Thomas Ostenfeld Larsen

Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium.

ABSTRACT In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosumisolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and threeP. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.

Review of secondary metabolites and mycotoxins from the Aspergillus niger group

AbstractFilamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread food and feed contaminants known but they are also some of the most important workhorses used by the biotechnological industry. The Nigri section consists of six commonly found species (excluding A. aculeatus and its close relatives) from which currently 145 different secondary metabolites have been isolated and/or detected. From a human and animal safety point of view, the mycotoxins ochratoxin A (from A. carbonarius and less frequently A. niger) and fumonisin B2 (from A. niger) are currently the most problematic compounds. Especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products where fumonisin-producing fusaria are not a problem, fumonisins pose a risk. Moreover, compounds such as malformins, naptho-γ-pyrones, and bicoumarins (kotanins) call for monitoring in food, feed, and biotechnology products as well as for a better toxicological evaluation, since they are often produced in large amounts by the black aspergilli. For chemical differentiation/identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested). Naptho-γ-pyrones are the compounds produced in the highest quantities and are produced by all six common species in the group (A. niger 134 of 140; A. tubingensis 169 of 177; A. acidus 44 of 47; A. carbonarius 40 of 40, A. brasiliensis 18 of 18; and A. ibericus three of three). FigureImage of Aspergillus niger growing on YES agar, and the resulting extract analysed by LCDAD-TOFMS

Ajoene, a Sulfur Rich Molecule from Garlic, Inhibits Genes Controlled by Quorum Sensing

ABSTRACT In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.

Characterization of volatile metabolites from 47 Penicillium taxa

Forty-seven different taxa within Penicillium were investigated for the production of volatile metabolites. Fungi were cultured on three agar media in Petri dishes. Volatiles were collected by diffusive sampling onto tubes with carbon black for 2 wk, and then extracted with diethylether. Ether extracts were analysed by gas chromatography (GC) and volatiles were characterized by mass spectrometry (MS) and Fourier transform infrared spectroscopy (FTIR). Their retention indices (RI) were compared with those of authentic compounds. In total, 196 different volatile metabolites were characterized from the taxa investigated. About 70 metabolites were assigned by MS as isomeric sesquiterpene hydrocarbons (C15H24), such as β-caryophyllene, α-bergamotene and thujopsene. However, the majority of sesquiterpenes have only been characterized by RI and mass spectral data generated at 70 eV, in a 9 peak index. Computer matching of spectral data with spectra in the NIST library showed that these compounds could not be assigned. A number of monoterpenes, alcohols, esters, ketones, alkenes and a few aromatic compounds were also produced. Geosmin and 2-methyl-isoborneol, reported as contributing to mouldy/earthy off flavours, were both produced by a number of different taxa, reported here for the first time. More than half of the metabolites were only detected from one species and nearly all taxa each produced a unique profile of volatile metabolites. Thus, the present study has demonstrated the potential of species of Penicillium as producers of a great diversity of volatile metabolites, likely to be useful in chemosystematics.

Dereplication of Microbial Natural Products by LC-DAD-TOFMS

Dereplication, the rapid identification of known compounds present in a mixture, is crucial to the fast discovery of novel natural products. Determining the elemental composition of compounds in mixtures and tentatively identifying natural products using MS/MS and UV/vis spectra is becoming easier with advances in analytical equipment and better compound databases. Here we demonstrate the use of LC-UV/vis-MS-based dereplication using data from UV/vis diode array detection and ESI+/ESI- time-of-flight MS for assignment of 719 microbial natural product and mycotoxin reference standards. ESI+ was the most versatile ionization method, detecting 93% of the compounds, although with 12% ionizing poorly. Using ESI+ alone, 56.1% of the compounds could be unambiguously assigned based on characteristic patterns of multiple adduct ions. Using ESI-, 36.4% of the compounds could have their molecular mass assigned unambiguously using multiple adduct ions, while a further 41% of the compounds were detected only as [M - H]-. The most reliable interpretations of conflicting ESI+ and ESI- data on a chromatographic peak were from the ionization polarity with the most intense ionization. Poor ionization was most common with small molecules (<200 Da). In ESI-, these were often polar and basic, while in ESI+ they were small aromatic acids or anthraquinones. No single ion-source settings could be applied over a m/z 60-2000 range. However, continuous switching among three settings (e.g., for 0.5 s each) during the chromatographic run allowed MS of both small labile molecules and large peptides, and pseudo MS/MS data on labile molecules since the settings for large molecules often induce fragmentation into small molecules.

