valinomycin and cereulide

Cereulide produced by Bacillus cereus sensu stricto and valinomycin synthesized mainly by Streptomyces spp. are natural dodecadepsipeptide ionophores that act as potassium transporters. Moreover, they comprise three repetitions of similar tetrapeptide motifs synthesized by nonribosomal peptide synthesis complexes. Resemblances in their structure find their reflections in the same way of action. The toxicity of valinomycin and cereulide is an effect of the disturbance of ionic equilibrium and transmembrane potential that may influence the whole organism and then cause fatal consequences. The vlm and ces operons encoding valinomycin and cereulide are both composed of two large, similar synthetase genes, one thioestrase gene and four other ORFs with unknown activities. In spite of the characterization of valinomycin and cereulide, genetic determinants encoding their biosynthesis have not yet been clarified.

Topics: Anti-Bacterial Agents; Depsipeptides; Ionophores; Operon; Valinomycin

We studied the effects of toxins, which inhibited the motility of boar spermatozoa, on rat liver mitochondria. The toxins studied were originally from bacteria isolated from moisture-damaged buildings where inhabitants exhibited symptoms, or from food causing poisoning. Some strains of Bacillus cereus and Streptomyces griseus produced potassium ionophoric peptides cereulide and valinomycin (Mikkola, et al., European Journal of Biochemistry 1999; 263: 112-117). Of interest is that channels were formed in black-lipid membranes (BLM) with a selectivity of K(+) > Na(+) at a concentration of 26 nM. Recently, bafilomycin A1--an inhibitor of V-H(+)ATPases--was found also to be a K(+)-specific ionophore active at nanomolar concentrations (Teplova, et al., J Bioenerg Biomembr 2007; 39: 321-329), while B. amyloliquefaciens produced amylosin, a cation channel-forming peptide with a higher selectivity for K(+) over Na(+) at around 200 nM concentrations (Mikkola, et al., Toxicon 2007; 49: 1158-1171). Of interest is that channels were formed in BLM with a selectivity of K(+) > Na(+) at a concentration of 26 nM. The ionophores and the channel-forming amylosin caused swelling of energized mitochondria due to uptake of K(+), loss of membrane potential, inhibition of maximal respiration rates due to loss of pyridine nucleotides, and inhibition of ATP synthesis. Various cell types may have different sensitivities to the effects of the ionophores. Thus, the mitochondrial membrane potential in neuronal cells was more sensitive to cereulide than in differentiated Paju cells (Teplova, et al., Acta Biochimica Polonica 2004; 51: 539-544). Swelling causes release of proapoptotic factors from mitochondria, which explains that undifferentiated neuronal cells were sensitive, while differentiated Paju cells were resistant, which probably is due to them having an increased expression of the antiapoptotic protein Bcl-2 and the neuroprotective stanniocalcin.

Topics: Animals; Bacterial Toxins; Depsipeptides; Enzyme Inhibitors; Macrolides; Mitochondria, Liver; Potassium; Valinomycin

Topics: Amphotericin B; Antifungal Agents; Candida albicans; Cryptococcus; Depsipeptides; Humans; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Valinomycin

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.

Topics: Antifungal Agents; Bacillus cereus; Depsipeptides; Fungi; Gene Deletion; Genes, Bacterial; Microbial Sensitivity Tests; Plasmids; Valinomycin

Cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties. Cereulide, produced by members of the Bacillus cereus group, is known mostly as emetic toxin, and no ecological function has been assigned. A comparative analysis of the antimicrobial activity of valinomycin produced by Streptomyces spp. and cereulide was performed at a pH range of pH 5.5 to pH 9.5, under anaerobic and aerobic conditions. Both compounds display pH-dependent activity against selected Gram-positive bacteria, including Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, Bacillus subtilis, and Bacillus cereus ATCC 10987. Notably, B. cereus strain ATCC 14579 and the emetic B. cereus strains F4810/72 and A529 showed reduced sensitivity to both compounds, with the latter two strains displaying full resistance to cereulide. Both compounds showed no activity against the selected Gram-negative bacteria. Antimicrobial activity against Gram-positive bacteria was highest at alkaline pH values, where the membrane potential (ΔΨ) is the main component of the proton motive force (PMF). Furthermore, inhibition of growth was observed in both aerobic and anaerobic conditions. Determination of the ΔΨ, using the membrane potential probe DiOC(2)(3) (in the presence of 50 mM KCl) in combination with flow cytometry, demonstrated for the first time the ability of cereulide to dissipate the ΔΨ in sensitive Gram-positive bacteria. The putative role of cereulide production in the ecology of emetic B. cereus is discussed.

