Food Contaminants

Chapter 1

Natural Products as Tools for Chemogenomic Analysis of Mycotoxin Biosynthesis and Fungal Stress-Response Systems Downloaded by 80.82.77.83 on December 24, 2017 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch001

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Bruce C. Campbell , Jong H. Kim , Jiujiang Yu , Russell J. Molyneux , Noreen Mahoney, Jeffrey D. Palumbo , Kathleen L. Chan , Deepak Bhatnagar, Thomas E. Cleveland , and William C. Nierman 1

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Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710 Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70124 The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850 The George Washington University, Washington, D.C. 20006

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Certain phenolic compounds with antioxidant properties inhibit aflatoxin biosynthesis in the fungus Aspergillus flavus, without affecting growth. Similarly, some of the same phenolics also inhibit biosynthesis of ochratoxin by A. alliaceous. Exposing A. flavus to oxidative stress, such as hydrogen peroxide, enhances aflatoxin biosynthesis. Bioassays with gene-deletion mutants of Saccharomyces cerevisiae, as a model fungus, showed phenolics and reactive oxygen species modulated the antioxidative stress-response system. Caffeic acid was selected as a chemogenomic tool to monitor expression profiling using A. flavus microarrays. These profiles showed that treatment of the fungus with caffeic acid resulted in significant down-regulation of almost all genes in the aflatoxin biosynthetic gene cluster. However, there was little change in expression by laeA and aflR, known regulatory genes of aflatoxin synthesis. Alternatively, a number of peroxiredoxin genes most closely related to alkyl

Siantar et al.; Food Contaminants ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by 80.82.77.83 on December 24, 2017 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch001

hydroperoxide reductases are dramatically up-regulated by the caffeic acid treatment. These enzymes are believed to reduce amounts of lipoperoxides. The lowering of such peroxides may reduce up-stream signaling from oxidative stress-response pathways that trigger aflatoxin biosynthesis. These results show antioxidative stress response genes are pivotal to modulating expression of the aflatoxin biosynthetic gene cluster.

Extremely low regulatory levels (parts per billion, ppb) of aflatoxin contamination significantly affect the economic value of a number of agricultural products, especially with regard to exportability. Certain tree nuts, almonds, pistachios, and walnuts, are particularly affected by these restrictions on levels of aflatoxin contamination. Practically all tree nut production in the United States is in California. The overall value of these tree nuts approaches $4 billion, annually. Moreover, approximately 40-60% of these nuts are exported to countries with strict regulatory thresholds at 1-4 ppb (7). These same restrictions also affect the marketability of other US agricultural products, such as corn, peanuts and cottonseed. As such, methods are needed to reduce or eliminate aflatoxin contamination of agricultural products for not only food safety, but also marketability purposes.

Aflatoxigenesis and Oxidative Stress Natural compounds that are components of agricultural products affected by aflatoxin contamination could play a role in preventing either fungal infection or aflatoxin biosynthesis. For example, a phenolic compound, gallic acid, discovered in the hydrolyzable tannins in walnut seedcoats was found to prevent aflatoxin biosynthesis by the fungus Aspergillusflavus(2). Bioassays involving gene deletion mutants of the yeast, Saccharomyces cerevisiae, as a model fungus, show that gallic acid acts as a potent antioxidant. Thesefindingssuggest there is an association between oxidative stress and aflatoxigenesis in A. flavus (3). Following the discovery of the anti-aflatoxigenic activity of hydrolyzable tannins in the walnut seedcoat, an effort was made to determine if the other tree nuts also possessed anti-aflatoxigenic natural compounds. In fact, a number of highly active compounds were found in all tree nuts (Figure 1). As found in walnuts, hydrolyzable tannins in general are quite inhibitory. Pentagalloyl glucose had the greatest activity, inhibiting aflatoxin biosynthesis >99%, relative to controls. Caffeic acid, a phenolic compound found in pistachio hulls, had the


