Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus

Abstract

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage–fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.

Introduction

Candida albicans is an important human opportunistic pathogen that can produce different types of infections ranging from superficial to systemic. Invasive candidiasis is an important cause of disease and mortality in immunosuppressed patients [1], [2], and the therapeutic arsenal is reduced and sometimes toxic [2], [3], [4]. Thus, the study of host response to Candida infections can be a very useful tool to discover new therapeutic strategies. Macrophages are crucial elements of the innate and adaptive immunity to systemic candidiasis [5], [6]. C. albicans is recognized by dedicated pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and lectins through its Candida-specific pathogen-associated molecular patterns (PAMPs) mainly mannan and β-glucan, present on its cell wall. Mannan and beta-glucan receptors, mannoside-binding lectins (such as Galectin-3), and Toll-like receptors 2, 4 and 6 recognize C. albicans cells and trigger the activation of macrophages (see recent reviews by Jouault et al. [7], Netea et al. [8] and Bourgeois et al. [9]). The recognition of C. albicans by macrophages causes phagocytosis and activation of cellular proinflammatory pathways (production of inflammatory mediators, toxic compounds like reactive oxygen (ROS) and reactive nitrogen (RNS) species). Macrophages also capture and process foreign antigens for their presentation to T cells, enabling host defense and immunological memory [10]. While macrophages display a wide variety of mechanisms to destroy the fungus, C. albicans attempts to survive the action of phagocytes by inhibiting the production of toxic compounds like NO [11], [12] by preventing phagolysosome fusion [13], [14], [15]; or modulating the pH of this compartment [15].

Macrophage murine cell line RAW 264.7 was used in this study, due to its ability to employ both oxidative and non-oxidative mechanisms to kill the fungus [16]. Macrophages activated by gamma-interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or lipopolysaccharide (LPS) produce two kinds of reactive products characterized by their cytotoxic activity: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI).

The introduction of proteomics has enabled the simultaneous analysis of changes in many proteins. Using a proteomic approach, we described the differential protein profile of control and murine macrophages interacting with live and heat-inactivated C. albicans cells and a pro- and anti-inflammatory effect was showed, respectively [17], [18]. 2D-Difference Gel Electrophoresis (2D-DIGE) [19] is a powerful analytical tool within the field of proteomics, allowing not only the relative quantitation of protein spot intensity across carefully matched gels, but also for detecting posttranslational modifications of the protein [20]. Because gel-based techniques have a bias toward abundant proteins, proteins in lower quantity are not often detected in the 2-DE analysis of total cellular proteins due to the complexity of these samples. The use of pre-fractionation methods by subcellular isolation or selective enrichment of a specific group of proteins provides an effective approach to eliminate this drawback. Thus in this work, we have combined cellular fractionation with 2D-DIGE technology to study cytosol, membrane, nucleus and cytoskeletal enriched protein fractions and analyze the differential protein profile of murine macrophages after 3 h of interaction with C. albicans.