Identification of Griseofulvin as an Inhibitor of Centrosomal Clustering in a Phenotype-Based Screen

A major drawback of cancer chemotherapy is the lack of tumor-specific targets which would allow for the selective eradication of malignant cells without affecting healthy tissues. In contrast with normal cells, most tumor cells contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. Many tumor cells regain mitotic stability after clonal selection by the coalescence of multiple centrosomes into two functional spindle poles. To overcome the limitations of current cancer treatments, we have developed a cell-based screening strategy to identify small molecules that inhibit centrosomal clustering and thus force tumor cells with supernumerary centrosomes to undergo multipolar mitoses, and subsequently, apoptosis. Using a chemotaxonomic selection of fungi from a large culture collection, a relatively small but diverse natural product extract library was generated. Screening of this compound library led to the identification of griseofulvin, which induced multipolar spindles by inhibition of centrosome coalescence, mitotic arrest, and subsequent cell death in tumor cell lines but not in diploid fibroblasts and keratinocytes with a normal centrosome content. The inhibition of centrosome clustering by griseofulvin was not restricted to mitotic cells but did occur during interphase as well. Whereas the formation of multipolar spindles was dynein-independent, depolymerization of interphase microtubules seemed to be mechanistically involved in centrosomal declustering. In summary, by taking advantage of the tumor-specific phenotype of centrosomal clustering, we have developed a screening strategy that might lead to the identification of drugs which selectively target tumor cells and spare healthy tissues.

Rate‐limited sorption and nonequilibrium transport of organic chemicals in low organic carbon aquifer materials

The rate-limited sorption and nonequilibrium transport of several hydrophobic organic chemicals in three low-organic carbon (< 0.025%) aquifer materials was investigated. Results of miscible displacement experiments performed at two pore water velocities and with very low solution-phase concentrations (30-60 {mu}g L{sup {minus}1}) were analyzed using a first-order mass transfer nonequilibrium model, as well as a model employing the local equilibrium assumption. Results of the analyses revealed sorption to be significantly rate limited, possibly by a diffusion-limited mechanism. The impact of rate-limited sorption on transport was dependent upon pore water velocity. The experiments performed at a faster velocity ({approximately} 1 cm/h) could be successfully simulated only with the nonequilibrium model, whereas the equilibrium model was adequate for the slower-velocity ({approximately} 0.2 cm/h) experiments. Comparison of experimental results to those reported in the literature revealed that time scale has a significant impact on the degree of nonequilibrium observed in, and on the values of rate constants determined from, experiments. Regression equations were developed for the two kinetics-associated parameters contained in the nonequilibrium model: first-order mass transfer constant and fraction of instantaneous sorption.

Accurate prediction of secondary metabolite gene clusters in filamentous fungi

Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association–based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.

Fumonisin and Ochratoxin Production in Industrial Aspergillus niger Strains

Aspergillus niger is perhaps the most important fungus used in biotechnology, and is also one of the most commonly encountered fungi contaminating foods and feedstuffs, and occurring in soil and indoor environments. Many of its industrial applications have been given GRAS status (generally regarded as safe). However, A. niger has the potential to produce two groups of potentially carcinogenic mycotoxins: fumonisins and ochratoxins. In this study all available industrial and many non-industrial strains of A. niger (180 strains) as well as 228 strains from 17 related black Aspergillus species were examined for mycotoxin production. None of the related 17 species of black Aspergilli produced fumonisins. Fumonisins (B2, B4, and B6) were detected in 81% of A. niger, and ochratoxin A in 17%, while 10% of the strains produced both mycotoxins. Among the industrial strains the same ratios were 83%, 33% and 26% respectively. Some of the most frequently used strains in industry NRRL 337, 3112 and 3122 produced both toxins and several strains used for citric acid production were among the best producers of fumonisins in pure agar culture. Most strains used for other biotechnological processes also produced fumonisins. Strains optimized through random mutagenesis usually maintained their mycotoxin production capability. Toxigenic strains were also able to produce the toxins on media suggested for citric acid production with most of the toxins found in the biomass, thereby questioning the use of the remaining biomass as animal feed. In conclusion it is recommended to use strains of A. niger with inactive or inactivated gene clusters for fumonisins and ochratoxins, or to choose isolates for biotechnological uses in related non-toxigenic species such as A. tubingensis, A. brasiliensis, A vadensis or A. acidus, which neither produce fumonisins nor ochratoxins.

Food as a Source for Quorum Sensing Inhibitors: Iberin from Horseradish Revealed as a Quorum Sensing Inhibitor of Pseudomonas aeruginosa

ABSTRACT Foods with health-promoting effects beyond nutritional values have been gaining increasing research focus in recent years, although not much has been published on this subject in relation to bacterial infections. With respect to treatment, a novel antimicrobial strategy, which is expected to transcend problems with selective pressures for antibiotic resistance, is to interrupt bacterial communication, also known as quorum sensing (QS), by means of signal antagonists, the so-called QS inhibitors (QSIs). Furthermore, QSI agents offer a potential solution to the deficiencies associated with use of traditional antibiotics to treat infections caused by bacterial biofilms and multidrug-resistant bacteria. Several QSIs of natural origin have been identified, and in this study, several common food products and plants were extracted and screened for QSI activity in an attempt to isolate and characterize previously unknown QSI compounds active against the common opportunistic pathogen Pseudomonas aeruginosa. Several extracts displayed activity, but horseradish exhibited the highest activity. Chromatographic separation led to the isolation of a potent QSI compound that was identified by liquid chromatography-diode array detector-mass spectrometry (LC-DAD-MS) and nuclear magnetic resonance (NMR) spectroscopy as iberin—an isothiocyanate produced by many members of the Brassicaceae family. Real-time PCR (RT-PCR) and DNA microarray studies showed that iberin specifically blocks expression of QS-regulated genes in P. aeruginosa.