Article pubs.acs.org/jpr
Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion Ana Gil-Bona, Lucía Monteoliva,* and Concha Gil
Downloaded by UNIV OF PRINCE EDWARD ISLAND on September 7, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.jproteome.5b00411
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Ctra. de Colmenar Viejo, 28034 Madrid, Spain S Supporting Information *
ABSTRACT: Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway. KEYWORDS: Candida albicans, ECM33, extracellular vesicles, LC−MS/MS analysis, secreted proteins, secreted aspartyl protease (Sap2), rapamycin
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INTRODUCTION Candida albicans is an opportunistic fungal pathogen of humans that inhabits the microbiota of healthy individuals and can cause superficial infections, such as oral or vaginal candidiasis, and invasive infections in immunocompromised patients.1 During both infections C. albicans uses a wide range of virulence factors and fitness traits, including the formation of biofilms, the morphological transition between yeast and hyphal forms, the expression of adhesins, and the secretion of hydrolytic enzymes.2,3 Biofilm formation on abiotic or biotic surfaces is connected to mucosal infection and infections that are related to medical devices. Biofilm formation is a very important virulence factor for the establishment of frequent candidiasis highly resistant to antifungal treatment and the host defense mechanism. The adherence of C. albicans to medical devices is mediated by cell wall proteins (CWPs) and is the first phase of biofilm formation. One factor that contributes to biofilm formation and to the process of virulence is the expression of hydrolytic enzymes that are most commonly associated with virulence.4 These enzymes contribute to colonization and infection by degrading host cell membranes to facilitate tissue invasion or to avoid or resist antimicrobial © XXXX American Chemical Society
attack by the host. The three most significant families of extracellular hydrolytic enzymes produced by C. albicans are the secreted aspartyl proteinases (Saps), phospholipase B enzymes (Plbs), and lipases (Lips). Within the Saps family, only Sap2 is significantly expressed in vitro when C. albicans is grown in the presence of bovine serum albumin (BSA) as the sole source of nitrogen, and sap2 mutants are unable to grow under these conditions.5 The secretome of C. albicans, defined as the fraction of the cell proteome secreted into the medium by the yeast, is composed of a large number of proteins involved in several vital processes. The proteomic analysis of the complete secretome under different conditions showed that C. albicans secretes different proteins to adapt to the environment.6,7 Recent studies have demonstrated that fungal organisms release many molecular components, such as pigments, polysaccharides, lipids, and proteins, into the extracellular medium via extracellular vesicles (EVs).8−11 These fungal EVs showed a diverse composition and the presence of proteins related to virulence and with immunological activity. Previous studies in Received: May 13, 2015
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DOI: 10.1021/acs.jproteome.5b00411 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Downloaded by UNIV OF PRINCE EDWARD ISLAND on September 7, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.jproteome.5b00411
Journal of Proteome Research Isolation of Extracellular Vesicles and Vesicles-Free Secretome
C. albicans showed that EVs transported cytoplasmic and other non-classically secreted proteins as well as cell wall-related proteins.12,13 Furthermore, inoculation of Galleria mellonella larvae with EVs of C. albicans, followed by challenge with C. albicans yeast cells, enhanced the survival of the larvae. The fungal cell wall is critical for virulence and pathogenicity, providing adhesive properties and a protective barrier and representing the initial point of interaction between the host and pathogen. The C. albicans cell wall is composed of β-1,6glucan (43−53%), β-1,3-glucan (30−39%), and chitin (2−6%) with different types of attached proteins, including glycosylphosphatidylinositol (GPI)-anchored proteins, such as the GPIlinked cell wall protein Ecm33 and Pir proteins.14,15 Previous studies have demonstrated the importance of Ecm33 in the biogenesis and maintenance of the C. albicans cell wall. The ecm33Δ/ecm33Δ mutant (RML2U) showed an altered cell wall structure and reduced adherence and damage to host cells. The formation of a catheter-associated biofilm in silicone elastomers was less dense and RML2U was avirulent in a murine model of systemic candidiasis.16−18 Furthermore, vaccination of mice with the RML2U strain protected them from a subsequent lethal infection with the virulent strain SC5314 in a systemic candidiasis model. The study of the cell surface of RML2U showed fewer cell wall organization- and biogenesis-related proteins at this cellular location.19 The lack of virulence of the ecm33 mutant might be related to a reduced secretion of hydrolytic enzymes, such as Saps and Plbs, among other factors, which contributed to the host-cell damage. To elucidate potential changes in the secretion pattern of virulence proteins from the ecm33 mutant as well as whether the mutant cell wall was implicated in the release of unexpected cell wall proteins into the medium, the protein composition of the complete RML2U secretome (EV-free medium and extracellular vesicles) was analyzed.
