Dataset Information

Carbon source-induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

ABSTRACT: 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.

INSTRUMENT(S): Q-Tof ultima

ORGANISM(S): Candida Albicans Sc5314

SUBMITTER: Clemens J. Heilmann

LAB HEAD: Clemens J. Heilmann

PROVIDER: PXD000008 | Pride | 2013-02-20

REPOSITORIES: Pride

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Carbon source-induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans.

Ene Iuliana V IV Heilmann Clemens J CJ Sorgo Alice G AG Walker Louise A LA de Koster Chris G CG Munro Carol A CA Klis Frans M FM Brown Alistair J P AJ

Proteomics 20121101 21

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We ...[more]

PMID: 22997008

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