Cyclic Hexadepsipeptides Research Articles

Characterization of mycotoxins produced by two Fusarium species responsible for postharvest rot of banana fruit

AbstractIn an open-air market in southern Italy, we noticed ‘Lady finger’ banana fruit imported from Costa Rica showing a severe rot, whose symptoms consisted of necrotic peel lesions with variable shape and size. Fusarium sacchari and F. proliferatum were consistently isolated from symptomatic fruit. In pathogenicity tests on ‘Lady finger’ banana fruit, F. proliferatum was more virulent than F. sacchari. Quantitative Time-of-Flight Mass Spectrometric analysis of secondary metabolites produced by isolates of these two Fusarium species on three different matrices (banana peel, barley and maize kernels) identified 11 mycotoxins. Seven of them (Fusaproliferin, Fumonisins A1, Fumonisins A2 and Fumonisins B1, Hydrolysed Fumonisin B1, Fusarin C and Moniliformin) were detected in matrices contaminated by F. proliferatum isolates. Fumonisin A1 was the prevalent mycotoxin in both maize kernels and banana peel, while Fumonisin A2 prevailed in barley kernels. Similarly, seven mycotoxins (the cyclic hexadepsipeptides Enniatins B2, B3 and B4, Fumonisins A1 and B2, Hydrolysed Fumonisin B1 and Fusarin C) were detected in matrices contaminated by F. sacchari isolates, but they were only in part the same as those produced by F. proliferatum isolates. Fusarin C prevailed in all three matrices colonized by F. sacchari. Fumonisin A1 was detected exclusively in maize kernels while Enniatins B3 and B4, Fumonisin B2 and Hydrolysed Fumonisin B1 were detected exclusively in barley kernels. Overall, F. proliferatum produced a higher amount of mycotoxins than F. sacchari. Moreover, in banana peel both species produced a lower number and amount of mycotoxins than in the other two matrices.

Environmental metabolomics characterization of modern stromatolites and annotation of ibhayipeptolides.

Lithified layers of complex microbial mats known as microbialites are ubiquitous in the fossil record, and modern forms are increasingly identified globally. A key challenge to developing an understanding of microbialite formation and environmental role is how to investigate complex and diverse communities in situ. We selected living, layered microbialites (stromatolites) in a peritidal environment near Schoenmakerskop, Eastern Cape, South Africa to conduct a spatial survey mapping the composition and small molecule production of the microbial communities from environmental samples. Substrate core samples were collected from nine sampling stations ranging from the upper point of the freshwater inflow to the lower marine interface where tidal overtopping takes place. Substrate cores provided material for parallel analyses of microbial community diversity by 16S rRNA gene amplicon sequencing and metabolomics using LC-MS2. Species and metabolite diversities were correlated, and prominent specialized metabolites were targeted for preliminary characterization. A new series of cyclic hexadepsipeptides, named ibhayipeptolides, was most abundant in substrate cores of submerged microbialites. These results demonstrate the detection and identification of metabolites from mass-limited environmental samples and contribute knowledge about microbialite chemistry and biology, which facilitates future targeted studies of specialized metabolite function and biosynthesis.

Toxicity of the emerging mycotoxins beauvericin and enniatins: A mini-review

Beauvericin and enniatins, emerging mycotoxins produced mainly by Fusarium species, are natural contaminants of cereals and cereal products. These mycotoxins are cyclic hexadepsipeptides with ionophore properties and their toxicity mechanism is related to their ability to transport cations across the cell membrane. Beauvericin and enniatins are cytotoxic, as they decrease cell viability, promote cell cycle arrest, and increase apoptosis and the generation of reactive oxygen species in several cell lines. They also cause changes at the transcriptomic level and have immunomodulatory effects in vitro and in vivo. Toxicokinetic results are scarce, and, despite its proven toxic effects in vitro, no regulation or risk assessment has yet been performed due to a lack of in vivo data. This mini-review aims to report the information available in the literature on studies of in vitro and in vivo toxic effects with beauvericin and enniatins, which are mycotoxins of increasing interest to animal and human health.