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Samples were isolated and analyzed according to Gil-Bona et al.12 In brief, all of the steps were carried out at 4 °C to avoid proteinase activity and vesicle rupture. Yeast cells were separated from culture supernatants by centrifugation at 5524g for 15 min. The resulting supernatants were collected and centrifuged again at 15 344g for 30 min to remove smaller debris. The supernatant was collected and concentrated using a Centricon Plus-70 centrifugal filter (cutoff filter 100 kDa, Millipore). The concentrated culture was centrifuged again at 4000g for 15 min and 15 000g for 30 min to remove smaller debris. The resulting supernatant was ultracentrifuged at 100 000g for 1 h. The supernatant was recovered for secretome analysis, and the pellet containing the EVs was washed and centrifuged again at 100 000g for 1 h. The pellet was used for the proteomic analysis or it was embedded in a fixative solution (as described later) for electron microscopy analysis. The flow through of the 100 kDa filter and the supernatant recovered from the first ultracentrifugation of the vesicles purification were concentrated approximately 20-fold using a 10 kDa cutoff filter (Millipore) to obtain C. albicans-secreted proteins that were soluble in the culture medium. The methanol/chloroform precipitation procedure based on the Wessel et al.21 protocol was used to precipitate and clean the sample. Three independent biological samples were performed. Protein Digestion
Two preparations were processed for proteomic analysis: (a) EVs and (b) EV-free supernatant proteome. The concentrated proteins of each sample were resuspended in 0.5 M triethylammonium bicarbonate (TEAB) and quantified using the Bradford protein assay. Aliquots of 13 μg of vesicles and EV-free supernatant samples were adjusted to the same volume of ammonium bicarbonate (NH4HCO3). All samples were reduced by adding 100 mM DTT for 30 min at 37 °C and alkylated with 55 mM iodacetamide for 20 min in the dark. Then, digestion was performed by adding recombinant sequencing-grade trypsin (Roche) 1:20 (w/w) overnight at 37 °C. Subsequently, the produced peptides were cleaned up with a POROS R2 column. The peptides were eluted with 80% acetonitrile (ACN) in 0.1% TFA, dried in a Speed-Vac, and resuspended in 0.1% formic acid. The samples were stored at −20 °C before the nano LC− MS/MS analysis.
MATERIAL AND METHODS
Microorganisms and Culture Conditions
C. albicans SC531420 is the wild type of RML2U mutant (ecm33Δ::hisG/ecm33Δ::hisG ura3Δ::imm434/ura3Δ::imm434::URA3) and the doubled-complemented strain RML4U (ecm33Δ::hisG::ECM33-cat/ecm33Δ::hisG::ECM33-cat ura3Δ::imm434/ura3Δ::imm434::URA3).17The laboratory strain Saccharomyces cerevisiae BY474 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (EUROSCARF) was used as control of nonbiofilm formation. Yeast strains were maintained on YPD (1% yeast extract, 2% peptone, and 2% glucose) agar plates at 30 °C. For extracellular proteome experiments, yeast cells were precultured in liquid synthetic defined (SD) medium (20 g/L glucose, 5 g/L ammonium sulfate, 1.7 g/L nitrogen base, and 2.2 g/L amino acids mix) with rotary shaking (200 rpm) at 30 °C, during 7 h. The preculture was used to inoculate flasks containing 1 L of SD medium adjusting OD600 to recover the culture at final OD600 of 4, 16 h later. For Sap2 activity assays, a single colony from each strain was pregrown overnight at 30 °C in SD medium, washed, and subcultured at an OD600 of 0.2 in YCB-BSA medium (23.4 g/L yeast carbon base and 4 g/L BSA, adjusted to pH 4.0 with HCl) and subsequently grown at 30 °C for different times. For biofilm assays Sabouraud liquid medium (1% peptone and 4% glucose) at 37 °C was used.
LTQ-Orbitrap Velos Analysis
Desalted peptides were concentrated (online) on a C18-A1 ASY 0.1 × 20 mm C18 RP precolumn (Thermo Scientific) and then separated on a Biosphere C18 RP-column [C18, inner diameter 75 μm, 15 cm long, 3 μm particle size (NanoSeparations)] and were eluted using a 150 min gradient (0− 140 min from 2 to 35% Buffer B, 140−150 min 35−95% Buffer B. Buffer A: 0.1% formic acid/2% ACN; Buffer B: 0.1% formic acid in ACN) at a flow-rate of 250 nL/min on a nanoEasy HPLC (Proxeon) coupled to a nanoelectrospay ion source (Proxeon). Mass spectra were acquired on the LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) in the positive ion mode. Full-scan MS spectra (m/z 400−1800) were acquired in the Orbitrap with a target value of 1 000 000 at a resolution of 30 000 at m/z 400, and the 15 most intense ions were selected for collision-induced dissociation (CID) fragmentation in the LTQ with a target value of 10 000 and normalized collision energy of 38%. Precursor ion charge-state B
DOI: 10.1021/acs.jproteome.5b00411 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel. The gel was stained with Coomassie blue (Quick Coomassie Stain, Generon). For silver staining, the gel was destained, fixed (40% MeOH, 10% acetic acid v/v), and stained by using the Silver Stain Kit (Bio-Rad). For Western blotting, 10% SDS-polyacrylamide gels were transferred to nitrocellulose membranes and blocked in 5% milk. Reversible Ponceau staining was applied to check equal protein quantity loading of gels and transferring to the membranes. Western blots were probed with anti-Sap2 (a gift from M. Monod, Centre Hospitalier Universitaire Vaudois, Switzerland) at 1:3000 and then with fluorescently labeled secondary antibodies: 1/2000 IRDye 800 goat antirabbit IgG (LI-COR Biosciences). The Western blotting was performed with the Odyssey system (LI-COR Biosciences, Nebraska). To detect BSA in the medium, 20 μL of the supernatant was loaded onto a 12% SDS-PAGE gel, and proteins were visualized with Coomassie staining.
screening and monoisotopic precursor selection were enabled. Singly charged ions and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 30 s.
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Protein Identification and Analysis
Protein identification from raw data was carried out using a licensed version of search engine MASCOT 2.3.0 with Proteome Discoverer software version 1.4.1.14 (Thermo Scientific). A database search was performed against the CGD21 database (6221 sequences, 2012). Search parameters were oxidized methionine as variable modification, carbamidomethyl cysteine as fixed modification, peptide mass tolerance 10 ppm, 1 missed trypsin cleavage site, and MS/MS fragment mass tolerance of 0.8 Da. In all protein identification, the FDR was
