Aflatoxin Biosynthetic Cluster Research Articles

Molecular analysis of aflR gene in Aspergillus flavus isolated from Iran

Aspergillus flavus produces the most potent carcinogens, aflatoxins, when it contaminates agricultural crops. aflR gene regulates aflatoxin-related genes and it has been identified in four species of A. flavus, A. parasiticus, A. sojae and A. oryzae. Contamination of agricultural commodities with aflatoxin is a grave risk to humans and animals’ health. Aflatoxin related genes are clustered in a 75 kb region of genome in A. flavus. Investigations obviously demonstrated that aflatoxin biosynthesis needs the aflR gene product and an entirely functional aflatoxin biosynthetic cluster. The purpose of the current study was to investigate the presence of the aflR gene in A. flavus. Material and methods. Forty-two A. flavus isolates including 10 references, 25 clinical and 7 environmental isolates were analyzed in this study. The isolates were identified by morphology. To characterize morphologically, the conidial arrangement, philiades, vesicles and conidiophores were observed microscopically. Using PCR, the aflR gene was amplified with primers aflR1 and aflR2. PCR were carried out to amplify an 800 bp DNA fragment of aflR gene. Some amplicons were sequenced. The sequences were searched in NCBI database and analyzed with MEGA5 software. Results and discussion. Out of 42 A. flavus isolates, an 800 bp band was amplified for 35 isolates. No band was observed for seven isolates including 4 clinical and 3 environmental isolates. Data analysis demonstrated that 100% of reference strains and 84% of clinical strains produced the expected fragment while it was only 57.14% for environmental isolates. The sequences had 100% identity with A. flavus aflR gene which was deposited in the NCBI database. Conclusion. In conclusion, molecular analysis of the aflR gene showed that this gene was not amplified from some strains of A.flavus; therefore, perhaps it lacks the gene or it is greatly abnormal. Additional researches are needed to verify whether the strains with lack of aflR gene have a loss of function in production of aflatoxin or other mechanisms of regulation exist

Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus.

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC50 value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC50 value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity.

A cytosine methyltransferase ortholog dmtA is involved in the sensitivity of Aspergillus flavus to environmental stresses

DNA methylation is an important epigenetic modification that depends on DNA methyltransferases (DMT). However, the filamentous fungus Aspergillus flavus has no detectable methylation, and role of a DMT homologue, DmtA, is undefined. Here we describe the role of the dmtA gene responding to changes in the environment by comparing knockout, point mutation, over-expression and wild type strains. Deletion of dmtA differentially affected conidia development in a media-dependent fashion, which suggests that dmtA plays an important role in conidiation. Furthermore, ΔdmtA strains lost the capacity to form the resistant structure, sclerotia, and alleviated sensitivity to several stress conditions, such as high osmotic pressure, hypoxia, low water activity and a high calcium concentration. We also noticed that deletion of dmtA and mutation C377S in DmtA negatively affected aflatoxin production and down regulated the expression of some early (fas-1, pksA, nor-1), middle and late (nor-A, ver-1, avnA, omtB) genes in the aflatoxin biosynthetic cluster. Finally, we found that all tested strains showed a similar phenotype when treated with 5-azacytidine. Our results indicate that the dmtA gene is important in regulation of aflatoxin biosynthesis and for A. flavus to adapt to stressful environments and for survival, although it may hold no apparent function in DNA methylation.

Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium

Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus. Temperature and water activity are the two key determinants in pre and post-harvest environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental to know which combinations can control or be conducive to aflatoxin contamination. Little information is available about the influence of these parameters on aflatoxin production on almonds. The objective of this study was to determine the influence of different combinations of temperature (20°C, 28°C, and 37°C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth, aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28°C and 0.96 aw; no fungal growth and AFB1 production were observed at 20°C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37°C AFB1 production was 70–90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28°C) and minimum (20°C and 37°C) AFB1 production. Conversely the two structural genes (aflD and aflO) were highly expressed only at maximum AFB1 production (28°C and 0.96–0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which is strictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO), but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are most likely subordinated to other regulatory processes acting at post-translational level.The results of this study are useful to select conditions that could be used in the almond processing chain to suppress aflatoxin production in this important product.

