biostudies-arrayexpressAndre NantelCandida albicanshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-34255The capacity to sense and transduce temperature signals pervades all aspects of biology, and temperature exerts powerful control over the development and virulence of diverse pathogens. In the leading fungal pathogen of humans, Candida albicans, temperature has a profound impact on morphogenesis, a key virulence trait. Many cues that induce the transition from yeast to filamentous growth are contingent on a minimum temperature of 37ºC, while further elevatation to 39ºC serves as an independent inducing cue. The molecular chaperone Hsp90 is a key regulator of C. albicans temperature-dependent morphogenesis, as induction of filamentous growth requires relief from Hsp90-mediated repression of the morphogenetic program. Compromise of Hsp90 function genetically, pharmacologically, or by elevated temperature induces filamentation in a manner that depends on protein kinase A (PKA) signaling, but is independent of the terminal transcription factor, Efg1. Here, we determine that despite morphological and regulatory differences, inhibition of Hsp90 induces a transcriptional profile similar to that induced by other filamentation cues, and does so in a manner that is independent of Efg1. Further, we identify Hms1 as a transcriptional regulator required for morphogenesis induced by elevated temperature or compromise of Hsp90 function. Hms1 functions downstream of the cyclin Pcl, and the cyclin-dependent kinase Pho85, both of which are required for temperature-dependent filamentation. Upon Hsp90 inhibition, Hms1 binds to DNA elements involved in filamentous growth, including UME6 and RBT5, and regulates their expression, providing a mechanism through which Pho85, Pcl1, and Hms1 govern morphogenesis. Consistent with the importance of morphogenetic flexibility with virulence, deletion of C. albicans HMS1 attenuates virulence in a metazoan model of infection. Thus, we establish a new mechanism through which Hsp90 orchestrates C. albicans morphogenesis, and define novel regulatory circuitry governing a temperature-dependent developmental program, with broad implications for temperature sensing and virulence of microbial pathogens. Two-color experimental design testing the effect of geldanamycin on wild type of delta-efg1 cells. RNA from each replicate came from independent cultures.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was isolated from quadruplicate independent biological samples using the RNeasy mini kit (Qiagen). Briefly, frozen cells were thawed out in RNeasy buffer RLT at a ratio of 3:1 [vol/vol] buffer/pellet. Resuspended cells were divided into 1 ml aliquots in 2 ml screw cap microcentrifuge tubes containing 0.6 ml of 0.5 mm diameter acid-washed glass beads. Samples were homogenized 6 times, for 5 minutes each, in a BeadBeater set at maximum speed.Labeling - 20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).Hybridization - The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. Hybridization was done with an Advalytix SlideBooster. The slides were washed twice with SSC-0.2% SDS, once with 0.1X SSC-0.2% SDS and 3 times with 0.1X SSC. A final wash with isopropanol was performed and slides were air-dried.Sample Processing - Cells were grown overnight in YPD at 30°C, diluted to OD600 of 0.2, and grown overnight again with or without 10 µM geldanamycin, as indicated in above. Cells were again diluted to OD600 of 0.2 in the same conditions and grown to mid-log phase.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsFeature Extraction - Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).Assay Data Transformation - ID_REF = <br>VALUE = log2 PRE_VALUE: log2 Lowess normalized test/reference (GdA treated/untreated, or mutant/wt, or mutant GdA/wild type GdA)<br>PRE_VALUE = Lowess normalized test/reference (GdA treated/untreated, or mutant/wt, or mutant GdA/wild type GdA)Image Adquisition - Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.UnknownTranscriptomicsGenomicsProteomics<h4>Background</h4>Temperature exerts powerful control over development and virulence of diverse pathogens. In the leading human fungal pathogen, Candida albicans, temperature governs morphogenesis, a key virulence trait. Many cues that induce the yeast to filament transition are contingent on a minimum of 37°C, whereas further elevation to 39°C serves as an independent inducer. The molecular chaperone Hsp90 is a key regulator of C. albicans temperature-dependent morphogenesis. Compromise of Hsp90 function genetically, pharmacologically, or by elevated temperature induces filamentation in a manner that depends on protein kinase A signaling but is independent of the terminal transcription factor, Efg1.