Proteomics Unravels Extracellular Vesicles as Carriers of Classical Cytoplasmic Proteins in Candida albicans Ana Gil-Bona, †,‡ Arancha Llama-Palacios, †,‡,∥ Claudia Marcela Parra, †,‡,⊥ Fernando Vivanco, § Ce ́ sar Nombela, †,‡,# Lucía Monteoliva,* ,†,‡ and Concha Gil †,‡ † Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain ‡ Instituto Ramó n y Cajal de Investigació n Sanitaria (IRYCIS), 28034 Madrid, Spain § IIS-Fundació n Jime ́ nez Díaz, Immunology, 28040 Madrid, Spain *S Supporting Information ABSTRACT: The commensal fungus Candida albicans secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture super- natants from C. albicans were separated into EVs and EV-free supernatant and analyzed by LC−MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by C. albicans secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model. KEYWORDS: Candida albicans, extracellular vesicles, LC−MS/MS analysis, secreted proteins, moonlighting proteins ■ INTRODUCTION Candida albicans is a commensal fungus in healthy humans and may cause different types of infections mainly in immunocom- promised patients. The pathogenicity of C. albicans is attributed to several virulence factors, such as the ability to evade host defenses, adherence, biofilm formation and the secretion of hydrolytic enzymes such as proteases and phospholipases. 1−3 Secreted proteins are important for the commensal to pathogenic change, not only because some of them are hydrolytic enzymes, but also because they are necessary for their adaptation to the environment, in this particular case, the host. In all living organisms, and in particular in yeast, the secreted proteins are involved in different vital processes including biofilm formation, tissue invasion, immune evasion, cell wall integrity maintenance and nutrient acquisition. 4,5 Therefore, studies of the “secretome”, as the part of the cell proteome secreted into the medium, are of outstanding interest. All eukaryotic cells have a classical secretory pathway that includes the endoplasmic reticulum (ER), the Golgi apparatus and a complex system of vesicles, to transport proteins to the plasma membrane or to the extracellular region, including the periplasmic space, the cell wall or the extracellular medium. 6 Although few works analyzing C. albicans proteins involved in these pathways have been reported, 4,7 the C. albicans genome contains all the orthologues to Saccharomyces cerevisiae genes involved in the classical secretory pathway, which is highly conserved. Proteins secreted through this pathway have an amino-terminal signal peptide that is responsible for directing them to the inside of the ER. This signal sequence has been used in genetic and bioinformatic studies to define the C. albicans secretome. 8,9 Several C. albicans virulence studies highlighted the importance of some families of proteins secreted through the secretory pathway as virulence factors, including secreted aspartyl proteases (Saps) and phospholipases (Plbs). 2 Proteins involved in cell wall synthesis, such as the exo-1,3-β-glucanase (Xog1) or the endoglucanase (Eng1), are also secreted by the classical protein secretory pathway. 10−12 In addition, an important group of plasma membrane and cell wall proteins are the glycosylphosphatidylinoditol (GPI)-anchored proteins such as Ecm33. 13,14 They are also secretory proteins with a signal peptide, serine- and threonine-rich region and a potential C-terminal domain for GPI anchor attachment. 15,16 However, proteins without a signal peptide have been detected in the extracellular medium of C. albicans and other yeast species as published in the last years. 17−23 Recent proteomic analysis of Special Issue: Environmental Impact on Health Received: July 30, 2014 Article pubs.acs.org/jpr © XXXX American Chemical Society A dx.doi.org/10.1021/pr5007944 | J. Proteome Res. XXXX, XXX, XXX−XXX