Cyclic hexadepsipeptides from the fermentation of Fusarium sp. DCJ-A and their cytotoxic activities

Beauvercin H (1), a new cyclic hexadepsipeptide, and two known ones (2 and 3) were isolated from the EtOH extract of the solid culture of Fusarium sp. Their structures were elucidated by spectroscopic analysis, including extensive 1D and 2D NMR techniques, as well as comparison with literature values. Additionally, compounds 1–3 were tested for their cytotoxic activities. The results showed that all isolated compounds exhibited cytotoxic activities against five human cancer cell lines with IC50 values ranging from 1.379 to 13.12 μM.

Genome-Mining-Guided Discovery and Characterization of the PKS-NRPS-Hybrid Polyoxyperuin Produced by a Marine-Derived Streptomycete

The azinothricin family comprises several cyclic hexadepsipeptides with diverse pharmacological bioactivities, including antimicrobial, antitumoral, and apoptosis induction. In this work, using a genome mining approach, a biosynthetic gene cluster encoding an azinothricin-like compound was identified from the Streptomyces sp. s120 genome sequence (pop BGC). Comparative MS analysis of extracts from the native producer and a knockout mutant led to the identification of metabolites corresponding to the pop BGC. Furthermore, regulatory elements of the BGC were identified. By overexpression of an LmbU-like transcriptional activator, the production yield of 1 and 2 was increased, enabling isolation and structure elucidation of polyoxyperuin A seco acid (1) and polyoxyperuin A (2) using high-resolution mass spectrometry and NMR spectroscopy. Compound 1 exhibited a low antibiotic effect against Micrococcus luteus, while 2 showed a strong Gram-positive antibiotic effect in a micro-broth-dilution assay.

Synthesis and Evaluation of Antimycobacterial and Antiplasmodial Activities of Hirsutellide A and Its Analogues.

Hirsutellide A is nature-derived cyclic hexadepsipeptide with reported antimycobacterial and antiplasmodial activities. To verify its structure, hirsutellide A was synthesized following a solution-phase peptide synthesis approach. A detailed analysis of the 1H and 13C NMR spectra of the synthesized compound revealed structural variation from what had been originally assigned for hirsutellide A, despite the use of identical building blocks. This variation occurred at the two allo-Ile moieties. To investigate the structure–activity relationship, the depsipeptide and peptide analogues of hirsutellide A were prepared and tested for antimycobacterial and antiplasmodial activities. The compounds displayed antiplasmodial potency against Plasmodium falciparum 3D7 while showing weak or no activity against Mycobacterium tuberculosis H37Rv. The drug-likeness of the series was assessed through in vitro absorption, distribution, metabolism, and excretion (ADME) profiling, revealing systematic differences between the pharmacokinetic properties of cyclic hexapeptides and hexadepsipeptides.

Meliponamycins: Antimicrobials from Stingless Bee-Associated Streptomyces sp.

Social insects establish complex interactions with microorganisms, some of which play defensive roles in colony protection. The important role of pollinators such as the stingless bee Melipona scutellaris in nature encouraged us to pursue efforts to study its associated microbiota. Here we describe the discovery of two novel cyclic hexadepsipeptides, meliponamycin A (1) and meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated from M. scutellaris nurse bees. Their structures were established by interpretation of NMR and MS data, and the absolute configuration of the constituent amino acids was determined by the advanced Marfey's method. Compounds 1 and 2 showed strong activity against the entomopathogen Paenibacillus larvae and human pathogens Staphylococcus aureus and Leishmania infantum.

Depsipeptide Aspergillicins Revealed by Chromatin Reader Protein Deletion.

