Corrado Fanelli

Natural functions of mycotoxins and control of their biosynthesis in fungi

Mycotoxins are harmful secondary metabolites produced by a range of widespread fungi belonging in the main to Fusarium, Aspergillus and Penicillium genera. But why should fungi produce toxins? And how is the biosynthesis of these toxins regulated? Several separate factors are now known to be capable of modulating mycotoxin synthesis; however, in this study, focussing just on mycotoxins whose regulatory mechanisms have already been established, we introduce a further factor based on a novel consideration. Various different mycotoxin biosynthetic pathways appear to share a common factor in that they are all susceptible to the influence of reactive oxygen species. In fact, when a fungus receives an external stimulus, it reacts by activating, through a well-defined signal cascade, a profound change in its lifestyle. This change usually leads to the activation of global gene regulators and, in particular, of transcription factors which modulate mycotoxin gene cluster expression. Some mycotoxins have a clear-cut role both in generating a pathogenetic process, i.e. fumonisins and some trichothecenes, and in competing with other organisms, i.e. patulin. In other cases, such as aflatoxins, more than one role can be hypothesised. In this review, we suggest an “oxidative stress theory of mycotoxin biosynthesis” to explain the role and the regulation of some of the above mentioned toxins.

Early detection of toxigenic fungi on maize by hyperspectral imaging analysis

Fungi can grow on many food commodities. Some fungal species, such as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Fusarium spp., can produce, under suitable conditions, mycotoxins, secondary metabolites which are toxic for humans and animals. Toxigenic fungi are a real issue, especially for the cereal industry. The aim of this work is to carry out a non destructive, hyperspectral imaging-based method to detect toxigenic fungi on maize kernels, and to discriminate between healthy and diseased kernels. A desktop spectral scanner equipped with an imaging based spectrometer ImSpector- Specim V10, working in the visible-near infrared spectral range (400-1000 nm) was used. The results show that the hyperspectral imaging is able to rapidly discriminate commercial maize kernels infected with toxigenic fungi from uninfected controls when traditional methods are not yet effective: i.e. from 48 h after inoculation with A. niger or A. flavus.

Modulation of Antioxidant Defense in Aspergillus parasiticus Is Involved in Aflatoxin Biosynthesis: a Role for the ApyapA Gene

ABSTRACT Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the β-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus. To better understand the role of ROS formation in toxin production, we generated a mutant (ΔApyapA) having the ApyapA gene deleted, given that ApyapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the ΔApyapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of ApyapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.

Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production

Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and β-glucan content. Mushroom β-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with β-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, β-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.

Lipoperoxidation and Aflatoxin Biosynthesis by Aspergillus parasiticus and A. flavus

Summary: The amount of aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus grown on various aged and non-aged seeds, kept at suitable conditions of temperature and moisture, is particularly related to the peroxide numbers of the seed oils. The addition of synthetic hydroperoxides to the cultures greatly increased aflatoxin production.

Study on the role of patulin on pathogenicity and virulence of Penicillium expansum

Although the antibacterial activity and toxicity to humans and animals of the mycotoxin patulin are well known, its role in the postharvest decay of apples by Penicillium expansum has never been investigated. In the present study the gene disruption technique was used to alter the sequence of 6-methyl-salicylic acid synthase, an enzyme involved in the first committed step of patulin biosynthesis. Thirty-nine mutants were obtained, however only two of them (M5 and M21) passed the sub-cultural and molecular confirmation tests. They proved to produce 33-41% less patulin than their wild-type (WT) strain, although no difference in the growth and morphology of the colony was observed. Moreover, the mutants showed a significantly reduced pathogenicity and virulence on artificially inoculated apples. In particular, a 33-34% and 47-54% reduction of disease incidence and severity were recorded for M5 and M21, respectively. As confirmation, when the biomass of the mutants was quantified in vivo by Real-time PCR, a significant difference was recorded as compared to the WT and even between mutants. Moreover, when patulin production potential of mutants was restored by exogenous application of the mycotoxin, their ability to cause the disease was not significantly different from that of WT. Finally, mutants showed an increased susceptibility to the application of the antioxidant quercetin, their pathogenicity and virulence being significantly reduced at only 1/100 of the concentration needed for the WT. Based on these findings, patulin seems to have a role in the development of blue mold decay on apples.

Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the “oxidative message” to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the ApyapA gene, a homologue of the yeast Yap-1, disables the fungus’s capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer AoloxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Aoyap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus.

Oxidant - antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis.

A close correlation between lipoperoxide formation in cells ofAspergillus parasiticus and aflatoxin biosynthesis has been established in rich and poor media in which oxidative stress was induced by addition of cumene hydroperoxide, a lipoperoxidation inducer. The presence of hydroperoxides of linoleic acid inA. parasiticus mycelia was analysed by liquid chromatography-mass spectrometry (LC-MS). This relation appears to be driven by activation of certain oxidative stress related transcription factors, such asyap1-like,skn7-like andhsf2-like. Activation of these factors then leads to the promotion of transcription of genes encoding antioxidant-related enzymes, such as superoxide dismutase, catalase and glutathione peroxidase.The incomplete seavenging of intracellular oxidation inA. parasiticus cells can lead to aflatoxin biosynthesis. The relationship between oxidative stress and aflatoxin biosynthesis is indicated by the high correlation among increased activity of lipoperoxidation and the antioxidant defence system with formation of aflatoxins.With regard to the relationship of oxidative stress and aflatoxin biosynthesis, the mechanism of action of butylated hydroxyl anisole (BHA), an antioxidant compound, in the control of aflatoxin biosynthesis was also investigated. Results indicate this compound can act,per se, by inhibiting lipoperoxidation and by inducing antioxidative defence responses of the fungal cell.

Role of lipoperoxidation in aflatoxin production

SummaryLipoperoxidation appears to play a role in inducing aflatoxin biosynthesis. In vitro, synthetic lipoperoxides greatly stimulate aflatoxin production when added to cultures of toxigenic strains of Aspergillus parasiticus or A. flavus. In vivo, the amount of toxin formed in sunflower seeds of different ages inoculated with A. parasiticus is directly related to the peroxide number of their oil content: the higher the peroxide number, the higher the aflatoxin production. In cultures of A. parasiticus carbon tetrachloride (CCl4) greatly stimulates aflatoxin biosynthesis. This effect might be due to the peroxidation of lipids of the endoplasmic reticulum of Aspergillus by the highly reactive CCl.3radicals formed by interaction with the NADPH-cytochrome P-450 system.

How Peroxisomes Affect Aflatoxin Biosynthesis in Aspergillus Flavus

In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.