Analysis of genes early expressed during Aspergillus flavus colonisation of hazelnut

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ. The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36 h in YES medium, and 72 h in hazelnut agar medium. Significantly, the amount of aflatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease, potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC. Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular, ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts.

Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis

Caffeic acid (3,4-dihydroxycinnamic acid, 12 mM) added to a fat-based growth medium reduces > 95% of aflatoxin production by Aspergillus flavus NRRL 3357, without affecting fungal growth. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated, ranging from a log 2 ratio of caffeic acid treated and untreated of − 1.12 (medium) to − 3.13 (high). The only exceptions were genes norB and the aflatoxin pathway regulator-gene, aflJ, which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA, also showed little change in expression levels between the fungal cohorts. Alternatively, expression of genes in metabolic pathways (i.e., amino acid biosynthesis, metabolism of aromatic compounds, etc.) increased (log 2 ratio > 1.5). The most notable up-regulation of A. flavus expression occurred in four genes that are orthologs of the Saccharomyces cerevisiae AHP1 family of genes. These genes encode alkyl hydroperoxide reductases that detoxify organic peroxides. These increases ranged from a log 2 ratio of 1.08 to 2.65 (moderate to high), according to real-time quantitative reverse transcription-PCR (qRT-PCR) assays. Based on responses of S. cerevisiae gene deletion mutants involved in oxidative stress response, caffeic, chlorogenic, gallic and ascorbic acids were potent antioxidants under oxidative stress induced by organic peroxides, tert-butyl and cumene hydroperoxides. Differential hypersensitivity to these peroxides and hydrogen peroxide occurred among different mutants in addition to their ability to recover with different antioxidants. These findings suggest antioxidants may trigger induction of genes encoding alkyl hydroperoxide reductases in A. flavus. The possibilities that induction of these genes protects the fungus from oxidizing agents (e.g., lipoperoxides, reactive oxygen species, etc.) produced during host-plant infection and this detoxification attenuates upstream signals triggering aflatoxigenesis are discussed.

Evolution of the aflatoxin gene cluster

WhyAspergillus species produce aflatoxin remains an unsolved question. In this report we suggest that evolution of the aflatoxin biosynthesis gene cluster has been a multistep process. More than 300 million years ago a primordial cluster of genes allowed production of anthraquinones that may have served as insect attractants to facilitate spore dispersal. Later adaptive evolutionary steps introduced genes into the cluster that encoded enzymes associated with fungal virulence. These genes may have allowed the otherwise saprophytic fungi to be better able to colonize living plants. Later, genes for production of aflatoxins B1 and G1 were added to the basal cluster. Loss of the ability to produce aflatoxin G1 occurred with the divergence ofA. flavus, a species that, perhaps, was more successful than its ancestors at colonizing plants. This logical progression in evolutionary development of the aflatoxin biosynthetic cluster fits the phylogenetic data as well as known chemical reactivity of the initially formed anthraquinone polyketide metabolites.

The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster

Aflatoxin contamination of food and feed is a major concern due to the carcinogenic properties of this mycotoxin. Previous studies using classical approaches have identified a cluster of genes responsible for aflatoxin production under the control of the pathway-specific transcriptional regulator aflR, but it is unknown whether aflR controls expression of other genes within the genome. Transcription profiling comparing wild type and DeltaaflR strains of Aspergillus parasiticus grown under conditions conducive for aflatoxin production identified only 23 upregulated genes in the wild type. These included 20 genes in the aflatoxin biosynthetic cluster, and three additional genes outside the aflatoxin biosynthetic cluster (nadA, hlyC, and niiA), all with AflR binding sites. This report is the first to demonstrate genes outside the biosynthetic cluster as being associated with aflR expression.