4 second greatest inhibitory activity. The remaining compounds isolated with antiaflatoxigenic activity were all phenolic compounds, the only exception being quinic acid. The discovery of these compounds having anti-aflatoxigenic activity further suggested that inhibiting oxidative stress in the fungus, in this case with anitoxidants, resulted in down-regulation of genes in the genetic pathways involved with aflatoxigenesis.

control

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pentagaltoyl glucose 0 walnut

caffeic acid

[ ] pistachio

lauryl gállate

H almond

3,4-digalloyl quinic acid (| 1.7% quinic acid chlorogenie acid vanillic acid gallic acid 4-hydroxybenzoic acid protocatechuic acid

mmmmm 30.7%

catee hln mmmmmxi 31.0% ellagic acid '//////////////. 20

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Anatoxin B1 (fig) Figure 1. Comparative anti-aflatoxigenic capacity of tree nut constituents. All assays used 2 mMof test compound in media containing agar and ground pistachio kernels. Spores ofAspergillus flavus NRRL3357 (-200) were spotted in the center of the plates and incubated at 30 °Cfor 5 days. *model compoundfor anacardic acids in pistachio.

Next, these tree nut constituents were tested against A. alliaceus, a fungus that produces a different mycotoxin, ochratoxin A. Like aflatoxin, thefirststeps in production of ochratoxin A also involve a polyketide synthase gene (pks). Hence, it seemed probable i f expression of pks is somehow increased under oxidative stress conditions, perhaps antioxidants might also inhibit ochratoxin A



5 biosynthesis. Interestingly, some of the tree nut constituents were quite antiochratoxigenic, but with some differences from those that were antiaflatoxigenic. For example, vanillic acid had the greatest anti-ochratoxigenic activity at >99%. Caffeic acid was ^94% inhibitory; whereas, gallic acid was only 40% inhibitory, and catechin, which was moderately anti-aflatoxigenic (Figure 1), actually stimulated ochratoxin A biosynthesis. Such results suggest there is some commonality in the association of oxidative stress and biosynthesis of certain oxygenated mycotoxins. But these preliminary results also indicate that chemical and genetic relationships between the antioxidative stress response and mycotoxigenesis may have certain independent and specific characteristics, depending on the mycotoxin and the fungal species. Further support of a relationship between oxidative stress and aflatoxigenesis is prior work showing that oxidative stress by peroxides stimulates aflatoxin biosynthesis in another aflatoxigenic fungus, A. parasiticus (4, 5). When we expose A. flavus to peroxide stress, it also results in almost doubling aflatoxin production over a 9-day period (Figure 2). Although the experiments with A. flavus and A. parasiticus involved artificial induction of oxidative stress, natural oxidative stress to the fungus can result from host-plant production of toxic reactive oxygen species (ROS). Such ROS molecules include superoxide, hydrogen peroxide, hydroxyl radicals or organic peroxides. ROS can cause cell death as a result of lipid peroxidation, protein denaturation, damage to DNA, etc. ROS molecules can be generated as by-products of metabolic respiratory mechanisms in response to infecting microorganisms or abiotic stresses, such as drought (6-8). As aflatoxin biosynthesis is initiated by a polyketide synthase and intermediates in its synthesis are highly oxygenated, aflatoxigenesis itself may be a potential fungal response to sequester toxic ROS and thus is a means of protectionfromoxidative stress and ROS (9). A number of cellular systems react to oxidative toxicity, some specifically to peroxides. Peroxiredoxins, peroxidases of the AhpC/TSA family, detoxify hydroperoxides by donation of electrons from NADPH (reduction) using thioredoxin/thiol-containing substances (70). Two transcription factors, Yaplp and Sknlp, have been identified that regulate the antioxidative stress-responses of yeast cells (77). Yaplp regulates amounts of GSH (reduced glutathione) vs. GSSG (oxidized glutathione) as a means of managing oxidative stress (72, 75). On the other hand, Sknlp regulates cellular thioredoxin/thiol homeostasis (14). Moreover, organic peroxides generated during fungal growth on lipid-containing substances (such as nut kernels) are likely sources of oxidative stress. The antiaflatoxigenic activity of natural antioxidants, such as gallic acid, may be due to the attenuation of signals that trigger up-stream fungal oxidative stress responses. It is known that inhibition of aflatoxin production in A. parasiticus is through activation of an /is/2-like transcription factor regulating antioxidative enzyme production (5).