Expression of biosynthetic gene clusters (BGCs) in filamentous fungi is highly regulated by epigenetic remodeling of chromatin structure. Two classes of histone modifying proteins, writers (which place modifications on histone tails) and erasers (which remove the modifications), have been used extensively to activate cryptic BGCs in fungi. Here, for the first time, we present activation of a cryptic BGC by a third category of histone modifying proteins, reader proteins that recognize histone tail modifications and commonly mediate writer and eraser activity. Loss of the reader SntB (Δ sntB) resulted in the synthesis of two cryptic cyclic hexa-depsipeptides, aspergillicin A and aspergillicin F, in the fungus Aspergillus flavus. Liquid chromatography, high resolution mass spectrometry, and NMR analysis coupled with bioinformatic analysis and gene deletion experiments revealed that a six adenylation (A) domain nonribosomal peptide synthetase (NRPS, called AgiA) and O-methyltransferase (AgiB) were required for metabolite formation. A proposed biosynthetic scheme illustrates the requirement for unusual NRPS domains, such as a starting condensation domain and a thiolesterase domain proposed to cyclize the depsipeptides. This latter activity has only been found in bacterial but not fungal NRPS. The agi BGC-unique to A. flavus and some closely related species (e.g., A. oryzae, A. arachidicola)-is located next to a conserved Aspergillus siderophore BGC syntenic to other fungi.

Fusarihexins A and B: Novel Cyclic Hexadepsipeptides from the Mangrove Endophytic Fungus Fusarium sp. R5 with Antifungal Activities.

Two novel cyclic hexadepsipeptides, fusarihexin A (1: ) and fusarihexin B (2: ), and two known compounds, cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Val) (3: ) and cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Ile) (4: ), were isolated from the marine mangrove endophytic fungus Fusarium sp. R5. Their chemical structures were elucidated on the basis of spectroscopic data and Marfey's analysis. In an in vitro bioassay, fusarihexin A (1: ) remarkably inhibited three plant pathogenic fungi: Colletotrichum gloeosporioides (Penz.) Sacc., which causes anthracnose in many fruits and vegetables, Colletotrichum musae (Berk. and M. A. Curtis) Arx, which causes crown rot and anthracnose in bananas, and Fusarium oxysporum Schlecht. f. sp. lycopersici (Sacc.) W. C. Snyder et H. N. Hansen, which causes Fusarium wilt and fruit rot in tomatoes. Fusarihexin B (2: ) strongly inhibited C. gloeosporioides and C. musae. The compounds were more potent than carbendazim, which is widely used as an agricultural and horticultural fungicide worldwide.

Mycotoxins and related Fusarium species in preharvest maize ear rot in Poland

This work presents a survey on mycotoxins (seasons 2013 and 2014) and Fusarium species (seasons from 1985 to 2014) in maize ear rot in Poland. Twelve mycotoxins were identified in maize kernel samples exhibiting symptoms of Fusarium ear rot or rotten kernels at the harvest in two locations in Poland during the seasons 2013 and 2014. This is the first complex survey on the co-occurrence of four Fusarium mycotoxin groups in maize kernels: the group of the mycohormone zearalenone; the group of trichothecenes – deoxynivalenol and nivalenol; the group of fumonisins; and the group of cyclic hexadepsipeptides – beauvericin and enniatins; and in addition, moniliformin. Four Fusarium species were identified in preharvest maize ear rot in the 2013 and 2014 harvests namely:<br /> F. graminearum, F. poae, F. subglutinans and F. verticillioides. Since 1985, eleven Fusarium species have been identified in 13 investigation seasons. Apart from those mentioned above, F. avenaceum, F. cerealis, F. culmorum and<br /> F. sporotrichioides were observed with irregular frequencies, and three species, i.e. F. proliferatum, F. tricinctum and F. equiseti, were identified sporadically. A significant increase of F. verticillioides frequency and a decrease of F. subglutinans frequency and changes of mycotoxin profile have been observed in the two decades since 1995.

