Sisters in structure but different in character, some benzaldehyde and cinnamaldehyde derivatives differentially tune Aspergillus flavus secondary metabolism

Abstract

Great are the expectations for a new generation of antimicrobials, and strenuous are the research efforts towards the exploration of diverse molecular scaffolds-possibly of natural origin - aimed at the synthesis of new compounds against the spread of hazardous fungi. Also high but winding are the paths leading to the definition of biological targets specifically fitting the drug's structural characteristics. The present study is addressed to inspect differential biological behaviours of cinnamaldehyde and benzaldehyde thiosemicarbazone scaffolds, exploiting the secondary metabolism of the mycotoxigenic phytopathogen Aspergillus flavus. Interestingly, owing to modifications on the parent chemical scaffold, some thiosemicarbazones displayed an increased specificity against one or more developmental processes (conidia germination, aflatoxin biosynthesis, sclerotia production) of A. flavus biology. Through the comparative analysis of results, the ligand-based screening strategy here described has allowed us to delineate which modifications are more promising for distinct purposes: from the control of mycotoxins contamination in food and feed commodities, to the environmental management of microbial pathogens, to the investigation of specific structure-activity features for new generation drug discovery.

Figure 1

Schematic representation of the structures of benzaldehyde (Benz), cinnamaldehyde (Cinn), their thiosemicarbazone derivatives BeTS, and CiTS, and their di-methylated variants BeTS-dm, and CiTS-dm.

Figure 2

In vitro anti-oxidant assay (DPPH assay). The scavenging activity of compounds was tested at increasing concentrations (from 5 to 50 μM), and expressed as inhibition percentage with respect to ascorbic acid (0.3 mM) scavenging activity (100%). Values were presented as mean ± S.D. (n = 3).

Figure 3

Biological activity of the tested compounds on A. flavus. The inhibitory activity of molecules on early development, biomass production and aflatoxin accumulation at 50 µM concentration was reported. Results were expressed as percentage with respect to control (0.5% DMSO-treated cultures). Different letters indicate statistically different values, at p value ≤ 0.01.

Figure 4

Effect of the tested compounds on sclerotia biogenesis. (A) The inhibitory activity of molecules on the sclerotia development in A. flavus cultures treated with 50 µM concentration. (B) Results were expressed as percentage with respect to 0.5% DMSO cultures (CNT). Different letters indicate statistically different values, at p value ≤ 0.01.

Figure 5

Asci production in W303 S. cerevisiae diploid strain treated with TSs. The effect of the tested thiosemicarbazones was compared with the effect of mHtcum. Values are expressed as percentage of asci with respect to the number of total yeast cells; statistically significant differences from the control (DMSO) were indicated with different letters (p ≤ 0.01).

Figure 6

Regulation of gene expression in A. flavus treated with di-methylated TSs. Relative transcript levels of genes involved in fungal development (VeA, NsdD, NsdC, aflrmtA and dmtA) and AFs biosynthesis (aflR and omtB) evaluated in the same cultural conditions used for proteomic analysis (CCM medium amended with 50 μM compounds). Results are means of 3 replicates ± S.D. from 3 independent experiments. Asterisks mark values significantly different between treatments and control (0.5% DMSO-treated cultures) at p ≤ 0.05 (t-test).

Figure 7

(A) Aflatoxin inhibition. BeTS-dm and CiTS-dm inhibitory effect on AF accumulation checked after 96 h exposure, before sampling mycelia for 2-DE analysis. (B) Differentially expressed proteins. Venn diagram indicating the overlap of differentially expressed spots between BeTS-dm (yellow circle) and CiTS-dm (blue circle) treatment, compared to the control (DMSO).

Figure 8

Hypothetical targets positioning of BeTS-dm and CiTS-dm along A. flavus secondary metabolism. Being ineffective on sclerotia biogenesis while highly inhibitory on AF accumulation, BeTS is suggested to intervene down-stream the bifurcation of secondary metabolism that divide AFs biosynthetic pathway from sclerotia developmental biogenesis. On the contrary, due to the containment effect of CiTS-dm on sclerotia and AF, its target could be expected enough up-stream the bifurcation to interfere with their production, or immediately after, in correspondence of a metabolic knot shared by (and controlling) both processes in A. flavus. Scheme created with Adobe Illustrator and Adobe Photoshop CS6.0 (Adobe Inc., San Jose, CA, USA).