extent of inhibition of former was greater than the latter; sorption values being 0.14 mmol/g Zn(II) and 0.27 mmol/g Co(II) at initial Zn(II) and Co(II) concentration of 1.5 mM each, pH 4.0 and 1mM Cu(II) as the interfering metal. Zn(II) and Co(II) were equally antagonistic to each others sorption; Zn(II) and Co(II) sorption being 0.23 and 0.24 mmol/g, respectively, at initial metal concentration of 1.5 mM each, pH 4.0 and 1mM interfering metal concentration. In contrast, Cu(II) sorption remained almost unaffected at lower concentrations of the competing metals. Thus, in binary system inhibition dominance observed was Cu(II)>Zn(II), Cu(II)>Co(II) and Zn(II) approximately Co(II), due to this the biosorbent exhibited net preference/affinity for Cu(II) sorption over Zn(II) or Co(II). Hence, the affinity series showed a trend of Cu(II)>Co(II)>Zn(II). In a ternary system, increasing Co(II) concentration exhibited protection against the inhibitory effect of Cu(II) on Zn(II) sorption. On the other hand, the inhibitory effect of Zn(II) and Cu(II) on Co(II) sorption was additive. The model equation for metal interactions was found to be valid within the design space.