Sequential Fractionation and Two-dimensional Gel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome

Abstract

The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and inte-

Enzyme secretion by ECM fungi and exploitation of mineral nutrients from soil organic matter

“…This can be at the outside the plasmalemma with GPI anchors or by binding to cell wall components after transglycosilation. Other binding forms that have been detected by sequential extraction of cell wall enzymes are weak ionic forces or disulfide bridges (Pitarch et al 2002;Rast et al 2003). Experimental evidence for the presence of relatively stable bonds of extracellular enzymes in intact ECMs is given by studies with excised ECM that after repeated washing steps in buffer maintained their activity (Pritsch et al 2004).…”

Decoding Serological Response to Candida Cell Wall Immunome into Novel Diagnostic, Prognostic, and Therapeutic Candidates for Systemic Candidiasis by Proteomic and Bioinformatic Analyses

“…Thereafter proteins were either visualized with silver nitrate (22,50) or electrotransferred to nitrocellulose membranes (51). M r and pI values were assigned after calibration of gels with internal 2-DE standards (Bio-Rad) using Melanie 3.0 software (GeneBio, Geneva, Switzerland).…”

“…Beyond encouraging chemical and/or enzymatic approaches for its extraction from intact cells or isolated walls (22)(23)(24)(25) and bypassing their inherent troubles (glucan and/or chitin sidechain residues and protein modifications (19,26)), an innovative stratagem, based on the analysis of proteins secreted from protoplasts in active cell wall regeneration, has also recently enabled this Achilles' heel to be successfully profiled (26 -28). This modus operandi draws on the observation that these wall biogenesis-related proteins are not retained into the nascent cell wall during the early stages of the regeneration process and are thus shed into the extracellular medium (26,29).…”

2D-immunoblotting analysis of Sporothrix schenckii cell wall

“…In addition, there are some reports dealing with the role of a cell wall glycoprotein of 70 kDa in adhesion of S. schenckii to host tissues and extracellular matrix components as well as fungal pathogenesis (Nascimento et al 2008, Ruiz-Baca et al 2009, Teixeira et al 2009). 2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al 2002, Gautam et al 2007, Young et al 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii.…”

“…2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al 2002, Gautam et al 2007, Young et al 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii.…”