2011 Annual Report
1a.Objectives (from AD-416)
To determine the molecular basis for the phenomenon of non-production of aflatoxin in certain members of the Aspergillus (A.)flavus group of the fungi with a view to understanding the global regulation of toxin synthesis and the convergent evolution of aflatoxin biosynthesis.
1b.Approach (from AD-416)
The present proposal is centered upon previously isolated Apergillus (A.) parasiticus sec- (for secondary metabolism minus) strains, that display altered morphology and sporulation. Recent work from our laboratory has shown that a mutation in aflR coding or promoter region is not responsible for the sec-phenotype. Yet, the aflR expression is lowered in the sec-strains. There is no expression of the aflatoxin pathway genes, and aflR overexpression does not reverse the sec- phenotype. Preliminary results have also revealed clear differences between the sec- and their parental sec+ (for secondary metabolism plus) total protein-profiles. Proteomics and microarray technology will be used to determine the differences between sec+ and sec- strains at the molecular level. These differences will provide insights into the global regulatory mechanisms governing aflatoxin synthesis.
Aflatoxins are carcinogenic secondary metabolites produced by the saprophytic fungi (ones that live on decaying or organic debris), Aspergillus (A.) flavus and A. parasiticus. Due to the health impacts of aflatoxins, strict food regulations are enforced to minimize exposure. However, the regulations also reduce the profitability of affected crops. In A. parasiticus (sec+) when normal development was thwarted, by forced repeated mycelial (fungal cell structures) transfer, the resulting isolate (sec-) permanently lost some of its normal developmental functions, including the ability to produce aflatoxins. It was determined that this loss of ability to produce aflatoxins was not due to any mutation in genes that govern the organisms ability to make the toxins. From other studies, we know that to produce aflatoxins, a set of 29 genes are turned on almost simultaneously to produce the necessary proteins (enzymes) required to make the toxin. The defects, if any, created by mycelial transfer in these atoxigenic isolates (sec-) remained to be determined. These (sec-) variants still produced transcripts (indication of gene expression) of aflatoxin biosynthesis pathway regulatory genes, aflR and aflJ. Which meant that the effect of mycelia treatment was at a global regulatory level ( i.e. regulation of several parameters). In efforts to understand regulation of aflatoxin synthesis, Expressed Sequence Tag (EST)-based microarrays (slides containing deoxyribonucleic acid sequences that correspond to all the active genes in the fungus) were used to identify genes differentially expressed in three strains of A. parasiticus; i.e., toxigenic wild-type strain (sec+), an atoxigenic mutant strain generated by maceration (sec-), and an atoxigenic strain generated by a treatment with the chemical 5-Azacytidine treatment (sec-5Az). Analysis under conditions that support aflatoxin formation by the fungus revealed 194 genes whose expression were statistically significant between the 3 strains. The regulatory gene (aflJ) was the only known aflatoxin synthesis related gene detected as significant in each strain for these conditions. Although we have found that there are two genes (aflJ and aflR) that regulate the genes needed for toxin synthesis, another protein (LaeA) has recently been identified as a key regulator of many secondary metabolites (compounds not required for growth, such as fungal toxins, mycotoxins). In this project, we have found that the protein LAEA binds to the two earlier identified regulatory proteins, AFLR and AFLJ, to affect the “turning on” of the toxin production. We now have a better understanding of the complex machinery that is needed by the fungus to allow it to start making the toxin. Through these studies, we hope to identify factors in the fungus that could be targeted for controlling aflatoxin formation, and consequently contamination of crops. Progress of cooperators was monitored through emails, telephone calls, visits to the labs, and interactions at scientific meetings.