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Figure 2. Aflatoxin production under oxidative stress. Oxidative stress was induced using 100 /iMteñ-butyl hydroperoxide. Culture conditions as in Figure 1.

Chemogenomics of Antioxidant Inhibition of Aflatoxigenesis In view of findings of ourselves and others that oxidative stress induces aflatoxin biosynthesis and antioxidants inhibit its biosynthesis, we wanted to learn more about the functional genomic basis of these associations. Using yeast deletion mutants we had already identified a number of fungal genes that play a role in oxidative stress responses, including ones showing gallic acid acts as an antioxidant, reducing H Ô -based oxidative stress (9). Using an A. flavus Expressed Sequence Tag (EST) database (75) we were able to identify some orthologs of genes identified in our yeast studies in A. flavus. We hypothesized that some of these orthologs also played a role in the oxidative stress response of A.flavus,and thus might be involved in the regulation of aflatoxin biosynthesis. In order to test our hypothesis we performed a comparison of the gene expression profiles of A. flavus under control conditions with that of fungi treated with one of the antioxidants already discovered that inhibits aflatoxigenesis. The effect of caffeic acid (12 mM) on aflatoxin production was 2

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evaluated over a 6-day period. Aflatoxin production in the treated cultures was reduced >95% compared to non-treated control plates (e.g., day 6: 46 ng vs. 1.7 jig aflatoxin in culture with caffeic acid). However, growth and mass of the fungal mat was practically identical between the treatments, showing that caffeic acid did not inhibit fungal growth. These features, potent anti-aflatoxigenic activity with minimal antifungal activity, made this compound a particularly useful chemogenomic tool for examining global gene expression in relation to aflatoxin biosynthesis and antioxidant treatment. We thus used caffeic acid to perform a comparative microarray hybridization analysis to directly identify genes in the functional genomic response of A. flavus during suppression of aflatoxin biosynthesis.

Gene Expression Profiles of AspergillusflavusTreated with an Antioxidant RNAs were isolated from control and caffeic acid treated fungi (4-day cultures) to compare quantitative changes in mRNAs between the treatments using microarray hybridizations. Changes in quantities of mRNAs were considered to reflect up-regulation (increase) or down-regulation (decrease) in expression of respective genes. Levels of change in gene expression were based on log transformed ratios of treated vs. control hybridizations, as follows: low > -1.0 to < 1.0); medium > -2.0 to < -1.0, or >1.0 to < 2.0; high < -2.0, or > 2.0. The more noteworthy results from this microarray analysis are summarized in Figure 3. A more detailed presentation of the microarray results is to be published elsewhere. 2

Down-regulation of Genes in the Aflatoxin Biosynthetic Cluster Practically all mRNAs of genes in the anatoxin biosynthetic cluster (16) showed significantly lower quantities (log ratios < -2.0) in the caffeic acid treatment. Examples from the microarray analysis included verB, a P450 monooxygenase involved in conversion of versicolorin B to A, and omtB, an Omethyltransferase converting demethylsterigmatocystin to sterigmatocystin (Figure 3). Declines in log ratios of other pathway genes in the microarray analysis ranged from low, -0.04, to high, -3.13, decreases. There was also a slight decline (-0.58) in expression of aflJ, a pathway transcription enhancer. We were unable to detect the pathway regulator gene, aflR, in the microarray analysis. Surprisingly, amounts of mRNA of a regulator gene for secondary metabolism, laeA, upstream from the cluster and reported to regulate aflatoxin biosynthesis (77), actually increased slightly (+0.23 microarray; +0.90 qRTPCR) (Figure 3). This "up-regulation of laeA suggests this gene does not, alone, govern levels of aflatoxin biosynthesis. 2

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8 Amino acid Aromatic Cell Antoxidation Regulator Aflatoxin btasyn. metabol. structure biosyn.