Topics: Aerobiosis; Anti-Bacterial Agents; Bacillus cereus; Bacterial Toxins; Depsipeptides; Flow Cytometry; Food Microbiology; Gram-Negative Bacteria; Gram-Positive Bacteria; Hydrogen-Ion Concentration; Membrane Potentials; Microbial Sensitivity Tests; Streptomyces; Valinomycin

Cereulide and valinomycin are both 36-membered cyclic depsipeptides with 12 stereogenic centers that have a very similar sequence of cyclo [-D-O-Leu-D-Ala-L-O-Val-L-Val-]3 and cyclo [-D-O-Val-D-Val-L-O-Ala-L-Val-]3, respectively. Cereulide is an emetic toxin produced by Bacillus cereus through an unusual non-ribosomal peptide synthesis (NRPS), whereas valinomycin, produced by Streptomyces fulvissimus, is a known antibiotic drug. Both compounds are known as K+-ion-selective ionophores and cause a potassium-dependent drop in the transmembrane potential of mitochondria, arising from the uptake of a K+-ion-charged ionophore complex. Such compounds may affect mitochondrial function. In the three-dimensional structure of cereulide and valinomycin, cereulide has a vertical and horizontal mirror-image-like structure as is the case in valinomycin. The only difference is the side chains which are linked to a similar framework. Through the current 1H NMR spectroscopy and metal-complexation studies, we found that cereulide had a higher complexation ability to metal ions compared to valinomycin. Cereulide exhibited the K+-ion-selective ionophore property at a lower concentration than valinomycin. X-ray crystallographic analyses of the cereulide and valinomycin H+ form were compared, and revealed that the higher structures of both compounds also showed similarity in the crystal structures. The structure of cereulide-H+ form was found to be in agreement with the structure obtained by a combination of NMR spectroscopy and molecular-mechanics calculations, which afforded reasonable dihedral angles at the local-minimum-energy conformation of the cereulide-K+-ion complex.

Topics: Anti-Bacterial Agents; Bacillus cereus; Bacterial Toxins; Crystallography, X-Ray; Depsipeptides; Emetics; Metals; Molecular Conformation; Potassium; Streptomyces; Valinomycin

Valinomycin and cereulide are bacterial toxins with closely similar chemical structure and properties but different toxic effects. Emetic poisoning is induced by cereulide but not by valinomycin. Both are specific potassium ionophores. Such compounds may affect mitochondrial functions. Both compounds cause a potassium-dependent drop in the transmembrane inner membrane potential due to the uptake of K+ as positively charged ionophore complex. Valinomycin is more potent than cereulide at high [K+] (>80 mM), whereas cereulide in contrast to valinomycin is active already at <1 mM. With cereulide, there is a substantial lag, while valinomycin acts without lag. Both ionophores induce mitochondrial swelling in the presence of K+, in the case of cereulide with a lag. These toxins strongly inhibited respiration at the level of complex IV when used at higher concentrations than that used for detection of ionophoretic transport of K+. At high [KCl] (120 mM), valinomycin was more potent than cereulide both as ionophore and inhibitor, but at low [KCl] (2.5 mM), cereulide was much more potent. Thus, valinomycin needed 20-30 mM KCl for substantial effects, cereulide only 1-3 mM K+, which is close to its level in blood serum. This explains the higher toxicity of cereulide at low concentrations with the positively charged potassium complex being accumulated in the cell by transport through the plasma membrane driven by the membrane potential. Furthermore, with similar concentrations, the final concentration of cereulide in the cells may become higher than that of valinomycin.

Topics: Animals; Bacterial Toxins; Depsipeptides; Dose-Response Relationship, Drug; Electron Transport; In Vitro Techniques; Ionophores; Male; Membrane Potentials; Mitochondria, Liver; Mitochondrial Swelling; Molecular Structure; Oxygen Consumption; Potassium Channels; Potassium Chloride; Rats; Rats, Wistar; Valinomycin

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.

Topics: Depsipeptides; Keto Acids; Molecular Structure; Peptide Synthases; Valinomycin

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.

Topics: 2,4-Dinitrophenol; Animals; Bacillus cereus; Bacterial Toxins; Biological Assay; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Respiration; Depsipeptides; Enterotoxins; Food Microbiology; Foodborne Diseases; Humans; Liver; Mitochondria, Liver; Oxygen Consumption; Rats; Reproducibility of Results; Sensitivity and Specificity; Staphylococcus aureus; Uncoupling Agents; Valinomycin

In this study we have investigated the impact of differentiation of neuronal cells on their sensitivity to microbial toxins. We used the human neural crest-derived tumor cell line Paju, which can be induced to differentiation in vitro by treatment with phorbol 12-myristate 13-acetate. Addition of the highly toxic potassium ionophores cereulide (4.5 and 9.0 ng/ml) or valinomycin (20 ng/ml), to cultures of undifferentiated Paju cells caused collapse of the mitochondrial membrane potential - measured with the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazole carbocyanine iodide (JC-1) followed by detachment of the cells and their apoptotic death. After induced differentiation of the Paju cells, their mitochondria retained the membrane potential upon exposure to the toxins and the cells displayed increased resistance to apoptosis as compared with undifferentiated cells. This effect may be caused by an elevated expression of the anti-apoptotic protein Bcl-2 and of the neuroprotective factor, stanniocalcin, in differentiated cells.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Cell Differentiation; Cell Line; Cell Proliferation; Depsipeptides; Fluorescent Dyes; Glycoproteins; Humans; Ionophores; Membrane Potentials; Neurons; Potassium; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Valinomycin

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.

Topics: Bacillus cereus; Bacterial Toxins; Chromatography, High Pressure Liquid; Depsipeptides; Mass Spectrometry; Peptides, Cyclic; Temperature; Valinomycin