Fusarium Cyclodepsipeptide Mycotoxins: Chemistry, Biosynthesis, and Occurrence.

Most of the fungi from the Fusarium genus are pathogenic to cereals, vegetables, and fruits and the products of their secondary metabolism mycotoxins may accumulate in foods and feeds. Non-ribosomal cyclodepsipeptides are one of the main mycotoxin groups and include beauvericins (BEAs), enniatins (ENNs), and beauvenniatins (BEAEs). When ingested, even small amounts of these metabolites significantly affect human and animal health. On the other hand, in view of their antimicrobial activities and cytotoxicity, they may be used as components in drug discovery and processing and are considered as suitable candidates for anti-cancer drugs. Therefore, it is crucial to expand the existing knowledge about cyclodepsipeptides and to search for new analogues of these compounds. The present manuscript aimed to highlight the extensive variability of cyclodepsipeptides by describing chemistry, biosynthesis, and occurrence of BEAs, ENNs, and BEAEs in foods and feeds. Moreover, the co-occurrence of Fusarium species was compared to the amounts of toxins in crops, vegetables, and fruits from different regions of the world.

Identification of autoinducing thiodepsipeptides from staphylococci enabled by native chemical ligation.

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.

Assessment of cytotoxic and genotoxic effects of enniatin-A in vitro.

Enniatin A (EN-A) is a Fusarium mycotoxin which is a common contaminant in grains and especially in maize and it causes serious loss of product. The aim of this study was to investigate the cytotoxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay in human cervix carcinoma (HeLa) cell line, and genotoxic effects of EN-A using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) and comet assays in human lymphocytes. The cells were treated with 0.07, 0.14, 0.29, 0.57, 1.15, 2.29, 4.59 and 9.17 µM concentrations of EN-A. It exhibited cytotoxic effects in HeLa cell lines especially when the concentrations were increased. The half-inhibitory value (IC50) was determined as 1.15 µM concentration for 24 h and 0.57 µM concentration for 48 h. However, EN-A failed to affect the frequency of CAs, SCEs and MN in human lymphocytes. Only a slight increase was observed in the frequency of SCEs at 0.57 µM concentration over 48 h. The replication (RI) and nuclear division (NDI) indices were not affected. On the contrary, EN-A decreased the mitotic index (MI) significantly at all concentrations compared to the negative control and solvent control (except at 0.29 µM for 24 h, and except at 0.14, 0.29 and 0.57 µM for 48 h). Treatments over 2.29 µM showed toxic effects in human lymphocytes. EN-A significantly increased comet tail intensity (except at 0.07 and 0.57 µM) in isolated human lymphocytes. The results of this study demonstrate that EN-A has an obvious cytotoxic effect especially when the EN-A concentration was increased. In addition, EN-A could exhibit a mild genotoxic effect.

Biological characteristics, bioactive components and antineoplastic properties of sporoderm-broken spores from wild Cordyceps cicadae.

BACKGROUND: Cordyceps cicadae, an entomogenous fungus has been used as a dietary therapeutic in traditional Chinese medicine for several millennia, in the form of powders and decoction. However, wild C. cicadae is notably scarce. To date, there is still a lack of comprehensive and deep studies on the biological characteristics, chemical profiles and antineoplastic mechanisms of C. cicadae, especially its spores. AIM OF THE STUDY: This study aimed to identify wild C. cicadae using rDNA-ITS sequences. Active constituents and volatile ingredients of C. cicadae sporoderm-broken spore powders (CCBSP) were elucidated using UPLC-ESI-Q-TOF-MS and GC-MS, respectively. The underlying anti-neoplastic mechanisms of CCBSP were further investigated in A549 lung carcinoma cells. RESULTS: Molecular phylogenetic analysis of nuclear rDNA sequences indicated that wild C. cicadae belonged to Paecilomyces cicadae. Eight primary compounds from CCBSP were identified by MS fragmentation ions including nucleosides, cordycepic acid, cordycepin, beauvericin and myriocin. In total, forty-nine volatile components representing 99.56% of CCBSP were clearly identified. CCBSP exhibited antiproliferative effects on A549 cells with IC50 value of 125.54 ± 2.71 µg/ml, blocking the cell cycle in the G2/M phase. The nuclear morphology exhibited typical characteristics of apoptosis by Hoechst fluorescent stain. AnnexinV-FITC/PI staining revealed that the number of apoptotic cells increased after CCBSP treatment. Furthermore, immunofluorescence experiments indicated that CCBSP lowered the expressions of ß-catenin and N-cadherin, which was accompanied by repressed Wnt/ß-catenin signalling and activation of caspase-mediated apoptosis pathways. CONCLUSIONS: rDNA-ITS sequencing enabled molecular identification of wild C. cicadae. Importantly, these findings provide the first evidence regarding the full-scale bioactive components and antineoplastic properties of CCBSP. These data highlight the significance of C. cicadae as a potential antineoplastic agent.