Erratum for: Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02 % trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2 % to 20 %, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Enniatin-containing solutions for oromucosal use: Quality-by-design ex-vivo transmucosal risk assessment of composition variability

Fusafungine, a mixture of the cyclic hexadepsipeptides enniatins, is currently on the market for the treatment of upper respiratory tract diseases because of its bacteriostatic and anti-inflammatory effects.In this study, a quality-by-design risk assessment was performed with two objectives: (i) investigate whether enniatins are able to permeate the mucosa and reach blood circulation, as the summary of product characteristics indicates this is not the case, and if so, to quantify their transmucosal kinetics and (ii) study the influence of excipient concentration variability on mucosal permeation.First, the concentration of the two main excipients isopropyl myristate and ethanol, known penetration enhancers, in several marketed samples was determined using GC-FID. Then, the transmucosal kinetics of the enniatins were quantitatively evaluated for different dose solutions, using porcine buccal mucosa in an ex-vivo in-vitro Franz diffusion cell set-up, with UHPLC-MS/MS bioanalytics.This study demonstrated that enniatins are capable of permeating the mucosa. However, no risk of a significant different transmucosal permeability with varying excipient concentrations was detected.

Inhibotory effect of cyclic hexadepsipeptides on the protiferation activity of human PC-3 cells and the role of Sal-like gene 4

Objective To investigate the inhibitory effects of cyclic hexadepsipeptides on human prostate cancer cells and the role of Sal-like gene 4 (SALL4).Methods Methyl thiazol tetrazolium (MTT) assay was used to detect proliferation rate of prostate cancer cells.Flow cytometry was used to detect apoptosis rate of prostate cancer cells.Western blotting was used to detect the Caspase-3,B cell lymphoma/leukemia-2 associated X protein (bax),B cell lymphoma/leukemia-2 (bcl-2) protein levels of prostate cancer cells.Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the SALL4 expression of prostate cancer cells.Results The survival rate of prostate cancer cells in cyclic hexadepsipeptides intervention after 24 h and 48 h was decreased [(32.40 ± 1.98) ％ and (38.20 ± 2.13) ％],and apoptosis rate increased [(48.43 ± 3.08) ％ and (50.08 ± 4.14) ％] as compared with the control group (P ＜0.01).The Caspase-3 (0.387 ±0.012) and bax protein (0.312 ±0.028) levels of prostate cancer cells in cyclic hexadepsipeptides intervention was increased after 24 h and 48 h as compared with the control group (P ＜ 0.05).The bcl-2 protein levels (0.312 ± 0.028) were decreased in cyclic hexadepsipeptides intervention group as compared with the C group (P ＜ 0.01),and the SALL4 had no significant change.Conclusion Cyclic hexadepsipeptides exert an inhibitory effect on the protiferation of prostate cancer cells probably by regulating Caspase-3,bax and bcl-2 levels. Key words: Prostate cancer; Cyclic hexadepsipeptides ; Sal-like gene 4 ; Caspase-3

Oleamycins A and B: new antibacterial cyclic hexadepsipeptides isolated from a terrestrial Streptomyces sp.

Oleamycins A and B: new antibacterial cyclic hexadepsipeptides isolated from a terrestrial Streptomyces sp.

Cyclic hexadepsipeptides in wheat field samples andesyn1 gene divergence among enniatin producingFusarium avenaceum strains

Fusarium avenaceum is one of the most important pathogenic species in agricultural and forest environments of moderate climate, particularly in cereals and legume pulse crops. Numerous mycotoxins can be synthesized by the species, with moniliformin and enniatins (ENN) being the prevailing metabolites. The aims of this work were to examine the amounts of ENN and beauvericin present in naturally contaminated field samples of wheat kernels and chaffs collected in Poland in 2005 and 2009 from heads infected withF. avenaceum, and to reveal the divergence of theesyn1 gene amongF. avenaceum strains of different origin. ENN-B and ENN-B1 were the major metabolites identified in wheat field samples. Chaff fractions contained significantly more mycotoxins than grain. Samples originating from 2005 were in general less contaminated with ENN than those from the 2009 season. The highest amount of ENN-B found in grain was 28,520 μg/kg. Beauvericin was only found in trace amounts in all the samples tested.F. avenaceum strains isolated from the analysed wheat samples were identified using species-specific DNA marker and translation elongation factor 1α (tef-1α) sequence analysis. A higher level of sequence polymorphism was revealed for the enniatin synthetase (esyn1) gene than ecorded bytef-1α analysis. Moreover, species known to be typical beauvericin producers, e.g.Fusarium oxysporum andFusarium proliferatum, were clustered into a separate branch on the dendrogram, apart from the strains of ENN-producing species, i.e.F. avenaceum andFusarium scirpi.

