Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome.

César Nombela,Concha Gil,Aida Pitarch,Miguel Sánchez

doi:10.1074/mcp.m200062-mcp200

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 integrated view of the cell wall proteome.

Highlights

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
After various washings of SDS-resistant cell walls, these were divided into two aliquots to extract cell wall proteins (CWPs) linked to the ␤-1,3-glucan network of the cell walls. (i) In the first aliquot, cell walls were treated with mild alkali, obtaining mainly an enriched fraction of proteins directly linked to ␤-1,3-glucan through their O-glycosyl side chains or by other alkali-sensitive linkages (Fig. 1, Fraction 2). (ii) In the second aliquot, a recombinant ␤-1,3-glucanase (Quantazyme ylg) was used to remove the ␤-1,3-glucan layer of the cell wall, releasing mainly an enriched fraction of mannoproteins indirectly attached to ␤-1,3glucan, via ␤-1,6-glucan, by a phosphodiester bridge (GPICWPs) and other CWPs anchored to ␤-1,3-glucan (Fig. 1, Fraction 3)
For several isoforms of a single protein, the minimum and maximum percentages of sequence coverage are only given. h Proteins analyzed by tandem mass spectrometry using a MALDI-TOF/TOF mass spectrometer. i Spots in-gel deglycosylated with PNGase F before trypsin digestion. j Peptide sequences that failed to match against C. albicans databases or partial short amino acid sequences that did not enable their positive identification. k Two of its peptides were identified as the sequences ESTVAGFLVGSEALYR and NDLTASQLSDKINDVR from S. cerevisiae Bgl2p, but not from C. albicans Bgl2p, notwithstanding the fact that C. albicans Bgl2p is present in databases and we have identified it here by peptide mass fingerprinting

Summary

Introduction

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. 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. The C. albicans cell wall is mainly composed of three components interconnected by covalent bonds: ␤-1,3- and ␤-1,6-

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Conclusion