Mycotoxin risk assessment for consumers of groundnut in domestic markets in Nigeria.

The fungal and multi-mycotoxin profiles of groundnuts sold in domestic markets in Nigeria as well as the associated risk to consumers were assessed in the present study. Four hundred fungal isolates representing mainly Aspergillus [58.6%: Aspergillus section Flavi (37.1%) and A. niger-clade (21.5%)], Penicillium (40.9%) and Fusarium (0.5%) were isolated from 82 (97.6%, n=84) groundnut samples collected from four agro-ecological zones (AEZs) of Nigeria. The incidence of aflatoxin-producing A. flavus isolates (71%) was significantly (p<0.05) higher in the groundnuts than that of the non-aflatoxigenic isolates (29%). Fifty-four fungal metabolites [including aflatoxins (AFB1, AFB2, AFG1, AFG2 and AFM1), beauvericin (BEAU), cyclopiazonic acid (CPA), moniliformin, nivalenol and ochratoxin A] and four bacterial metabolites were detected in the groundnuts by liquid chromatography tandem mass spectrometry. Aflatoxins (39%; max: 2076µg/kg; mean: 216µg/kg) were detected in more samples than any other mycotoxin. About 25, 23 and 14% of the samples respectively were above the 2µg/kg AFB1, 4 and 20µg/kg total aflatoxin limits of the European Union and US FDA respectively. The mean margins of exposure of AFB1 and total aflatoxins for adult consumers were 1665 and 908, respectively, while mean estimated daily intake values for infants, children and adults were <0.1% for BEAU and 4% for CPA. Consumers of mycotoxin contaminated groundnuts in Nigeria may therefore be at a risk of liver cancer in addition to other combinatory effects of mycotoxin/metabolite cocktails. There is need for increased targeted interventions in the groundnut value chain in Nigeria for public health benefits.

Chemodiversity of cereulide, the emetic toxin of Bacillus cereus.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Phylogenetic and chemical diversity of three chemotypes of bloom-forming lyngbya species (Cyanobacteria: Oscillatoriales) from reefs of southeastern Florida.

The cyanobacterial genus Lyngbya includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries. Lyngbya spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct Lyngbya species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three Lyngbya species with three distinct Lyngbya chemotypes. One species, identified as Lyngbya cf. confervoides via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two Lyngbya species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three Lyngbya chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming Lyngbya species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.

Survey of maize from south-western Nigeria for zearalenone, alpha- and beta-zearalenols, fumonisin B1 and enniatins produced by Fusarium species.

