Name: Control of carbon assimilation by Candida albicans
Keywords: Not available

Dataset Information

Control of carbon assimilation by Candida albicans

ABSTRACT: Not available

INSTRUMENT(S): instrument model, Voyager-DE STR

ORGANISM(S): Candida Albicans (yeast)

TISSUE(S): Logarithmic Phase Culture, Cell Culture

SUBMITTER: David Stead

PROVIDER: PRD000035 | Pride | 2013-07-08

REPOSITORIES: Pride

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			Action	DRS
	PRIDE_Exp_Complete_Ac_3186.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_3187.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_3188.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_3189.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_3190.xml.gz	Xml

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Publications

The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans.

Sandai Doblin D Yin Zhikang Z Selway Laura L Stead David D Walker Janet J Leach Michelle D MD Bohovych Iryna I Ene Iuliana V IV Kastora Stavroula S Budge Susan S Munro Carol A CA Odds Frank C FC Gow Neil A R NA Brown Alistair J P AJ

mBio 20121211 6

Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose trigge ...[more]

PMID: 23232717

Similar Datasets

Project description:Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

Project description:2.3 Cell wall and secretome preparation. C. albicans RM1000 cells grown to mid-exponential phase were harvested by centrifugation. The resulting supernatant was sterile-filtered and then concentrated on 10-kDcut-off filters (Amicon Ultra-15 Centrifugal filter units, Millipore) as described previously [31]. To analyse the wall proteome, cell walls were isolated by hot SDS-extraction. The cell pellet obtained was used to prepare cell walls as described previously [36]. Briefly, the finely ground cell pellet was washed several times with PBS, and then subjected to breakage in a FastPrep bead beater (Savant Instruments Inc., Farmingdale, NY, USA) with glass beads (0.25-0.50 mm, 12-16 runs for 45 sec at speed 6) in the presence of a protease inhibitor cocktail. Full breakage was controlled by light-microscopic inspection. The pellet was washed several times with 1 M NaCl and stored overnight at 4°C. The following day, the pellet was washed several times with MilliQ-water and then boiled four times for 10 min in SDS extraction buffer (150 mM NaCl, 2% (w/v) SDS, 100 mM Na-EDTA, 100 mM β-mercaptoethanol, 50 mM Tris-HCl, pH 7.8), washed with MilliQ-water and lyophilized overnight. The resulting purified wall pellets were either stored at -80°C or directly reduced and S-alkylated [37]. 2.4 Mass spectrometric analyses of cell wall proteomes and secretomes. Lyophilized cell walls were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 (1 h at 55C). After cooling to room temperature and centrifugation, the supernatant was discarded. The reduced proteins were alkylated with 65 mM iodoacetamide in 100 mM NH4HCO3 for 45 min at room temperature in the dark. The samples were quenched with 55 mM dithiothreitol in 100 mM NH4HCO3 for 5 min. Subsequently, the samples were washed six times with 50 mM NH4HCO3 and either frozen in liquid nitrogen and stored at -80C or digested using 2 µg Trypsin Gold (Promega, Madison,WI) from a 1 µg/µl stock solution for 18 h at 37°C. The concentrated secretome samples were treated similarly, with reduction and alkylation on 10-kD cut-off spin filters (Amicon, Billerica, MA) [32]. The resulting tryptic digests were desalted using a C18 tip column (Varian, Palo Alto, CA) according to the manufacturer’s instructions. After evaporation of acetonitrile with a Speedvac (Genevac, Ipswich, England) the peptide concentration was determined at 205 nm using a NanoDrop ND-1000 (Isogen Life science, IJsselstein, The Netherlands) [38]. Each sample was diluted with 0.1% trifluoroacetic acid to a final concentration of 25 ng/µl, and 10 µl per run were injected onto an Ultimate 2000 nano-HPLC system (LC Packings, Amsterdam, The Netherlands) equipped with a PepMap100 C18 reversed phase column (75 µm inner diameter, 25 cm length; Dionex, Sunnyvale, CA). An elution flow rate of 0.3 µl/min was used along a linear gradient with increasing acetonitrile concentration over 45 min. The eluting peptides were directly ionized by electrospray in a Q-TOF (Micromass, Whyttenshawe, UK). Survey scans were acquired from m/z 350-1200. For low energy collision-induced dissociation (MS/MS), the most intense ions were selected in a data-dependent mode. 2.5 Data processing and analysis. After processing with the MaxEnt3 algorithm (MasslynxProteinlynx), the spectra were converted into pkl (peak list) files (available on PRIDE: www.ebi.ac.uk/pride: accession numbers 22716-22721) . Proteins were identified using MASCOT server 2.3.02 (Matrix Science, UK) and a database (6210 entries) consisting of a complete ORF translation of C. albicans and a list of regularly encountered contaminants. Enzyme was set to trypsin and allowing two miscleavages were allowed. Carbamidomethyl (C) was used as a fixed modification and Oxidation (M) as a variable modification. Mass tolerance and MS/MS tolerance were set to and a tolerance of 0.3 Da0.5 Da. Detected masses were charge deconvoluted to +1. Based on probabilistic MASCOT scoring a P value of < 0.05 was considered significant for peptide identification. At least three independently obtained biological samples were analysed for each condition and each was subjected to two MS/MS runs.