Unknown,Transcriptomics,Genomics,Proteomics

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Transcription orofiling of Saccharomyces cerevisiae upon exposure to aaxitoxin

ABSTRACT: Saxitoxin is a potent neurotoxin produced by several species of dinoflagellates and cyanobacteria. The molecular target of saxitoxin in higher eukaryotes is the voltage-gated sodium channel; however, its target in lower eukaryotic organisms remains unknown. The goal of this study was to obtain the transcriptional fingerprint of the model lower eukaryote Saccharomyces cerevisiae upon exposure to saxitoxin to identify potential genes suitable for biomarker development. Microarray analyses identified multiple genes associated with copper and iron homeostasis and sulfur metabolism as significantly differentially expressed upon exposure to saxitoxin; these results were verified with quantitative reverse-transcriptase PCR (qRT-PCR). Additionally, the qRT-PCR assays were used to generate expression profiles in a subset of the differentially regulated genes across multiple exposure times and concentrations, the results of which demonstrated that overall, genes tended to respond in a consistent manner to the toxin. In general, the genes encoding the metallothioneins CUP1 and CRS5 were induced following exposure to saxitoxin, while those encoding the ferric/cupric reductase FRE1 and the copper uptake transporter CTR1 were repressed. The gene encoding the multicopper ferroxidase FET3, part of the high-affinity iron uptake system, was also induced in all treatments, along with the STR3 gene, which codes for the cystathionine beta-lyase found in the methionine biosynthetic pathway. Experiment Overall Design: Cultures of S. cerevisiae S288C were grown to OD600=1.0 in YPD (1% yeast extract, 2% peptone, 2% dextrose). Four hundred microliters from individual 10 mL overnight cultures were transferred to sterile tubes and saxitoxin added to a final concentration of 16 ÂµM. Sample were mixed gently and incubated at 30Â° C for 45 min. Controls consisted of deionized water. Three biological replicates were performed under these conditions for microarray analysis. Cells were harvested via sequential centrifugation and placed immediately in liquid nitrogen.Total RNA was extracted from frozen cell pellets using a protocol that combined the hot acid phenol method with the RNEasy kit. Total RNA was processed by the Affymetrix Core Facility at the University of Tennessee (Knoxville, TN) following the protocols of the Affymetrix GeneChip Expression Analysis Technical Manual. MAS5.0 was used to provide present/absent calls; genes absent in all six samples were then removed from the data set. Intensity values for all transcripts were obtained from the scanned image files of the chips. The intensity values of the six arrays were background-corrected and normalized with the GC-RMA algorithm.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Kathleen Cusick

PROVIDER: E-GEOD-16652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mechanism of action of antimicrobial peptides (AMPs) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMPs is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast Saccharomyces cerevisiae to characterize the antifungal effect of two unrelated AMPs. Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene onthology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW related genes, including those of MAPK signaling pathways, revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as ∆ecm33 or ∆ssd1. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, ARG genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking ARG genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (ARG1) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene IPT1 was extended to the peptides studied here. Reinforcement of CW is a general response common after exposure to distinct AMPs, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMPs. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMPs, was also independently confirmed. We carried out the characterization of the transcriptome of S. cerevisiae after exposure to PAF26 and melittin peptides. The global transcriptome response to peptides was undertaken by treating S. cerevisiae FY1679 cells in the logarithmic growth phase to sub-lethal concentrations (5 µM) of either PAF26 (PAF samples) or melittin (MEL samples) for 3 hours. Control treatment (CTR samples) consisted in cells exposed to buffer in the absence of peptide. Three biological replicates were conducted for each treatment. DNA macroarrays representing 6,020 yeast genes were hybridized with labelled cDNAs from cells.

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Transcription orofiling of Saccharomyces cerevisiae upon exposure to aaxitoxin

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