Streptogramins – Two are better than one!

Streptogramins are potent drugs against numerous highly resistant pathogens and therefore are used as antibiotics of last-resort human therapy. They consist of a mixture of two different types of chemical substances – the group A streptogramins, which are polyunsaturated macrolactones, and the group B streptogramins, representing cyclic hexadepsipeptides. Streptogramins are unique in their mode of action: each component alone exhibits a moderate bacteriostatic activity by binding to the bacterial 50S ribosomal subunit and thereby blocking translation, whereas the synergic combination of both substances is up to hundred fold more effective than the single compounds, resulting in a bactericidal activity. The streptogramin biosynthetic genes are organized as large antibiotic superclusters. These clusters harbour numerous regulatory genes, which encode different types of regulators that together form a complex hierarchical signalling system, which governs the regulation of streptogramin biosynthesis. Resistance is also regulated by this cascade. However, whereas resistance against streptogramins is quite well understood in diverse pathogenic organisms, only little is known about how the natural producer strains protect themselves against these toxic compounds.Here, we give an overview about the recent advances in streptogramin investigations with a main focus on the best-studied representatives, pristinamycin and virginiamycin. We concentrate on the biosynthesis of these compounds, their regulation and resistance determinants as well as their application in medicine and food industry.

Effects of 14C-labelled precursor feeding on production of beauvericin, enniatins H, I, and MK1688 by Fusarium oxysporum KFCC11363P

The effects of ¹⁴C-labelled precursor feeding on the production of cyclic hexadepsipeptides were investigated by the mycelium of F. oxysporum KFCC11363P producing beauvericin along with enniatins H, I, and MK1688. Most ¹⁴C-phenylalanine and ¹⁴C-valine were incorporated easily into the biosynthetic pathway of ¹⁴C-labelled beauvericin in vivo by the mycelium. However, only a small amount of ¹⁴C-labelled enniatins could be detected by feeding of ¹⁴C-valine. When L-valine was fed as a precursor to the mycelium at large scale, the level of beauvericin increased to maximum followed by enniatins H and I. Feeding of L-valine did not affect the production of enniatin MK1688. These results suggest that L-valine feeding led to the production of D-hydroxyisovaleic acid in the mycelium and specifically enhanced the production of cyclic hexadepsipeptides containing D-hydroxyisovaleic acid, such as beauvericin and enniatins H and I.

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.

Statistical optimization of culture conditions for the production of enniatins H, I, and MK1688 by Fusarium oxysporum KFCC 11363P

The aim of this study was to optimize the culture conditions for the production of biological cyclic hexadepsipeptides (enniatins H, I and MK1688) from Fusarium oxysporum KFCC 11363P. Tests of 10 complete or chemically defined liquid culture media revealed that Fusarium defined medium was the best for the production of enniatins (produced amounts: enniatin H, 185.4 mg/L; enniatin I, 349.1mg/L; enniatin MK1688, 541.1mg/L; and total enniatins, 1075.6 mg/L). On the eighth day after inoculation, the maximal production of enniatins was observed at 25°C in Fusarium defined medium. The optimal carbon and nitrogen sources for producing biological cyclic hexadepsipeptides (enniatins H, I, and MK1688) were sucrose and NaNO(3), respectively, and their optimal concentrations were determined by the principle of response surface methodology. It was confirmed that using the optimized growth medium compositions increased the amounts of enniatins H, I, and MK1688, and total enniatins produced to 695.2, 882.4, 824.8, and 2398.5mg/L, respectively. These findings will assist in formulating microbiological media useful for enniatin research.