Raimo Mikkola

Inhibition of human natural killer cell activity by cereulide, an emetic toxin from Bacillus cereus

The lipophilic toxin, cereulide, emitted by emetic food poisoning causing strains of Bacillus cereus, is a powerful mitochondria toxin. It is highly lipophilic and rapidly absorbed from the gut into the bloodstream. We tested how this toxin influences natural killer (NK) cells, which are important effectors in defence against infections and malignancy. Cereulide inhibited cytotoxicity and cytokine production of natural killer cells, caused swelling of natural killer cell mitochondria, and eventually induced natural killer cell apoptosis. The suppressive effect on cytotoxicity was fast and toxic concentration low, 20–30 μg/l. As the emesis causing concentration of cereulide is around 10 μg/kg of total body mass, our results suggest that emesis causing or even lower doses of cereulide may also have a systemic natural killer cell suppressive effect.

A new method for in vitro detection of microbially produced mitochondrial toxins

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Frigoribacterium faeni gen. nov., sp. nov., a novel psychrophilic genus of the family Microbacteriaceae.

The taxonomic position of five actinobacterial strains isolated from dust, an animal shed, the air inside a museum and soil was investigated using a polyphasic approach. The growth characteristics were unusual for actinomycetes. Optimal growth was at temperatures ranging from 2 to 10 degrees C. After small-step adaptation (5 degrees C steps) to higher temperatures, the strains were also able to grow at 20 degrees C. Cell wall analyses revealed that the organisms showed a hitherto undescribed, new group B-type peptidoglycan [type B2beta according to Schleifer & Kandler (1972), but with lysine instead of ornithine]. All strains contained menaquinone MK-9. Mycolic acids were not detected. Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were detected in the polar lipid extracts. The main fatty acids were 12-methyl-tetradecanoic acid (15:0 anteiso), 12-methyl-tetradecenoic acid (15:1 anteiso), 14-methyl-pentadecanoic acid (16:0 iso) and 14-methyl-hexadecanoic acid (17:0 iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso-pentadecane (15:0 anteiso-DMA). The G+C content of DNA was approximately 71 mol%. The results of 16S rRNA gene sequence comparisons revealed that the strains represent a new lineage in the suborder Micrococcineae and the family Microbacteriaceae of the order Actinomycetales. On the basis of these results the new genus Frigoribacterium gen. nov. is proposed, harbouring the new species Frigoribacterium faeni sp. nov. (type strain = 801T = DSM 10309T).

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

ABSTRACT Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen−1). The same exposure concentration depleted the boar sperm cells of NADH2. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψm) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.

Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin

Aim:  To screen and characterize toxic, heat‐stable substances produced by food borne strains from Bacillus subtilis group.

20-Residue and 11-residue peptaibols from the fungus Trichoderma longibrachiatum are synergistic in forming Na + /K + -permeable channels and adverse action towards mammalian cells

Certain species of the filamentous fungal genus Trichoderma (e.g. Trichoderma longibrachiatum and Trichoderma citrinoviride) are among the emerging clinical pathogens and also the most common species in the indoor space of mould‐damaged buildings. The molecules involved in its pathology are not known. In the present study, we report that 0.5–2.6 wt% of the T. longibrachiatum mycelial biomass consisted of thermostable secondary metabolites mitochondriotoxic to mammalian cells. These were identified by LC/MS as one 11‐residue and eight 20‐residue peptaibols, AcAib‐Asn‐Leu/Ile‐Leu/Ile‐Aib‐Pro‐Leu/Ile‐Leu/Ile‐Aib‐Pro‐Leuol/Ileol (1175 Da) and AcAib‐Ala‐Aib‐Ala‐Aib‐Ala/Aib‐Gln‐Aib‐Val/Iva‐Aib‐Gly‐Leu/Ile‐Aib‐Pro‐Val/Iva‐Aib‐Val/Iva/Aib‐Gln/Glu‐Gln‐Pheol(1936–1965 Da) (Aib, α‐aminoisobutyric acid; Ac, acetyl; Ileol, isoleucinol; Iva, isovaline; Leuol, leucinol; Pheol, phenylalaninol). The toxic effects on boar sperm cells depended on these peptaibols, named trilongins. The trilongins formed voltage dependent, Na+/K+ permeable channels in biomembranes. The permeability ratios for Na+ ions, relative to K+, of the 11‐residue trilongin channel (0.95 : 1) and the 20‐residue trilongin channel (0.8 : 1) were higher than those of alamethicin. The combined 11‐residue and 20‐residue trilongins generated channels that remained in an open state for a longer time than those formed by either one of the peptaibols alone. Corresponding synergy was observed in toxicokinetics. With 11‐residue and 20‐residue trilongins combined 1 : 2 w/w, an effective median concentration (EC50) of 0.6 μg·mL−1 was reached within 30 min, and the EC50 shifted down to 0.2 μg·mL−1 upon extended exposure. By contrast, with 11‐residue or 20‐residue trilonging separately in 30 min of exposure, the EC50 values were 15 and 3 μg·mL−1, respectively, and shifted down to 1.5 and 0.4 μg·mL−1 upon extended exposure. This is the first report on ion‐channel forming peptaibols with synergistic toxicity from T. longibrachiatum strains isolated from clinical samples.