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i—in—F

L-Ivs L-his

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ahpC1

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i r~i r

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/ / / / / / omtB

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H Array-log Ratio 2

0PCR-Log Ratio 2

Figure 3. Summary of more notable results ofgene expression analyses of Aspergillus flavus treated with caffeic acid, compared to controls. Histograms represent level of log transformed ratios (left axis) of differences in amounts of mRNAs (positive or negative) between treated and controlfungi after a 4-day period. Differences are shown based on microarray analyses (dotted bars) and quantitative real-time PCR (qRT-PCR, hatched bars). Classes ofgenes are shown within brackets at the top of the graph and include genes involved in growth and aflatoxin biosynthesis. 2

Other Down-regulated Transcripts Thirty-six other transcripts, in addition to those in the aflatoxin biosynthetic pathway, showed some degree of down-regulation (log < -1.0) in the microarray analysis of the caffeic acid treatment. Most of these genes were involved in lipid metabolism, cell wall structure/integrity, molecular transport/pumping, and redox homeotasis. In addition hypothetical proteins and various genes were involved in a variety of functions, such as mitochondrial metabolism, DNA mismatch repair, etc. 2

Up-regulated Transcripts The mRNAs of 28 genes showed some increases (log ratio > 1.0) resulting from the caffeic acid treatment. These genes were in 3 main categories, including various enzymes in amino acid metabolism, metabolism of aromatic compounds 2


9 (Figure 3) and glutathione-S-transferases, in addition to a number of hypothetical proteins, an FAD-binding protein, a ribosomal protein, and others.


Up-regulation of Peroxiredoxin Genes As stated above, a more thorough presentation of the comparative expression analyses from the caffeic acid treatment of A. flavus will be presented elsewhere. However, to summarize here some of thefindingsnoted include only minor changes in genes involved in sugar utilization. But perhaps the most notable changes of all in our microarray analyses included up-regulation of four peroxiredoxin genes showing 38% to 50% sequence identity and 58% to 69% sequence homology, at the amino acid level, to Ahplp (alkyl hydroperoxide reductase) of S. cerevisiae. Alkyl hydroperoxide reductases are important in detoxifying organic and lipid peroxides. Although expression levels of these peroxiredoxin genes were low in the microarray analysis, qRT-PCR (real-time quantitative reverse transcription-PCR) analysis showed there were dramatic increases in NAFAC53TV (ahpCl), NAFCN83TV (ahpC2) (Figure 3) and two others, NAGAD20TV and NAFDD05TV.

Discussion We observed that aflatoxin production in A.flavusis greatly enhanced when incubated under oxidative stress induced by the organic peroxide, tert-butyl hydroperoxide (f-BuOOH). Also, we found that A. flavus already stressed by exposure to i-BuOOH and then treated with tannic acid, a commercial hydrolysable tannin containing only gallic acid moieties, reduced aflatoxin levels well below those observed without induced oxidative stress (unpublished results). Reverberi et al (5) have shown that filtrates from the mushroom Lentinula edodes inhibit aflatoxin production by A. parasiticus, wherein the mode of action appeared to be delayed activation of aflR, the pathway regulator gene, and nor A, an aflatoxin biosynthetic pathway gene. This filtrate is believed to contain a polysaccharide having lectin-like properties. Interestingly, treatment with this filtrate also triggers up-regulation for the antioxidant enzymes superoxide dismutase> catalase, and glutathione peroxidase, accompanied by activation of a yeast /w/2-like antioxidative transcription factor (5). Based on microarray expression profiling, we were able to elucidate the functional genomic basis for the anti-aflatoxigenic activity of antioxidants. Expression was repressed for almost all genes in the aflatoxin biosynthetic gene cluster in A. flavus treated with caffeic acid. The mode of action of this antiaflatoxigenic activity results from attenuation of the oxidative stress response to organic peroxides. Perhaps most notable in our analysis is induction of putative



