Aspergillus section Flavi produce the aflatoxins, secondary metabolites toxic to humans and animals. Why do these fungi produce aflatoxins? They do not have a clear role in pathogenicity or in niche competition. Aspergillus employs a considerable amount of energy to synthesize them: more than 20 enzymatic catalyzes are needed. Within the A. flavus species, all opportunistic pathogens of maize, more than half of the natural population are atoxigenic, indicating that aflatoxins are not so obviously linked to an enhancement of population fitness. The perspective changes in A. parasiticus, pathogen to peanuts, where more than 90% of the natural population produce the four aflatoxins. In this chapter, we aim to discuss our recent hypothesis that aflatoxins act as antioxidants providing more time to Aspergillus to “escape” an exploited substrate, that in the meanwhile is “fully charged” with reactive oxygen species and oxylipins.
- resilience to stress
- host adaptation
The species belonging to the section Flavi of the genus
2. The role of oxidative stress in modulating aflatoxin synthesis
Oxidative stress is a condition which organisms must cope with since the process used for producing energy (namely ATP) involves a very oxidizing molecule: the oxygen . Thus, aerobic organisms have evolved means for facing this stress by building up a complex antioxidant system composed of structures, proteins (enzymes) and small metabolites. The ability to control this system enables organisms to face oxidative stress and, indeed, using it to “boost” some pathways (e.g., the defense in the plants) . Aflatoxins are among these: they are synthesized in response to oxidative stress conditions  and, as we aimed to clarify within this chapter, can act as antioxidants to enhance the survival ability of these fungi.
2.1 Reactive oxygen species (ROS)
Free radicals are, by definition, very reactive chemical species, due to their presence of one or more unpaired electrons in valence orbitals. This condition makes them highly reactive molecules, energized and unstable; free radicals will try to to give up or, as more commonly happens, to acquire an electron at the expense of another to obtain a stable configuration.
In living systems, spontaneously forming radicals are numerous, and those of greater biological interest, the so-called ROS, are those molecules in which the unpaired electron is found on O2, such as, for example, superoxide (·O2-), hydroxyl (.OH), hydroperoxyl (·OOH), peroxyl (·OOR) and alkoxy (.RO) radicals. Oxygen is found in nature in the form of diatomic molecules that have two unpaired electrons of parallel spins arranged on two different orbitals (triplet), and therefore possessing characteristics paramagnetic. The fact of having uncoupled electrons makes O2 particularly prone to forming covalent bonds but, in the case of incomplete reduction, ROS may be generated. These react quickly with other compounds to acquire the electrons necessary for their chemical stability, losing, in turn, their electrons and becoming radicals themselves, thus triggering a chain reaction. Once that process starts, it is determined in the cell a cascade of reactions that often begins with the peroxidation of lipids membrane (oxidation of the hydrocarbon chain), resulting in its destabilization, and which proceeds with the oxidation of other cellular components (such as proteins and DNA), to the point of causing the deconstruction of the entire cell.
The reactions in which the radical molecules can take part are many and vary significantly, for example, depending on: (i) the compartment or organelle cell in which they originate, (ii) of the antioxidant systems present, (iii) of the molecules that they attack, (iv) the water and nutritional conditions of the cell. Also, non-radical molecules, such as hydrogen peroxide (H2O2), can trigger responses that lead to the formation of ROS: the Haber-Weiss reaction, for example, produces hydroxyl radicals starting from H2O2 and O2-. The cells of photosynthetic organisms are more subject to oxidative damage since they have concentrations of very high O2 since, not only do they use it during breathing, but they also generate it with photosynthesis. In fact, they have membrane thylakoids composed mainly of polyunsaturated lipids (molecules subject to reactions of peroxidation) and, by means of photosynthetic pigments, absorb light energy, the excess of which favors the production of ROS. In its ground state, O2 is relatively not very dangerous because, although it can give rise to excited states reactive and free radicals (during photosynthesis, for example), its utilization proceeds expeditiously by means of a route in stages, in which a reduction to H2O involving four electrons and during which intermediates can be generated partially reduced reactive species. In fungi as well as in other organisms, ROS can be produced in a tightly regulated way by the NADPH oxidase complex (NOX in fungi; ). This complex controls, upon stimuli, the formation of anion superoxide and controls several processes in hyphal growth and development [11, 12] and in mycotoxin synthesis too .
2.2 Antioxidant responses
If, as just described, the formation of free radicals can cause serious damage at the cellular level, which can sometimes lead the cell to death, it is equally true that the aerobic cells have evolved and developed efficient ROS control and detoxification systems. The latter are known as antioxidant systems and can be enzymatic and non-enzymatic in nature. The systems non-enzymatic include molecules such as: α-tocopherol, β-carotene, compounds phenolics, ascorbate, glutathione; the enzymatic ones involve: superoxide dismutases (SOD; EC 188.8.131.52), which catalyzes the dismutation of O2.- in H2O2, together to others which eliminate the H2O2 such as catalases (CAT; EC 184.108.40.206), peroxidases, glutathione peroxidases (GP; EC 220.127.116.11), (which uses glutathione as an electron donor - GSH, reduced form, and GSSG, oxidized form) and ascorbate peroxidases (APX; EC 18.104.22.168; ASA, reduced form of ascorbic acid, and DHA, oxidized form). All the enzymes described are found in multiple forms (isoforms) that can be classified, for example, based on their metallic cofactor. The latter can also be found in different cellular compartments (such as cytosol and apoplast) or organelles [mitochondrion, chloroplast (in plants), peroxisome and vacuole]. Some of them catalyze the same reaction and can use different substrates as electron donors. In fungi, these antioxidant capacities are tightly controlled by transcriptional regulators. Main transcription factors that in fungi “react” to ROS are msn2–4 , skn7  and Yap-1 . Notably, Yap-1 orthologue ApyapA can control aflatoxin biosynthesis .
