Transcription profiling by array of C. albicans to investigate antifungal resistance
ABSTRACT: A pre-culture in NGY (overnight 30°C) was used to inoculate RPMI-1640 medium to an initial optical density at 600 nm (OD600) of 0.05. Cells were then grown to mid exponential phase (OD600 = 0.50.6) at the appropriate temperature (25°C or 35°C) before the culture was
split into two aliquots and fluconazole (32 ?g ml1 final concentration) added to one; the other serving as the control. After 2 h the cells were harvested by centrifugation and flash
frozen in liquid nitrogen. Three independent cultures were grown for RNA extraction for each isolate at each condition. RNA was prepared from cell pellets with TRIZOL® reagent (Invitrogen) and a mechanical disruption method.
Project description:Based on studies in S. pombe (Chen et al., 2003), we predicted that conditions which activate C. albicans Hog1 would result in the induction of a common set of genes that are regulated by this SAPK (Smith et al., 2004). Hence in this study we compared the global transcriptional responses of wild-type and hog1 C. albicans cells to environmental stresses that are known to activate the Hog1 SAPK. We compared the homozygous hog1/hog1 null mutant (JC50) with the isogenic HOG1 reintegrant (hog1/hog1/HOG1: JC52) because this controlled for any secondary mutations that might have been introduced during the construction of the null mutant. We have shown that this reintegrant is indistinguishable from its parental wild-type strain RM1000 (HOG1/HOG1) with respect to their stress phenotypes (Smith et al., 2004) and their expression of stress genes (Supplementary Data). Three conditions were chosen for transcript profiling: osmotic stress imposed by 0.3 M NaCl, oxidative stress imposed by 5 mM H2O2, and heavy metal stress imposed by 0.5 mM CdSO4. Each of these treatments stimulates the phosphorylation and nuclear accumulation of this Hog1 SAPK within a 10-min time frame (Smith et al., 2004). Furthermore, significant differences in stress regulated gene expression are observed within this time scale (Enjalbert et al., 2003; Smith et al., 2004). Hence, we analyzed the C. albicans transcriptome after a 10-min exposure to each stress condition. Although some stress genes might be missed by analyzing a single time point, most C. albicans stress genes are induced within 10 min (Enjalbert et al., 2003). At least four independent biological replicates were analyzed for each condition
Project description:We investigated how Candida albicans adapts its transcriptional pattern to a sudden availability of glucose in the extracellular medium, having previously grown on a non-fermentable carbon source.
Project description:C. albicans (strain CAI4-CIp10) was grown according to CRISP SOP. An overnight culture was started from a single colony in YPD-Tris,(100mM Tris.HCl) pH 7.4 and incubated overnight at 30C in shaking incubator. The next day, 500 ul culture was inoculated into 50 ml YPD-Tris, pH 7.4. The following day a fresh culture was inoculated in YPD-Tris to an OD600 of 0.2 and grown to OD600 of 0.8 at 30C in a shaking incubator. At this point the culture was split, diluted back to OD600 of 0.2 in fresh medium and stress agents added at time =0 (XS = 5mM H2O2, OS = 1M NaCl or a combination, OSXS). After 10 min the cultures were harvested by centrifugation and cell pellets flash frozen in liquid nitrogen. Three independent cultures were grown for RNA extraction for each isolate at each condition.<br><br>The control sample was obtained from cells with no stress agents added, harvested at time=0. RNA was extracted and transcript profiling carried out by microarray analysis using custom microarrays (Eurogentec).
Project description:Transcript profiling was performed using a wild-type C. albicans strain (CaI8+CIp10). Cells were cultured in 50 ml SC-pH3.0 to a cell density of 1x10^7 cells per ml and then either treated with control (0 mM acetic acid) or stress inducing (20 mM acetic acid) doses. Cells from the same culture were harvested after 300 min treatment. Three independent biological replicates were obtained for each condition.