10 alkyl hydroperoxide reductases (AHP1) by caffeic acid. These AHP1 orthologs in A. flavus probably counteract upstream signals (i.e., those involved with oxidative stress) that activate the aflatoxin biosynthetic pathway genes. As such, these enzymes play an important role in anti-aflatoxigenesis as well as tolerance to organic peroxides generated by fatty acid/lipid-rich substrates. Metabolism of lipids/fatty acids stimulates aflatoxin production (18, 19). Repression of lipases may be associated with the anti-aflatoxigenic activity resulting from treatment with caffeic acid. Caffeic acid may also inhibit enzymes involved in oxidation of cellular molecules (e.g., lipids, proteins, DNA, etc.), which, in turn, may subdue signaling in pathways involved in oxidative stress responses. For example, methyl gállate inhibits aflatoxin biosynthesis (2) and also inhibits peroxide-induced oxidative stress in yeast (3). This compound was recently found to directly inhibit cyclooxygenase-2 (COX-2), an enzyme involved in lipid hydroperoxide formation (20). It has been known for some time that certain lipid peroxides from the host-plant are capable of modulating aflatoxin production (21). Thus disruption of normal lipoxygenase activity, either in host-plant tissues or in the fungus (e.g., by inclusion of antioxidants in the culture medium), alters aflatoxin biosynthesis. Disruption of lipoperoxide production by the antioxidative activity of caffeic acid is probably the major biological component for its ability to inhibit aflatoxin biosynthesis. The levels of caffeic acid we added to the culture medium resulted in almost complete repression of aflatoxin biosynthesis but did not affect fungal growth. The microarray profiles concurred with this observation in that genes involved in amino acid metabolic pathways and aromatic metabolism were up-regulated by the caffeic acid treatment. The up-regulation of these metabolic enzymes actually suggests that our caffeic acid treatments had a positive effect on fungal growth.

Conclusion Oxidative stress triggers aflatoxin production in A. flavus and other aflatoxigenic species of aspergilli studied to date. Caffeic acid (and other phenolic anitoxidants) is a potent inhibitor of aflatoxigenesis and perhaps production of other mycotoxins (e.g., ochratoxin A). Our functional genomic studies show that the mechanism for this antimycotoxigenic activity appears to involve attenuation of oxidative stress responses. The oxidative stress response pathways appear to provide upstream signals that induce aflatoxin production. Further functional genomic studies should enable elucidation of the genes involved in providing these signals that are linked to up-regulation of aflatoxin production. In summary, merging of natural product chemistry with genomics has provided us with a powerful "chemogenomic" process. Use of such a chemogenomic approach will enable us to probe the functional genomic


11 responses of fungi in a specific and measured fashion. This process will help in developing methods of preventing aflatoxin contamination„either through chemical treatments, breeding of resistant crops or through some other form of genetic manipulation, as target genes involved in repressing aflatoxin production are identified.


Acknowledgements We thank Gary Payne, North Carolina State University, for his integral role in developing genomic tools used in this study. We also thank Yan Yu, TIGR, for technical assistance with microarray hybridizations. This research was conducted under USDA-ARS CRIS Projects 5325-42000-032-00D and 643541420-004-00D.

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16. Yu, J.; Chang, P.-K.; Ehrlich, K. C.; Cary, J. W.; Bhatnagar, D.; Cleveland, T. E.; Payne, G. A.; Linz, J. E.; Woloshuk, C. P.; Bennett, J. W. Appl. Environ. Microbiol. 2004, 70, 1253-1262. 17. Bok, J. W.; Keller, N . P. Eukaryot. Cell 2004, 3, 527-535. 18. Fanelli, C.; Fabbri, A. A. Mycopathologia 1989, 107, 115-120. 19. Fanelli, C.; Fabbri, A. A.; Finotti, E.; Passi, S. J. Gen. Microbiol. 1983,129, 1721-1723. 20. Kim, S. J.; Jin, M ; Lee, E.; Moon, T. C.; Quan, Z.; Yang, J. J.; Son, K. H.; Kim, K. U.; Son, J. K.; Chang, H. W. Arch. Pharm. Res. 2006, 29, 874-878. 21. Burow, G. B.; Nesbitt, T. C.; Dunlap, J.; Keller, N . P. Mol. Plant Microbe Interact. 1997, 10, 380-387.


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