<h4>Results</h4>Here, we establish that despite morphological and regulatory differences, inhibition of Hsp90 induces a transcriptional profile similar to that induced by other filamentation cues and does so independently of Efg1. Further, we identify Hms1 as a transcriptional regulator required for morphogenesis induced by elevated temperature or Hsp90 compromise. Hms1 functions downstream of the cyclin Pcl1 and the cyclin-dependent kinase Pho85, both of which are required for temperature-dependent filamentation. Upon Hsp90 inhibition, Hms1 binds to DNA elements involved in filamentous growth, including UME6 and RBT5, and regulates their expression, providing a mechanism through which Pho85, Pcl1, and Hms1 govern morphogenesis. Consistent with the importance of morphogenetic flexibility for virulence, deletion of C. albicans HMS1 attenuates virulence in a metazoan model of infection.<h4>Conclusions</h4>Thus, we establish a new mechanism through which Hsp90 orchestrates C. albicans morphogenesis, and define novel regulatory circuitry governing a temperature-dependent developmental program, with broad implications for temperature sensing and virulence of microbial pathogens.transcription profiling by arrayCandida albicansPho85, Pcl1, and Hms1 signaling governs Candida albicans morphogenesis induced by high temperature or Hsp90 compromise.Shapiro RS, Sellam A, Tebbji F, Whiteway M, Nantel A, Cowen LELeah CowenMalcolm WhitewayRebecca ShapiroAndre NantelAdnane SellamfalsePho85, Pcl1, and Hms1 Signaling Governs Candida albicans Morphogenesis Induced by Elevated Temperature or Hsp90 Compromise [mRNA]The capacity to sense and transduce temperature signals pervades all aspects of biology, and temperature exerts powerful control over the development and virulence of diverse pathogens. In the leading fungal pathogen of humans, Candida albicans, temperature has a profound impact on morphogenesis, a key virulence trait. Many cues that induce the transition from yeast to filamentous growth are contingent on a minimum temperature of 37ºC, while further elevatation to 39ºC serves as an independent inducing cue. The molecular chaperone Hsp90 is a key regulator of C. albicans temperature-dependent morphogenesis, as induction of filamentous growth requires relief from Hsp90-mediated repression of the morphogenetic program. Compromise of Hsp90 function genetically, pharmacologically, or by elevated temperature induces filamentation in a manner that depends on protein kinase A (PKA) signaling, but is independent of the terminal transcription factor, Efg1. Here, we determine that despite morphological and regulatory differences, inhibition of Hsp90 induces a transcriptional profile similar to that induced by other filamentation cues, and does so in a manner that is independent of Efg1. Further, we identify Hms1 as a transcriptional regulator required for morphogenesis induced by elevated temperature or compromise of Hsp90 function. Hms1 functions downstream of the cyclin Pcl, and the cyclin-dependent kinase Pho85, both of which are required for temperature-dependent filamentation. Upon Hsp90 inhibition, Hms1 binds to DNA elements involved in filamentous growth, including UME6 and RBT5, and regulates their expression, providing a mechanism through which Pho85, Pcl1, and Hms1 govern morphogenesis. Consistent with the importance of morphogenetic flexibility with virulence, deletion of C. albicans HMS1 attenuates virulence in a metazoan model of infection. Thus, we establish a new mechanism through which Hsp90 orchestrates C. albicans morphogenesis, and define novel regulatory circuitry governing a temperature-dependent developmental program, with broad implications for temperature sensing and virulence of microbial pathogens. Two-color experimental design testing the effect of geldanamycin on wild type of delta-efg1 cells. RNA from each replicate came from independent cultures.2012-03-20T00:00:00Z2023-08-17T23:46:42.875Z2022-02-03T15:42:56.148ZE-GEOD-34255GSE3425522365851EFO_000276810.1016/j.cub.2012.01.062