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. 2017 Jul 12;9(7):219.
doi: 10.3390/toxins9070219.

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus

Kurin Iimura  1 , Tomohiro Furukawa  2 , Toshiyoshi Yamamoto  3 , Lumi Negishi  4 , Michio Suzuki  5 , Shohei Sakuda  6
Affiliations

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus

Kurin Iimura et al. Toxins (Basel). .

Abstract

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors.

Keywords: Aspergillus flavus; aflatoxin; diketopiperazine; glutathione S-transferase; inhibitor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of diketopiperazines with aflatoxin-production inhibitory activity.
Figure 2
Figure 2
Binding experiments with cyclo(l-Ala-l-Pro)-immobilized Sepharose beads and preparation of His-AfGST. (a) Cyclo(l-Ala-l-Pro)-binding protein was purified from the cell lysate of A. flavus using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Compared with the competitive inhibition condition (+), a strong protein band (arrow) was observed under the non-competitive inhibition condition (−). (b) Recombinant His-AfGST was expressed in E. coli and purified by a Ni Sepharose 6 Fast Flow affinity resin column. (c) His-AfGST was incubated with cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. His-AfGST bound to the beads was detected with anti-His antibody. Band intensity observed under the non-competitive inhibition condition (−) was greater than that observed under the competitive inhibition condition (+).
Figure 3
Figure 3
Time courses of aflatoxin B1 production and mRNA level of the gene encoding AfGST. Strain IMF 47798 was cultured in a PDB liquid medium for 4 days, and the time courses of aflatoxin B1 production (a) and the mRNA level of the gene encoding AfGST (b) were measured. Error bars show the standard deviations. n = 4 or 5, ** p < 0.01 between timepoints.
Figure 4
Figure 4
Effects of ethacrynic acid on aflatoxin production and fungal growth. The bar graph and polygonal line graph show aflatoxin B1 production and dry mycelial weight, respectively. Error bars show standard deviations. n = 3 or 4, ** p < 0.01, * p < 0.05 vs. control.
Figure 5
Figure 5
Effects of cyclo(l-Ala-l-Pro) (2 mM) and ethacrynic acid (100 μM) on GST activities of His-AfGST (a) and Schistoma japonicum GST (rShGST) (b). Error bars show standard deviations. n = 3 or 4, ** p < 0.01 vs. control.
Figure 6
Figure 6
Effects of cyclo(l-Ala-l-Pro) (a) and ethacrynic acid (b) on the mRNA level of aflR. Error bars show standard deviations. n = 3 (b) or 4 (a), * p < 0.05, ** p < 0.01, vs. control.
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