A survey for the natural occurrence of Fusarium mycotoxins in maize for human consumption in four south-western states of Nigeria using High Performance Liquid Chromatography coupled with Mass Spectroscopy (HPLC/MS) showed that 93.4% of the samples were contaminated with zearalenone (ZON), alpha- and beta-zearalenols (alpha- and beta-ZOL), fumonisin B(1) (FB(1)) or enniatins (ENNs). The fractions of contaminated samples were 73% for FB(1) (mean:117 microg kg(-1), range:10-760 microg kg(-1)); 57% for ZON (mean:49 microg kg(-1), range:115-779 microg kg(-1)) and 13% for alpha-ZOL (mean: 63.6 microg kg(-1), range:32-181 microg kg(-1)), while ENNs A1, B and B(1) were present in 3, 7 and 3% of the samples respectively. There was no beta-ZOL present above the quantification limits of 50 microg kg(-1). Only the FB(1) content was significantly different at the 95% confidence level among the four states. The Fusarium species most frequently isolated from maize seeds were F. verticillioides (70%), followed by F. sporotrichioides (42%), F. graminearum (30%), F. pallidoroseum (15%), F. compactum (12%), F. proliferatum (12%), F. equiseti (9%), F. acuminatum (8%) and F. subglutinans (4%). This is the first report of the occurrence of alpha-zearalenol and enniatins in Nigerian maize.

[Beauvericin: chemical and biological aspects and occurrence]. / Bovericin: kemizam, bioloski aspekti i rasirenost.

Beauvericin (BEA) is a cyclic hexadepsipeptide produced by Beauveria bassiana, Paecilomyces fumosoroseus, Paecilomyces tenuipes, Polyporus sulphurous, and a variety of Fusarium species. This mycotoxin shows antimicrobial, insecticidal, cytotoxic, and apoptotic activity. It is the most potent specific inhibitor of cholesterol acyltransferase and possesses ionophoric properties. BEA increases ion permeability in biological membranes by forming a complex with essential cations (Ca2+, Na+, K+), which may affect the ionic homeostasis. BEA has been frequently found in maize samples in Europe, USA and Africa and co-contamination with other Fusarium toxins such as fumonisins, and moniliformin was also found. There is only one report of BEA occurrence and co-occurrence with fumonisin B1, fumonisin B2 and ochratoxin A in Croatia. Biological activity of BEA may increase the toxicity of other mycotoxins that co-occur with BEA in food. The role of BEA in the development of human and animal mycotoxicosis is still unknown.

Circular dichroism analysis of destruxins from Metarhizium anisopliae.

Destruxins are secondary metabolites secreted by Metarhizium anisopliae [Y. Kodaira, Toxic substances to insects, produced by Aspergillus ochraceus and Oopsra destructor, Agric. Biol. Chem., 25 (1961) 261-262. D.W. Roberts, Toxins from the entomogenous fungus Metarhizium anisoplaie: Isolation from submerged cultures, J. Invertebr. Pathol., 14 (1969) 82-88. D.W. Roberts, Toxins from the entomogenic fungi in microbial control of pest and plant disease, Academic press, New York, 1981, pp441-464.]. In recent research, other than being used as insecticides, destruxins exhibited great potential in therapeutical applications such as antitumor, antivirus, and animal cell immunization effectiveness, etc. In this study, the conformations purified destruxins were determined by circular dichroism (CD). The results indicated that these cyclic peptides have the type I beta-turn conformation. In addition, different types of destruxins exhibited different CD spectra in acetonitrile. Therefore, these characters can be used as fingerprints to identify each type of destruxin. To further investigate the interactions among destruxins, various combinations of destruxins in 10 mM phosphate-buffered saline (PBS) were also studied by CD. The results strongly suggested that destruxins might work independently in vivo. To our knowledge, this is the first report presenting the CD analysis of purified destruxins.

Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not?

It appears to be a general belief that stable isotopically labeled (SIL) internal standards yield better assay performance results for quantitative bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays than does any other internal standard. In this article we describe our experiences with structural analogues and SIL internal standards and their merits and demerits. SIL internal standards are the first choice, but deuterium-labeled compounds may demonstrate unexpected behavior, such as different retention times or recoveries, than the analyte. In addition, a SIL internal standard with identical chemical properties as the analyte may cover up assay problems with stability, recovery, and ion suppression. Since SIL internal standards are not always available or are very expensive, structural analogues can be used, however, with consideration of several issues, which are usually displayed during method validation.