2.3 Oxidative stress, antioxidant system and aflatoxin synthesis
Oxidants are continuously produced within and outside fungal cells. In some way they can fuel cells to switch metabolic pathways  or differentiation patterns . Inter alia, in A. flavus and A. parasiticus ROS boost aflatoxin formation [19, 20, 21]. In the past we showed that several oxidants amended to culture as well as increase of cell ROS were able to trigger aflatoxin synthesis . Intriguingly, even external oxidants augment the titer of cell oxidants. How can these oxidants turn into “aflatoxins”? which is the “mediator” of the opening of the complex aflatoxin pathway? Our group and John Linz group demonstrated that ApYapA can orchestrate their synthesis . Notably, ApYapA, similarly to its orthologue Yap-1 of
3. The role of oxylipins in the
Aspergillussect. Flavi lifestyle
3.1 Discovery of oxylipins
Understanding the evolution of fungal pathogenesis requires the treatment of some lipid molecules that mediate the fungus-host interaction.
First evidence on the existence of oxylipins dates to 1987, when Champe et al., demonstrate the role of precocious sexual inducer (psi), later called oxylipins, in Aspergillus nidulans. Psi factors inhibit asexual sporulation and stimulate premature sexual sporulation, acting as hormone-like molecule .
Oxylipins derive from free fatty acids or from fatty acids present into membrane phospholipids. Fatty acids included in membranes, during the plant-pathogen interaction, are released by lipase action. Lipases are considered as virulence factors in plant pathogenic fungi .
Oxylipin oxidation may happen by two routes: the radical and the enzymatic. In fact, during the first steps of infection the production of radical species favors the accumulation of Reactive Oxygen Species (ROS). Superoxide anion (·O2-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH) can spontaneously oxidize the free fatty acids. The second oxylipins synthesis route, i.e., enzymatic, in fungi involves the action of dioxygenases (DOX), lipoxygenases (LOX) and cyclooxygenases (COX) that convert free fatty acids in oxylipins .
The crosstalk established during a plant-pathogenic fungus interaction, therefore, involves a lipases-LOX concerted activity, that carries towards the oxylipin biogenesis .
3.2 Oxylipins in host-pathogen interaction
Oxylipins act as modulator of many signal transduction pathways, both in plant and fungi because the chemical structure as well as the main synthesis routes of oxylipins are common between the two kingdoms. For that reason, several authors defined the oxylipins as the common language between hosts and pathogens . But why do hosts and pathogens produce oxylipins?
Plant can release oxylipins as 9S-HPODE (9S-hydroperoxyoctadecadienoic acid) and 13S-HPODE (13S-hydroperoxyoctadecadienoic acid) able to influence the development in the Aspergilli, in addition, but at the same time they act in the regulation of plant defense and development.
The analysis of the phenotypes derived from the mutant of ppo-genes shown that in
As previously introduced, the oxylipins produced by the plant may influence the fungal lifestyle and being chemically similar to the fungal oxylipins they can substitute them. That was demonstrated in one study, where the maize lipoxygenase Zm-LOX3 cloned in
The development of the fungus depends on cell densities when the cell density is high
Although the role of oxylipins in development and host-pathogen communication is recognized, little is known about their perception. Mammal oxylipins are sensing by G protein-coupled receptors (GPCRs), but in fungi GPCRs are not actually identified also if in
GPCR-mediated signaling seems to be linked to pathogenesis, therefore it is hypothesized that they could be potential targets for disease control .
4. Evolving a strategy for enhancing the resilience to stress: the role of aflatoxins
Aflatoxins have been linked to oxidative stress since the 1980s , their synthesis being convincingly linked to increase in the presence of oxidants,
A recent research by Finotti et al.  aims at elucidating this aspect. Finotti explored the intrinsic potential (Figure 3) of the four main AFs congeners (B1, B2, G1, G2) as scavengers of reactive oxygen species (ROS). In this work, 2,2′-Azobis, 2-amidinopropane (APAB) was used to generate oxidants
Fungal species belonging to the
Several young and “less” young scientists have a minor but important role in this play call “why Aspergillus produces aflatoxins”: I reserve a special thanks to Corrado Fanelli, Anna Adele Fabbri, Enrico Finotti, Alessandra Ricelli, Marzia Scarpari, Marta Punelli and Valeria Scala
Conflict of interest
“The authors declare no conflict of interest.”