In vitro toxicity of cereulide on porcine pancreatic Langerhans islets

Cereulide is a K(+) ionophore cytotoxic and mitochondriotoxic to primary cells and cell lines of human and other mammalian origins. It is a heat-stable, highly lipophilic (logK(ow) 5.96) peptide (1152 g mol(-1)) produced by certain strains of Bacillus cereus, a bacterium connected to emetic food poisonings. In this study the pancreatic toxicity of purified cereulide, and cereulide-containing bacterial extracts, was studied using fetal porcine Langerhans islets in culture. Exposure to 1ngml(-1) of purified cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. Cell extracts of cereulide-positive B. cereus strains connected to food poisoning or isolated from foodstuffs were toxic, corresponding to their measured cereulide content. Extracts of B. cereus strains producing or not producing the B. cereus diarrheal toxin, but no cereulide, were tolerated by the porcine islet cultures up to concentrations 1000-fold higher compared to extracts from strains containing cereulide, and up to exposure times of 7d. Cereulide thus was identified as the B. cereus-produced substance toxic towards porcine fetal Langerhans islets and beta cells.

Inhibition of Human NK Cell Function by Valinomycin, a Toxin from Streptomyces griseus in Indoor Air

ABSTRACT Streptomyces griseus strains isolated from indoor dust have been shown to synthesize valinomycin. In this report, we show that human peripheral blood lymphocytes treated with small doses (30 ng ml−1) of pure valinomycin or high-pressure liquid chromatography-pure valinomycin from S. griseus quickly show mitochondrial swelling and reduced NK cell activity. Larger doses (>100 ng/ml−1) induced NK cell apoptosis within 2 days. Within 2 h, the toxin at 100 ng ml−1 dramatically inhibited interleukin-15 (IL-15)- and IL-18-induced granulocyte-macrophage colony-stimulating factor and gamma interferon (IFN-γ) production by NK cells. However, IFN-γ production induced by a combination of IL-15 and IL-18 was somewhat less sensitive to valinomycin, suggesting a protective effect of the cytokine combination against valinomycin. Thus, valinomycin in very small doses may profoundly alter the immune response by reducing NK cell cytotoxicity and cytokine production.

Boar spermatozoa as a biosensor for detecting toxic substances in indoor dust and aerosols.

The presence, quantity and origins of potentially toxic airborne substances were searched in moisture damaged indoor environments, where building related ill health symptoms were suspected and reference sites with no health complaints. Boar spermatozoa were used as the toxicity sensor. Indoor aerosols and dusts were collected from kindergartens, schools, offices and residences (n=25) by electrostatic filtering, vacuuming, wiping from elevated surfaces and from the interior of personal computers. Toxicity was measured from the ethanol or methanol extracts of the dusts and aerosols. EC(50) was expressed as the lowest concentration of the airborne substance that inhibited motility of >50% of the exposed sperm cells compared to vehicle control, within 30 min, 1 day or 3-4 days of exposure. Remarkably toxic aerosols (EC(50) <or=6 μg ml(-1)) were found from 11 sites, all of these were sites with known or suspected for building related ill health. Toxic microbial cultures were obtained from subsamples of the toxic aerosols/dusts. From these cereulide, amylosin, valinomycin and a novel indoor toxin, stephacidin B were identified and toxicities measured. Airborn dispersal of valinomycin from Streptomyces griseus cultures was evaluated using a flow-through chamber. Significant amounts of valinomycin (LC-MS assay) and toxicity (boar sperm motility assay) were carried by air and were after 14 days mainly recovered from the interior surfaces of the flow chamber.

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate

Aims:  To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms.