Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

Abstract

Background: Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis.

Methods: We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death.

Results: We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis.

Conclusions: In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.

Figure 1

Real-time RT-PCR for selected genes from the microarray hybridization analysis. S. cerevisiae was grown for 9 hours in liquid YPD at 30°C and the cells (~ 2 x 10⁷ cells ml^-1) were transferred to fresh liquid YPD and exposed to propolis 0.125% for 5 or 10 minutes. The relative quantitation of RLF2 (A), PDR15 (B), TIM10 (C), SNQ2 (D), VMA7 (E), VMA21 (F) was performed using TAF10 as normalizer. Gene expression was determined by a standard curve (i.e., C_T –values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments using completely independent biological replicates. The values above the bars are mean of the log2-ratio obtained in the microarray hybridization experiments.

Figure 2

Design of the union network generated from repressed- and induced-associated gene PPPI networks. Green nodes indicate proteins of genes that were induced in the array, while red nodes are associated to proteins of genes that were repressed in the array.